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This is to certify that the

thesis entitled

DEVELOPMENT OF LASER PHOTOBLEACHING SYSTEM
FOR USE WITH UNIQUE MICROSCOPY TECHNIQUES

presented by

Mark N . Melkerson

has been accepted towards fulfillment
of the requirements for

Masters Wdcgree in .Menhanical. Engineering

./“ . '

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Major Hofessor

Date 73W!“ O; /7d>b

0-7639 MS U i: an Affirmative Action/Equal Opportunity Institution

 

 

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“ your record. FINES will
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DEVELOPMENT OF LASER PHOTOBLEACHING SYSTEM
FOR USE WITH UNIQUE MICROSCOPY TECHNIQUES

by

Mark N. Melkerson

A Thesis

Submitted to
Michigan State University
in Partial Fulfillment of the Requirements
for the Degree of

MASTER OF SCIENCE

Department of Mechanical Engineering

1986

zesex

42

ABSTRACT

DEVELOPMENT OF LASER PHOTOBLEACHING SYSTEM
FOR USE WITH UNIQUE MICROSCOPY TECHNIQUES

By

Mark N. Melkerson

An experimental laser photobleaching system has been developed. The
system is functional and preliminary results have been obtained. The
laser system was applied to four microscopy techniques. Two of the
techniques were designed to select single bilayer vesicles "in situ"
prior to their use in the quantitative determination of membrane
characteristics. The developed fluorescence contrast technique and
comparison technique for selection of single bilayer vesicles were
unsuccessful. The other two techniques were applications of fluores-
cence recovery after photobleaching (FRAP) for the determination of
lateral diffusion coefficients of fluorescently labeled molecules
present in membranes. The spot technique predicted lateral diffusion
coefficients for rhodamine (4.6 x 10-611) in water ranging from (0.2 to
1.36 x 10-6) cmzlsec. These lateral diffusion coefficients agree in

6 cmzlsec). The multi-point

magnitude with published values (1.2 x 10"
technique was unsuccessful in predicting lateral diffusion coefficients

for rhodamine (4.6 x lO-GM) in water.

Dedicated to my Dad, my Mom, and my Uncle Walt

iii

ACKNOWLEDGMENTS

I would like to express my appreciation to the following people
whose help was invaluable in the completion of my thesis: Leonard J.
Eisele -- for his help with the design and machining of the system
fixtures; Ronald K. Kraus -- for his help with the system instru-
mentation; Robert E. Rose -- for his help with the heat exchanger and
the electrical work; Ronald L. Haas -- for his help with the laser; John
F. Holland -- for his help with the laser system design and software;
Melvin S. Schindler -- for his help with fluorescence recovery theory
and application; Carol J. Bishop -- for her help with the word process-
ing of the thesis; Gerna J. Rubenstein -- for her help with the editing
of the thesis; Lyla J. Melkerson-Watson -- for her help with the
photography; and John J. McGrath -- for his support and advice.

I‘would especially like to thank Serge Weiss for his work with the
quantization applications of the laser photobleaching system.

One last note of thanks goes to Meridian Instruments Incorporated,

Okemos, Michigan for their help with the laser system design and
controlling software.

iv

TABLE OF CONTENTS

Page

LIST OF TABLES . vii
LIST OF FIGURES viii
NOMENCLATURE x
1.0 INTRODUCTION: PHOTOBLEACHING SYSTEMS AND MICROSCOPY l

TECHNIQUES

1.1 Motivation L

1.2 Background 2

1.3 Objectives 7

2.0 ANALYSIS 9
2.1 Theoretical 9

2.2 Experimental L4

3.0 FRAP LASER SYSTEM 21
3.1 Introduction 2i

3.2 Hardware 23

3.3 Software 33

3.4 Operational Characteristics 40

4.0 APPLICATION OF SYSTEM 49
4.1 Quantization Techniques A9

4.2 FRAP Techniques 58

4.3 Application Results to

)

5.0 DISCUSSION 72
6.0 CONCLUSIONS 84

7.0 SUGGESTIONS FOR FUTURE WORK 06

APPENDIX

???’?>?’>
@U‘J-‘WNH

APPENDIX B

3.2

APPENDIX C

APPENDIX D

APPENDIX E

APPENDIX F

APPENDIX G

REFERENCES

Laser Operation and Care
Laser Start Up

Laser Shut Down

Mode Selection

Cleaning and Peaking Output
Lasing -- Multi-Line

Lasing -- Single-Line

Laser Photobleaching System Programs
Control Only Operating System
Program LOPERA

Subroutine MPOSIT

Subroutine CONVRT

Subroutine MOVEM

Subroutine CLOCIN

Interrupt Service Routine DTACHA
Subroutine FPLACE

Subroutine TRANSV

Subroutine SHUTTR

Subroutine OCTREP

Data Acquisition and Control Operating System
Program LASCON

Subroutine VALUES

Subroutine CPARAM

Subroutine INSHUT

Subroutine TRANSV

Subroutine SHUTTR

Subroutine OCTREP

Subroutine SETIME

Subroutine NAMEF

Subroutine HELPME

Subroutine LPARAM

Subroutine EXPRUN

Interrupt Service Routine ATDC13
Interrupt Service Routine LTIME
Subroutine INTDAT

Subroutine LASET

Subroutine STNDBY

Beam Alignment Operations

Solution to Diffusion Problem
Liposome Preparation
BLM Preparation

Determination of Diffusion Coefficient Using
Least Squares Fit

vi

Page

90
9O
90
91
92
95
96

98

98

98
106
109
109
112
100
101
103
103
104
114
114
116
119
121
122
123
123
125
128
130
132
133
137
138
139
141
143
144

149
152
154

155

156

TABLE 3.

TABLE 3 .

TABLE 3.

TABLE 4.

TABLE 4.

TABLE 5.

LIST OF TABLES

Laser System Intensity Levels

Intensity Comparisons

Beam Reduction Results

Mean Fluorescent Intensities of Microspheres
Diffusion Coefficients

Bleaching Energy Densities

vii

Page
27
42
44
64
7O

82

Figure

Figure

Figure

Figure

Figure

Figure
Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

1.1

1.2

2.1

2.2

2.3

3.1

3.2

3.3

3.4

3.5

4.1

4.2

4.3

4.4

4.5

4.6

LIST OF FIGURES

Membrane structure of
reproduced from (46).
Distribution of normalized phase contrast intensi-
ties versus radius for liposomes reproduced from

(27).

a) a liposome b) a BLM;

a) diffusion only
c) combined diffusion and flow; repro-

Normalized fluorescence recovery
b) flow only
duced from (1).

I1/2/‘0 °r ’F ' T1/2

for a Gaussian beam; reproduced from (1).

Factor 1(7D I ltF) versus K

A graphic illustration of the log-log superposition
method; reproduced from (1).

Components of the laser photobleaching system.
Heat exchanger.

Neutral Density Filter Positioning Unit - solenoid
mechanism.

Optical path.

Fluorescence Response Curve for green excitation,
produced from (41).

re-

Intensity detection of a) phase contrast b) fluores-

cent contrast.
BLM formation apparatus.
BLM formation stages.

Phase contrast and fluorescent contrast results:

Page

15

l7

19

21
24

28

3O

42

51

55
55

61

x-fluorescent contrast data; - Servuss and Boroske phase

contrast data; background reproduced from (27)

Long term exposure response of fluorescent micro-
spheres reproduced from data supplied by (39).

Long term exposure response of liposomes reproduced
from data supplied by (39).

viii

66

67

Page

Figure 4.7 Typical recovery responses for spot - FRAP tech- 69
nique.
Figure 5.1 Low frequency drift of the photometer output. 77

ix

NOMENCLATURE

-attenuation factor with respect to total output power
-initial concentration of fluorescently labeled molecules
-concentration of fluorescently labeled molecules
-latera1 diffusion coefficient

-fluorescence intensity

-normalized fluorescence intensity

-bleaching intensity

-amount of bleaching

-immobile fraction

-superscript denoting a measured value

-summation index

-Legendre Polynomial of order n

-total laser power

-quantum efficiency (absorption*emission*detection)
-position

-bleaching time

-time

-bulk flow velocity

-effective beam diameter (e.2 height, 86%)

-extent of bleaching (O < a < 1)

-partial derivative with respect to x

-ration of Il/thi where i - D,F

-effective beam radius (9-2 height, 86%)
-characteristic time for diffusion

-characteristic time for flow

1.0 Introduction

1.1 Motivation

The development and research described in this thesis were conducted
at the Bioengineering Transport Processes (BTP) laboratory of Michigan
State University. The project was to adapt and develop the experimental
instrumentation needed to to study membrane changes related to freezing
injury and to determine the number of molecular layers present in the
membranes of models of biological cells. This development and research
were part of a study relating the transport properties of cell membranes
to model cell membranes during freezing and thawing.

‘The number of molecular layers and changes in membranes affect the
behavior of biological cells and models during freezing. As water in
the environment surrounding a cell freezes solutes separate from the
crystal structure of the ice. The increase in ice surnmnuflng the
cells causes the solutes to become more concentrated in the remaining
liquid. 13"; increased solute concentration in the "local" environment
causes the cell to attempt to regain an equilibrium between the
chemical potential inside and outside of the cell. The dynamic response
for such a situation is governed by the permeability characteristics of
the cell or cell models.

The permeability of a membrane to water dependsnnxnithe amount of
resistance encountered in crossing the membrane during freezing or
thawing. An increase in the number molecular layers present in a
membrane decreases its permeability to water across the membrane due to

the increased resistance from each layer (42). Changes in mobility of

molecules or lesions in membranes also affect permeability. Leakage
eliminates the resistance to crossing the membrane and quickly restores
equilibrium. Effects on permeability of water across membranes, hence

freezing and thawing of cells, due to changes in mobility of molecules

from changes of phase in membranes described in (47).

From these effects on permeability of membranes it is apparent that
the incorporation of this additional information on the number of

molecular layers and molecular mobility into models of freezing and

thawing would better estimate the actual processes.

1.2 Background

The models used in this study are liposomes and bilayer lipid

membranes (BLMs). Liposomes and BLMs are comprised of a double layer

(bilayer) of phospholipids. (See Figure 1.1.) Phospholipids form

these bilayers because of their hydrophobic "tail" portions of the

molecule and hydrophilic "head” groups of the molecule. (See Figure
1.1.)

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Membrane structure of

a) a liposome b) a BLM, reproduced
from (46).

There are many techniques for determining the number of bilayers
and the lateral diffusion coefficients in the phospholipid membranes of
liposomes. The determination of the number of bilayers (lamellea) in
liposomes is usually accomplished by qualitative means. The most widely
used technique is visual. It uses phase contrast microscopy to choose
liposomes with the faintest (least visible) membranes. The results from
this visual technique for choosing liposomes varies greatly depending
on the observer, the light intensity used, and the magnification used.

Liposomes and BLMs can be considered reasonable models for the study
of membrane characteristics in biological cells for three reasons.
First, phospholipids comprise 50 to 75 percent of cell membranes.
Secondly, liposomes are generally spherical and trap solvents within
themselves much like a biological cell. Finally the phospholipids
making up liposomes and BLMs are extracted from biological materials

such as egg yolk.

One quantitative technique for determining the number of bilayers in
liposomes was developed by Servuss and Boroske (27,38). It also uses
phase contrast. It eliminates the subjective "visual sehunfion" but
still deadjildith the contrast intensity of the membrane. The contrast
intensity of liposomes is measured by a photometer system, normalized
and then plotted versus the liposome radius. The results of their work
is shown in Figure 1.2. Servuss and Boroske proposed that each
grouping was a different number of lamellea (bilayers)in liposomes. The
grouping with the lowest normalized define the unilamellar liposomes.

This technique provided some indication that quantization of the number

of bilayers was possible. But to determine the actual number of
bilayers present in any liposome the differences in the normalized

intensities need to be more distinct.

 

 

 

 

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radius for liposomes reproduced from (27).

Indications that a fluorescence technique for determining the number
of lamellea in liposomes could be possible came from observations of
others. Chan and Webb (28) used fluorescently labeled phosolipid
multilayers to measure increased emission intensities for increasing
numbers of lipid layers. Tamm and McConnell (35) have determined that

fluorescent intensity doubles when a second fluorescently labeled

monolayer is applied to a single fluorescently labeled monolayer. Fahey
and Webb (6) stated that the number of bilayers in an unilamellar
fluorescently labeled liposome chosen by visual techniques could be
determined by comparing it to a similarly labeled BLM. These observa-
tions suggested that a fluorescence method to measure the number of
bilayers in liposome membranes could be developed.

Among the fluorescence methods for studying membrane characteristics
there are two which determine lateral diffusion coefficients: Fluor-
escence Correlation Spectroscopy (FCS) and Fluorescence Recovery After
Photobleaching (FRAP)(1). The FCS method is based on a statistical de-
termination of the motion of fluorescently labeled molecules in mem-
branes. This method was rejected for this study because the associated
analysis requires an extensive background in statistical probability.
The FRAP method is based on the fluorescence intensity recovery due to
the diffusion of fluorescently labeled molecules in a membrane. This
method was chosen because the associated analysis is based on the
diffusion equation.

The FRAP method for determining the lateral diffusion coefficients
of molecules can be outlined as follows (1). Fluorescently labeled
samples with a low molecular concentration of fluorescent probe are
observed on a microscope. A focused low intensity beam (at the proper
wavelength) is used to excite the fluorescing material to a higher
energy level. The fluorescent material emits energy at a longer
wavelength as it returns to its ground energy level . This emitted
energy is detected by a photometer and recorded. This initial intensity
can be related to the initial concentration of fluorescently labeled

molecules in the sample excited by the focused beam. The fluorescent

sample is then excited by a brief high energy pulsezuleOO-1000O
times greater than the observing intensity excitatflnn This pulse
eliminates the ability of the probe to emit energy. Unable to emit
energy the spot under the focused beam appears black..This condition of
blackness is called "photobleaching". The black region emits almost no
energy initially. It begins to emit energy as non-bleached fluorescent
molecules diffuse into the region under the focused beam at the initial
observing intensity. The affected fluorescent molecules diffuse out of
the region at the same time. This recovery of the fluorescence emission
intensity is monitored and recorded. Ideally the fluorescence intensity
should recover to its initial level. The lateral diffusion coefficients
of molecules can be determined from these fluorescence recoveries.
Lateral diffusion coefficients can be determined by using any of
the four general FRAP techniques. These FRAP techniques are called the
spot, multi-point, normal-mode, and periodic pattern techniques. The
spot technique (1) is the earliest of the four. This technique is
workable but involves some complex mathematical analysischmeto the
possibility of bulk motion of the sample. Bulk motion:hnxpduces a
non-homogeneous term involving the velocity of the sample to the
diffusion equation. Many researchers have avoided this problem by
affixing the cells in place or using sealed samples of fluorescent
solutions. This leaves the velocity term zero. The zero velocity term
reduces the problem to just a simple diffusion equatflnrfbr the con-
centration of fluorescently labeled molecules. This simplification does

not work for systems where the velocity term was not zero.

The other three FRAP techniques (multi-point, normal-mode, and
periodic pattern) are attempts to account for the bulk motion pos-
sibilitqn. 'The multi-point technique (2) accounts for the bulk motion
possibility by monitoring the fluorescent recovery at a number points
including the bleached spot. If the membrane does move, the shift in
intensity can be detected while still tracking the recovery. The
normal-mode FRAP technique (44) monitors the fluorescent intensity along
a line which passes through the entire cell or liposome. In this tech-
nique a spot on the very edge of the membrane, where the line of
excitation is normal to the surface, is bleached. The recovery of
points along this line are monitored and any bulk motion can be ac-
counted for. In the periodic pattern technique (15) many lines.ane
bleached. The recovery of these lines across the plane of the sample

are monitored to account for the possibility of bulk motion.

1.3 Objectives

There were three main objectives for the study described in this
thesis: 3) produce an operational laser photobleaching system opera-
tional, b) verify some photobleaching techniques, and c) develop
fluorescence technique for the quantization of the number of bilayers in
liposomes.

Producing an operational laser photobleaching system operational
required a decision on which FRAP technique to use. Tfluenmlti-point
ZFRAP technique has been selected as the system model for two reasons.
It allows for the determination of lateral diffusion coefficients with
the existence of bulk flow of the sample. 2) It can also perform the

spot FRAP technique with only minor software modifications. Other

factors con-tributing to the decision were: a) the microscope used by
Koppel (2) was identical to the one in BTP laboratory, b) a system using
the normal-mode FRAP technique existed at MSU (45), and c) budget
limitations.

Meeting this first objective required: a) gaining experience on an
existing FRAP laser system (48), b) ordering all required equipment, c)
writing necessary software, and d) interfacing the equipment and
software.

The objective of verifying a photobleaching technique was to show
that a laser photobleaching system could perform the determination of
lateral diffusion coefficients using the spot and multi-point FRAP
techniques. Verification beyond establishing that the system was
functional would be left to others.

The last major objective of this study was to develop fluorescence
microscopy techniques to determine the number of bilayers in liposomes.
This determination was to include investigating: a) the phase contrast
technique used by Servuss and Boroske (27,38), b) designing a "fluores-
cence contrast" technique and c) designing a technique for a fluores-
cence comparison of liposomes to a known phospholipid single bilayer.

These three major objectives outlined the initial goals of this
study. As in most developmental and experimental work mmmenmdifi-
cations of these goals were made. The remainder of this study presents
an analysis of FRAP theory, a description of the laser photobleaching
system and applications, results, conclusion, and suggestions for future

work.

2.0 Analysis

In this section the theoretical and experiment bases for the
Fluorescence Recovery After Photobleaching (FRAP) microscopy technique
are presented. The majority of the content of this presentation is
extracted from a detailed analysis by Axelrod, et al. (1). Their
analysis has been used by many researchers using FRAP systems. (2,9 ,
18,19). Also the presentation includes the solution of the diffusion

equation required by the FRAP technique used in this study.

2.1 Theoretical

FRAP theory is based on the lateral transport (diffusion and/or
convective flow) of bleached and non-bleached fluorescently labeled
molecules into and out of a defined region of a membrane. Fluorescently
labeled molecules allow the researcher a convenient method for measuring
the concentration at a given position and time. Fluorescence and
concentration of a spherical membrane are related by the following

relationship (1)

Fx(t) =- (qlA) I I(r) CK(r.t) a2: (2.1)

The equation is valid for time t 2 O, with zero denoting time just after
bleaching. The term q is the product of all the quantum efficiencies of
light absorption, emission, and detection (0 < q < 1). The attenuation
factor of'tflue beam during observation with respect to the total inten-
sity is represented by the term A. The bleaching intensity is denoted

by I(r). (1.5.10). The concentration CK(r,t) of fluorescently labeled

molecules at position r and time t is the solution to an equation
describing the lateral transport of fluorescently labeled molecules. The
subscript K denotes a dependence on the amount of bleaching, K = aI(O)T.
Here a is a constant characterizing the extent of bleaching (O<a<1) and
T is bleaching time. Integrating the product of the bleaching intensity
and concentration over a defined region gives the total possible
fluorescence. The total fluorescence multiplied by thetmuummm ef-
ficiency and attenuation factor gives the predicted fluorescence at
time, t, and location, r, of a spherical membrane.

The application of the fluorescence equation 2.1 requires an
expression for both bleaching intensity and concentration. The bleach-
ing intensity I(r) for properly aligned lasers has been shown to have a
Gaussian profile (1,5,10). An expression for the bleaching intensity is

given by

2P
I(r) - —§ exp (4:21.12) (2.2)
1w
Here w, the effective laser beam size, is the half widthtunmaining
86.5% of the beam (efl2 height). Po is the total laser power. (1,5). The
first group of terms represents the total power applied per area. The
last term describes the exponential nature of the intensity with
increasing distance (rZw) from beam application position.
Obtaining an expression for the concentration C(r,t) involves

solving an equation describing the lateral transport of a single type of

fluorophore. This is expressed by the equation

3C 2 3C
8t - DV C - V0 3: (2.3)

10

D is the lateral diffusion coefficient, and V0 is the uniform flow
velocity in the x direction. (1). The boundary condition imposed on
this equation is that the concentration at large distance from the
bleached area but still on the membrane is finite, C(0,t) I Co’ where Co
is initial concentration of fluorescently labeled molecules. The
initial condition depends on the assumption that photobleaching a
fluorophore to a nonfluorescent one is an irreversible first-order
reaction with rate constant aI(r) (l/sec) with no chemical regeneration.

(1,6,11). The initial condition is given by

C(r,o) - Co expl-a TI(r)] (2.4)

In this equation a is a constant characterizing the extent of bleaching
and T is bleaching interval (1).

Methods for solving the lateral transport problem vary greatly among
researchers. (1,12,13,14). Many researchers other than Axelrod, et a1.
only solve the concentration equation for the simplified case where the
flow is assumed to be zero. (12,13,15). The differential equation

remaining after the assumption Vo 3 O is simply the diffusion equation:

3C 2
at IN C (2.5)
Since the case V0 = O is also a solution presented by Axelrod, et al.

the equation (2.5) was solved here as a verification of their analysis.
The diffusion equation is solved for a spherical coordinate system using
separation of variables. The solution is presented in terms of Legendre
polynomials. The solution obtained for the diffushn1equathm1is the

following:

11

O
Q_ a (2n + 1)
co l/2(l + c0800) + IE: 2(n + 1) [cosoo Pn(cosoo)
n-l
2
- Pn-l (cosoo)] Pn (coa0)e_u(n+l)(nlp )t (2.6)

Here (oz/D) is the characteristic time for diffusion. The term 90
describes the circular bleached region and p is the membrane radius. The
expressions Pn are Legendre polynomials of degree n. For mathematical
expressions for these polynomials see (16,17). A detailed analysis of
the solution of the lateral transport equation for the case of dif-
fusion only can be found in Appendix D. Identical solutnnuiof the
diffusion equation can be found in many references (12,13,14).

Expressions obtained for bleaching intensity and concentration allow
for the calculation of the theoretical fluorescence. Again this
presentatdxni refers to the solutions presented by Axelrod as do most
other researchers working with FRAP systems (2-4,6-9,18,19).

Axelrod presents solutions of the fluorescence equathn:2.1 for
three possible modes of transport. These modes of transport are
diffusion only with Vo 3 0, flow only with D 8 O, and combined diffusion
and flow. In all cases the solution assumes that the beam has a
Gaussian profile. Only the solutions for each mode are presented. For

derivations see reference (1).

Diffusion Only (VG-O)

 

The fluorescence is given by

12

Fxhz) . (q Paco/A) Z (-K)nln!] [1 + n(l + ZterH—l (2.7)
n-O

In the equation TD 8 w2/4D is the characteristic diffusion time, and K =

aI(O)T is the amount of bleaching (a non-dimensional energy density).

Uniform Flow Only (D=O)

 

The expression for fluorescence in this case is the following:

0
PRO.) - (q Paco/A) Z (- x>“/<n + 1)! epr- 2nl(n + l)l(tltl,)2 (2.8)
n=0

Here, I I w/Vo is the characteristic time for uniform flow.

F

Combined flow and diffusion

The fluorescence is shown to be expressed as:

° (- x)“ exp{- 2 (calf nl[1 + n (1 + 21:15)]
F(t)-(chlA)
K o 0 :E: n! [l + n(l + 2t/rn)l

n80
(2.9)

where T a w/V and T a w2/4D. In all cases qP (C /A i F t) describes
F o o o 0 K
the fluorescence before bleaching.
In all three cases it is difficult to use or plot typical results
for the fluorescence equation 2.1 without introducing a method to

normalize them. A convenient way to normalize the expressions for

fluorescence is to use the following

F (t) - FK(0)

(t) = o _ (2.10)
K FK( ) FK(0)

 

f

13

Here, FK(O) is the fluorescence immediately after bleaching and FK(0) is
tflue fluorescence at very long times after bleaching (1). The term for
fluorescence immediately after bleaching is expressed by

-1 -x
PK (0) = (q Paco/A) K (1 - e

) (2.11)

.3150 from (1). The fluorescence at long times approaches the fluores-

cence before bleaching and is approximated by the following

17‘“) 3 Pr") = q Paco/A (2.12)
In both FK(O) and Fx(m) the beam profile is Gaussian (1). Typical plots
of normalized fluorescence for each mode of transport can be seen in
Figure 2.1, reproduced from (1).

It is the normalized fluorescence equation (2.10) that allows for
the determination of the transport parameters, D and V0. The methods

of solution are presented as part of the experimental analysis.

2.2 Experimental

Up to this point only the theoretical basis for FRAP systems has
been discussed. This theoretical basis was presented to develop avenues
for calculating the transport parameters,l)(latera1cfiifushnicoef-
ficient) auni Vo (bulk flow velocity), from measured fluorescence time
histories. The experimental theory describes the collection and
manipulation of fluorescence time histories. llzis this experimental
theory which was used as a guideline for the development of a laser
photobleaching system. Again the experimental theory presented is from
Axelrod, et al.(1), which has been used by many others. (3,4,6, 7,

18,19).

14

 

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mp-
10

amt ,

05-

 

 

 

 

Normalized fluorescence recovery. a) diffusion only, b)
flow only, c) combined diffusion and flow; reproduced
from (1).

15

As stated earlier, the normalized fluorescence equation (2.10)
allows the experimental determination of the transport parameters, D and
V0. The parameters may be obtained by using measured flhnnescence
intensities instead of the theoretical fluorescence intensity expres—
sion terms contained in equation (2.10). Rewriting Equation (2.10) in

terms of measured values leaves

f" (c) - K (2.13)

K m m
FRO") - FK(0)

F"(t) - F:(0)

 

The superscript m denotes measured intensities. The subscript K denotes
a dependence of each term on the amount of bleaching (non-dimensional
energy density, K 8 -aI(0)T) (1).

Values for the diffusion and the flow velocity can be. determined
using either of two methods presented by Axelrod, et. al (1). The first
is essentially a three point fit of the normalized data. This method is
most tuneful uduni FK(m) (nun be measured or estimated and the nnturn of

transport is known. The first point to determine is the time t the

1/2'

m . . . . . . . . .
time when fK(t) = 0.5. .‘m1v1ng for this: time rmmirnn an ilnml no

I O I s O ‘m
process tn) find the time from the fluorescence intensity history lr(t)

\

which provides the value 0.5 for Equation 2.)). Thu next stop is to
find the value for the non-dimensional energy density (amount of

bleaching) K. The value of K is found by determining Fz;(())/f':j(-)

and solving for K in equation (2.11),tdwne FE(-) hithe fhunescence
before bleaching. The third point of the data fit: is to calculate the

lateral transport coefficients by using one of the following (1):

Diffusion coefficient: D = (w2/4I (2.14)

l/2)7n

Flow velocity: v = (w/I )7F (2.|5)
0

1/2
16

In both equations, w is the effective laser beam width. The constants

7 and 7 are given by 7 / and 7 HF, where 1.’ and t

D F D . T1/2 II) F ' I1/2 D F
are characteristic times for diffusion and flow. The values for 7D and

7 are found using the value for K determined in step 2 and Figure 2.2

F
reproduced from Axelrod, et al. (1).

 
 

4......

 
 

 

l 1 l IJILLI l 1

° 7 a a
Figure 2.- Factor 7(7D Tl/Z/TD or 7F 11/2/ID) versus K

for a Gaussian beam; reproduced from (1).

l7

This first experimental solution method also allows for the calcu-
lation of the immobile fraction of the fluorophore. If some of the
fluorophore is immobile fluorescence at large times, F:(m) will be
less than the fluorescence before bleaching F2130). Thus the mobile

fraction is given by (1) as

-
[me — 112(0)]
3 i (2.16)

[rim - 4(0)] .

 

The second experimental solution method presented by Axelrod et al.
(1) to determine the transport parameters is a graphical method. It is
based upon the observation that plots of [F:(t) - F:(0)] vs.t:and
fK(t) vs. t/t are different by two multiplicative factors: an intensity
scale and time scale. The multiplicative factors when plotted in
log-log form show up as orthogonal displacements along the intensity and
time axes. Superposition of the two plots determines the displacements
of the intensity and time scales. These displacements give the amount
of fluorescence recovery and the characteristic time of experiment.

The superposition method for determining the characteristic time and
mobile fraction is described only briefly here. See (Axelrod) et al.
(1) for further details. The characteristic time is flmnuitnrsuper-
positioning the point t/T 8 1 of the theoretical log-log plot of fK(t)
vs. t/t with the time axis of the log-log plot of the experimental
[F$(t) - F:(O)] vs. t. The displacement of these log-log plots

along tdua time axes gives the experimental characteristic time I. The

transport parameters of D or V0 can then be calculated from this

18

characteristic time. Likewise, the superposition of the point fK(t) = 1
on the log-log plots with [172(0) - F:(O)] gives the amount of
recovery F1120») - F:(O). It is from this amount of recovery that the
mobile fraction can be calculated.

Figure 2.3, reproduced from (1), illustrates this graphical method using

the superposition of experimental data and the theoretical values of

 

 

 

 

    
   

 

 

 

 

 

 

fK(t).
LO‘~4‘§§BLEACH
LEVEL
I
s ._ .
U IL
0) I l
U z:
5 :5: . :
Q—s I
33 gw/- l
IL - : I
L I
_- __________ J
loo“)
1 1 llLllll 1 411111
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
24 48‘”?
TIME (s)

Fig. 2.3 A graphic illustration of the log-log superposition method;
reproduced from (1).

Besides providing percent recovery and characteristic time this
second method of solution allows for the determination of the mode of
transport. The characteristic time found is compared to the theoretical

. . . . . 2
expressions for characteristic time for diffu51on (TD - w /4D) and flow

(IF - w/Vo). If the mode of transport is that of flow the charac-

teristic time will be proportional to w, the effective laser beam size.

19

If the mode is diffusion then it will be proportional to w2. For
mixtures of diffusion and flow, small w tends to make diffusion dominant
and large w tends to make flow dominant. The theoretical analysis and
experimental analysis of Axelrod et al. (1) as presented above supplied
the guidelines needed for the development of the laser photobleaching

system.

20

21

3.0 FRAP Laser System
3.1 Introduction
The main objective of this work was the development of a laser photo-
bleaching system. In this section the laser photobleaching system will
be briefly described as a whole. Its individual components will be
’ described in detail. After the description of all the components, a
presentation of the laser photobleaching system operation and character-
stics will be made.

The laser photobleaching system which has been developed can be seen

as a block diagram in Figure 3.1.

 

 

ADI-3A

 

 

POP ”/03

 

 

 

 

 

 

 

1| .
' Jiillll
II ”WITH {1 L15“:

 

 

 

 

 

 

 

 

 

 

 

 

Fig. 3.1 Components of the laser photobleaching system

The system consists of two major divisions: system hardware and the
other the system software. The hardware division consists of energy
generation, beam manipulation, and light detection. The software
division contains two operating systems. One is for control only. The
other is for data acquisition and control of the laser photobleaching
system.

The energy generation system (hardware) produces the necessary
means to excite fluorescent probes. The laser provides the proper
wavelength needed to excite a particular fluorophore. The power supply
regulates the current to the laser. The purpose of the heat exchanger
is to cool the plasma tube of the laser and power supply and to prevent
damage due to contaminates.

The beam manipulation system includes all components dealing with
the optical hardware. The optical hardware deals with considerations of
attenuation, direction, filtration, fixation, position, protection, and
reduction. The attenuation grouping controls intensity levels needed
for observation and bleaching. The direction system determines the path
of the laser beam into the microscope and then to the sample. The
filtration system eliminates any unwanted wavelengths being passed along
the optical path of the beam. The fixation of the laser system assures
precise alignment of the optical path of the laser and microscope. The
[masitioning group includes the hardware required to select the desired
location for bleaching and observing. The protection system protects
both the sensitive photodetection equipment and the operators of system.
The reductixni system allows the desired beam diameter to be selectable

at the membrane surface.

22

The light detection system is the most sensitive of the laser
photobleaching hardware. This system collects and relays fluorescence
intensities. It is this collected and relayed data on which FRAP theory
is based.

The software division of the laser photobleaching system consists of
a control only system and the other a data acquisition and control
system. Both are menu-driven operating systems. These operating
systems are interactive "user friendly" programs and subroutines which
allow the reasearcher to select experimental parameters. The control
portion of both operating systems are a set of subroutines which
control the instrumentation of the system in real-time. Subroutines
which collect and store experimental data in real-time form the data

acquisition portion of the second operating system.

3.2 Hardware
In this section each hardware grouping, will be described in
greater detail.

The energy generation group contains three components. The first
component is a Spectra-Physics argon laser model 164-05. The second is
the Spectra-Physics power supply model 265 for the laser. The last
component is a heat exchanger similar to that developed by Edwards
(20).

The heat exchanger is the only portion of the energy generation
system designed and built as part of the system development. This

component has a recirculating cooling loop which includes a pump, the

23

laser, a heat exchanger, a filter, a deionizer, and a reservoir. See
Figure 3.2. The outside loop of this heat exchanger is an open loop

which uses filtered tap water.

 

Figure 3.2 Heat Exchanger.

The pump of the heat exchanging unit is a close coupled turbine pump
(Burks pump model SCTSM). It provides a water flow rate of 4.5 gallons
per minute at a pressure of 70 psi.

The water from the laser is cooled with a shell and tube heat
exchanger (Basco model 500). It is a single pass, counterflow unit. The

recirculating cooling water for the laser and its power supply passes

24

through the shell side of the unit. The filtered top water flows
through the tube side removing the heat generated by the laser and power
supply.

The recijnnilating cooling water of the heat exchanger unit is both
filtered and deionized. The water filters are Ameteck Plymouth Supreme
model PS-Sl filters. These filters surpass the 25 um limit for parti-
cles set by Spectra-Physics. The deionization is handled by a Barnstead
Cartridge deionizeru It not only deionizes the water, it also removes
oxygen, scale , and other contaminant.

The heat exchanger components are connected with general pumping
fixtures and polyvinyl tubing. The exchanger is connected to the laser
and power supply with 5/8" garden hose.

The heat exchanger water flow rate and water pressure are both
monitored and adjustable. The flow rate is measured by Hedland flow
meters in both loops of the heat exchanger. The flow rate (minimum 2.5
gal/min) is adjusted by opening or closing water valves in each loop.
The pressure is monitored before and after the laser by 100 psi gauges
from 0.8. Gauge. The pressure is adjusted to the required operating
range (30-50 psi) by opening or closing a valve which bleeds off water
from the pump.

‘Water temperatures throughout the heat exchanger can be monitored.
Thermocouples and a ten channel Omega Digicator Box can determine
temperature in five locations. These locations include inlet and
outlet to the laser, inlet and outlet to the heat exchanger for the tap

water, and the reservoir.

25

The heat exchanger assures proper cooling of the laser and power
supply. It also prevents damage from contaminants, excessive pressure,
and other sources.

The remaining portion of the energy generation system provides a
flexibility to the laser photobleaching system. The model 164-05 laser
has prism selectable wavelengths from 351.1-1090 nm. This range of
wavelengths allows the use of many different fluorescent probes. The
power supply can generate a measured maximum of 4W total output across
all wavelengths. This output capacity implies that the laser can emit
enough power at multiple wavelength settings to perform FRAP experiments
using many different fluorescent probes.

The beam manipulation system contains many different groups of
equipment which control the delivery of the beam to the membrane
sample. The first task performed by the manipulation system is attenua-
tion of the laser beam. It is accomplished either by adjusting the laser
power supply or by placing reflecting neutral density filters (Melles
Griot) into the beam path. Attenuation using the power supply is
limited to controlling power or current requirements of the laser.
Therefore the power supply is only used to set the maximum level from
which the beam will be further attenuated using the neutral density
filters.

The laser intensilnrzis controlled by positioning different combi-
nations of neutral density filters in the beam path. The optical
densities of the filters used in the positioning unit are 0.3, 1.0, 2.0
enui 3.0. The percentages of the beam transmitted by these filters are

50%, 10%, 1% and 0.1% respectively. Further attenuation is obtained by

26

adding optical densities of neutral density filters combined in series.
Transmittance percentages are found by multiplying corresponding
percentages since T = 10-D.

The neutral density filters are positioned by a solenoid activated
mechanism. See Figure 3.3. The positioning unit is activated by
applying a voltage to an appropriate solenoid. This pulls the filter out
of the beam path. The positioning unit has four such mechanisms. They
permit 16 different filter combinations or 14 different intensity

levels. The intensity levels for the laser at a wavelength of 514.5 nm

producing 500 mW of power are listed in Table 3.1.

TABLE 3.1: LASER SYSTEM INTENSITY LEVELS

INTENSITY LEVEL POWER TRANSMITTED

 

 

Observation Levels

1 250 mW
2 500 mW
3 2.5 uW
4 5 pH
5 25 uW
6 50 pH
Bleaching Levels
7 250 pW
8 500 uw
9 2.5 mW
10 5 mW
11 25 mW
12 50 mW
13 250 mW
14 500 mW

*Based on maximum output of 500 mw at wavelength of 51&.5 nm.

27

 

 

/‘ SOLENOID

Ill N D FILTER

/ PIVOTING ROD

. d
L BLOCKING ROD

 

 

 

 

 

 

 

 

 

 

Fig. 3.3: Neutral Density Filter Positioning Unit Solenoid Mechanism

The neutral density filter positioning unit. was «designed
for computer control. The concept used in designing the unit was to
make use of the transistor transitor logic (TTL) capability of a
(parallel line unit (DEC DRVll) in an LSI-ll/OZ computer. The parallel
line unit can apply a high logic level (z5V) or a low logic level (zOV)
voltage to any of 16 output channels. The use of TTL capability Us
control the filter position keeps the four D/A channels open for other
equipment requiring variable voltages as inputs. The use of the
parallel line unit also provides the response needed for real time to
position the filters for observing and bleaching.

The electronic circuit used to control the solenoids of the neutral
density filter positioning unit was designed by Joseph Peplinskizni
undergraduate working in the BTP laboratory. Each circuit requires a

separate TTL channel from the DRVll to control the applicatnnxof a

28

voltage to a solenoid. Applying a high logic level (35V) activates a
solenoid to pull a filter out of the path. Each of the circuits acts
independently allowing any combination of the four solenoids to be
activated.

The interfacing of the neutral density filter positioning unit and
the parallel line unit of the computer was a joint effort. The
original interfacing of the neutral density filter positioning unit was
done by Joe Peplinski. His interfacing box splits the 16 output
channels into two portions, a 10 line ribbon cable for the positioning
unit and a 6 line ribbon cable for other laboratory equipment. The 10
line ribbon cable connects directly to the positioning unit.

The addition of computer controlled shutters required that the 10
TTL channels be split to complete the interfacing. The Electronic and
Computer Services (Department of Engineering Research, Michigan State
University) built the signal splitting board and connecting cables. The
signal splitting board allowed ten TTL channels on a ribbon cable to be
split to service the neutral density filters, the laser shutter, and the
photomultiplier shutter. The connecting cables permit the matching of
the electrical connectors for each of those units.

The task of directing the beam was left to three different types of
mirrors. The mirrors used and their optical path can be seen in Figure
3.4. The first mirror used to direct the beam from the lasertx>the
Ziess Universal microscope is a planar front-surfaced mirror (Optics for

Research MU-Sl). These two inch diameter mirrors are capable of

29

 

5 Is. 3’ /'

Figure 3.4 Optical path.

reflecting over the spectral range of 200 nm to 2 pm, with reflectance
of 80-90%. Three of these planar mirrors manipulate the beam through
three 90 degree direction changes. The scanning mirror, a 5 mm diameter
mirror (General Scanning M2-0505-00) mounted on a servo-driven motor,
directs the beam into the microscope. It allows the beam to scan across
a given region of the sample membrane. The third type of mirror, is the
dichroic mirror. These mirrors are contained in the epi-illumation
attachment of the Ziess microscope. These dichroic mirrors are select-
able for the wavelength being used. They come as part of filter sets
which will be described later. The dichroic mirror is used to re-direct
the beam to the membrane sample.

Filtration of the beam is performed in the epi-illumination at-
tachment of the microscope by three filter sets. The filter sets
(Ziess) used are set #487702 for ultra-violet excitation, set #487709

for blue-excitation, and set #487714 for green-excitation. The fourth

30

position of the epi-illuminator contains only a dichroic mirror for the
visible light range. The filter sets consist of an excitation filter, a
dichroic mirror, and.at least one barrier filter. Operating the laser
at a single wavelength (514.5 nm) eliminates the need for an excitation
filter.

The dichroic mirrors reflect wavelengths below a cutoff while longer
wavelengths are transmitted through the reflective coating. This
filtering property of dichroic mirrors is useful for fluorescence work
because emission is always at a longer wavelength than the wavelength
required for excitation. The laser beam with an excitation wavelength
shorter than the cutoff is reflected down to the membrane sample by the
dichroic mirror. Emitted light from the sample membrane at any wave-
length longer than the cutoff passes through the dichroic mirror to the
light detection system.

Barrier filters are placed in the optical path after the dichroic
miranirs and before the light detection unit. These filters prevent the

unwanted reflections of excitation wavelengths from reaching the light
detection unit. Thus only the intensity of emission from the fluores-
cencing sample will be measured.

The fixation group of the equipment prevents misallignment of the
laser beam optical path and the microscope optical path. Each piece
of equipment is affixed to a Micro-G model 71-151 vibrathnadamping
optical table. The optical table has air activated supports which must
be leveled so the table is supported equally. The optical surface has
pre-tapped holes with 2 inch centers for mounting equipment to the
table. Mounting equipment to the table insures that it is on the same

reference plane in space.

31

The mounting fixtures required for each piece of the equipment were
machined by Leonard J. Eisele, who also aided in their design. The
laser is held in place by two mounting plates which permit movement in
the x-y plane of the optical table. The neutral density filter unit is
mounted with two supports. These were designed to allow movement in
three dimensions. The planar mirrors are fixed to the optical table by
mounting brackets which allow adjustment in the x-y plane of the table.
The scanning mirror mounts permit rotation about the z axis as well as
three dimensional movement with respect to the table. The final
mounting fixture allows for rotation about the z axis and facilitates
three dimensional adjustment of the microscope.

The positioning of the beam depends on the mounting fixtures of the
equipment. Having the equipment affixed to the optical table provides a
fixed reference frame. It is from this reference frame that the optical
path of time laser beam can be precisely matched to the optical path of
the microscope. See Appendix C for detailed positioning procedures.

The optical path of the laser beam is kept parallel or normal to the
reference plane by using the planar mirrors. The planar mirrors are
positioned using adjustable mirror mounts (Oriel #1750).

The scanning mirror system is the most critical component for
positioning the bean» The scanning mirror redirects the beam into the
microscope and controls the position of the beam on the membrane sample.
Its position is controlled by a galvanometer (Frequency Control
Products model ALS 200 PS) with a resonant frequency of 200 Hz at.a
maximum of 15 degrees of deflection. The positioning galvanometer is
rated at: 23 sensitivity of 20 mA/degree and a repeatability of 0.05%.

The galvanometer is externally controlled by the digital/analog (D/A)

32

channel A from an LSI-ll/OZ computer. The controlling electronics for
the scanning mirror system were developed in conjunction1mhfliflec-
tronics and Computer Services (ECS, of the DeparUmnn:of Engineering
Research, Michigan State University).

The laser photobleaching system includes many features which protect
the laser and its operators. The first protection feature is that the
laser can be shut down from various sources. These sources include the
wall circuit breaker, power supply key, and power supply circuit
breakers. Simply turning off the heat exchanger pump is also a pro-
tective feature, but is an extreme emergency measure. A second
operator safety feature is that the beam can be blocked or attenuated.
Blockage of the beam is accomplished by manipulating (Vincent Associates
model ZGL) shutters located at the laser and at the photomultiplier.
These shutters are controlled by a Uniblitz electronic control unit
(Vincent Associates model 325B). This control unit can open or close
two shutters either manually or under computer control.TNmebeam is
attenuated by the neutral density filters either manually or under
computer control. A final protective feature is shielding which blocks
stray reflections of laser beam.

The laser itself is protected by the power supply. The power supply
shuts down if the key is not in place and turned on. It also reacts to
an insufficient supply of cooling water, and impropertnunent and
voltage levels. In addition, the power supply sounds warning signals
for high cooling water temperature and low gas pressure.

One of the most critical tasks of the beam manipulation system is
the reduction of the beam. The beam has to be reduced from 1.25 mm to

.approximatelor 1 micrometer when the sample is in focus on the micro-

33

scope. The beam is reduced by inserting a convex.lens (40 mm focal
length (Optics for Research #LL-ZS-40) in the beam path so that its
focal point closely matches the focal point of the focusing objective
(Ziess 25X Neofluor). A diffraction limited,l.5’ndcrometer spot is
produced when a sample is in focus, due to an image of a point seen by
the objective. "Spot" diameters of the laser beam measured to be 1-4
micrometers are obtained by changing the manification power of the
objectives.

The last hardware system controls light detection. This light
detection system consists of two parts, a photometer (Ziess MPM 01K) and
a measuring amplifier support box (Electronic and Computer Services,
MSU).

The photometer is comprised of a photomultiplier tube,zn1elec-
tronics housing, a mounting housing, and a protecthmashutter. The
photomultiplier tube (Hamamatsu/Ziess # R928HA) detects light energy and
converts it into a proportional current. The electronics of the
photometer changes this proportional current into a voltage, O-l.6V dc.
This voltage is then used as an input for the amplifying support box.

The measuring amplifier support box was built according to require-
ments supplied by Ziess. The support box supplies power for the
photomultiplier tube, PMT electronics, and hinged mirror control of the
photometer housing. This box amplifies the output voltage supplied by
the PMT electronics, to a O-lOV dc scale. The support box also controls
the gain of the photomultiplier tube with both a fine gain (O-le) and

a decade gain (1x, 10x, 100x, 1000x). It also supplies various output

34

signals on a O-lOV dc scale on digital display, banana plug, and BNC
port. The latter two allow for strip chart or A/D records of the output
from the photomultiplier tube.

The mounting housing for the photometer contains selectable pinhole
stops and a hinged viewing mirror. The pinhole stops (Ziess 4T1380)
have diameters ranging from 50 pm to 12 mm. These diameters correspond
to 1.6 pratu) .38 mm measured diameters at the sample membrane using a
20x objective. The hinged viewing mirror selects the path of the
emitted light of the sample. One position of the mirror sends the
emitted light to an observing eyepiece another position sends the
emission to the photomultiplier tube. The position of this mirror is

controlled by a manual foot pedal switch.

3.3 Software

As mentioned earlier the software of the lasers/photobleaching
system developed here consists of two operating systems. In this
sectixni, the capabilities and.development of each of these systems is
described in detail.

The laser photobleaching can be controlled by two different operat-
ing systems, written in FORTRAN IV. The first operating system
controls the laser system only. The control<nfly'sysmanis used for
aligning, initializing, and testing the laser photobleaching system.

The run image of control only system is named LOPERA.SAV. This run
image is composed of the programs and subroutines contained on the
following files: LOPERA.FOR, MPOSIT.FOR, FPLACE.FOR, CLOCIN.FOR,
DTACHA.FOR. The actual contents of these files are reprinted in

Appendix B.

35

The control only operating system is based on programs and sub-
routines contained in the five files listed above. This system is menu
driven by the program LOPERA contained on LOPERA.FOR. The program
LOPERA allows for the selection of intensity levels, shutter conditions,
miscellaneous options, or exiting.

The subroutines FPLACE, TRANSV, SHUTTR, and OCTREP in the file
FPLACE.FOR control intensity level selection and shutter conditions.
The subroutine FPLACE called by LOPERA allows selection of the six
observing intensity levels and eight bleaching intensity levels pre-
sented in table 3.1. The subroutine TRANSV called by FPLACE determines
the percentage of the beam transmitted for the chosen intensity levels.
The subroutine OCTREP also called by FPLACE determines the octal values
needed to activate the proper TTL (transistor tranistor logic) channels
of the parallel line unit to remove the neutral density filters from the
beam path. The subroutine SHUTTR allows the user to select from four
shutter conditions. The shutter conditions are 1) both open, 2) both
closed, 3) only the laser shutter opened, and 4) only the PMT shutter
opened. SHUTTR also provides octal values needed to activate the TTL
channels of the parallel line unit for the chosen shutter condition .

The subroutines MPOSIT, CONVRT, and MOVEM of the MPOSIT.FOR file
select mirror settings. The subroutine MPOSIT called from LOPERA
selects operational modes for the scanning mirror. One mode changes the
mirror position until the desired position is reached. The other mode
scans the sample over a given range for a specified number of passes at
a selectable rate. The scanning mode of the subroutine MPOSIT is
supported by the subroutine CONVRT. The subroutine CONVRT converts

desired deflection voltages corresponding to a certain mirror position

36

into integer equivalents for the BIA (digital/analog) converter. The
analog signal produced is directed out of channel A of the D/A board.
This signal is used as the external input to the scanning mirror control
box.

The subroutine MOVEM is used in the position mode of the subroutine
.MPOSIT. The subroutine MOVEM increases or decreases the D/A output of
channel A to the scanning mirror control box until the desired beam
location is reached.

The files CLOCIN.FOR and DTACHA.FOR must be linked with the file
MPOSIT to control the mirror positioning or scanning. The subroutine
CLOCIN called by MPOSIT selects real-time clock settings to control the
rate of scanning. These settings include operating frequency, mode of
operation, and duration of operation.

The subroutine DTACHA called by MPOSIT is actually an Interrupt
Service Routine (ISR) An interrupt service routine allows the computer
to interrupt a running program temporarily. It then completes a task
and resumes the running program at the point of interruption. For more
information about ISRs consult (21-24). The tasks performed by the ISR
DTACHA include outputting desired voltage from D/A channel A and
counting the number of scans.

The second operating system for the laser photobleaching system is
designed for real-time data acquisition and control. The name of the
run image for this acquisition and control operating system is LASCON.
SAV. This run image consists of the programs and subroutines found in
these files: LASCON.FOR, INSHUT.FOR, EXPRUN.FOR, LASET.FOR, CPARAM.FOR,
LPARAM.FOR, NAMEF.FOR, SETIME.FOR, LTIME.FOR, ATDC13.FOR, STNDBY.FOR,

and HELPME.FOR. The contents of these files may be found in Appendix B.

37

The program LASCON, contained in the LASCON.FOR file, is the
interactive menu of the acquisition and control system. This program
contains and defines most of the initial and default variables. LASCON
allows the user to change parameters, list parameters, name output
files, run FRAP experiments, adjust laser system, go to a stand by mode,
ask for help, or exit the program.

The subroutine CPARAM, in CPARAM.FOR file, permits changes of the
parameters of the output file name, the intensity levels, the shutter
conditions, and the experimental time settings.

The subroutine NAMEF, contained in NAMEF.FOR, can be called by
LASCON or CPARAM. It allows the output file name to be varied fnmn
FTN10.DAT to FTN99.DAT. The default output file name is FTN10.DAT.

The subroutine INSHUT, called by CPARAM, is contained in INSHUT.FOR.
This subroutine permits the user to select the observing intensity
level, bleaching intensity level, and shutter conditions. The sub-
routine INSHUT calls the subroutines TRANSV, OCTREP, and SHUTTR. INSHUT
also allows the user to select one of six observing intensities and one
of eight bleaching intensities presented in Table 3.1 . The subroutine
TRANSV, called by INSHUT matches the chosen intensity level with the
fraction of the beam transmitted to the sample. The transmission
fraction is written into the output file and to the screen. The
subroutine OCTREP determines the octal values which the TTL channels of
the computer parallel line unit need to activate the neutral density
filters. The SHUTTR subroutine allows the selection of the four
possible shutter conditions and provides the octal.values needed to

activate these shutters.

38

The selection of the experimental times is handled by the SETIME
subroutine called under CPARAM. SETIME permits the user to select
length of bleaching, length of experimental run, and the time interval
between the collection of data. The bleaching and run times set the
parameters for the line time clock the computer's . The collection time
interval specifies the of real time clock parameters. These clock
parameters define values needed to prepare the programs interrupt
service routines. For more information on interrupt service routines
refer to (21-24).

LASCON program lists the existing parameters when the subroutine
LPARAM is called. This subroutine is contained in the file LPARAM.FOR.
LPARAM is also called by the subroutine CPARAM.

The subroutine STNDBY called by LASCON puts the laser photobleaching
system into a "safe" mode. This "safe" mode lets the user leave the
system unattended without having to power down the laser photobleaching
system. In this mode the laser is attenuated to its lowest level and
the photomultiplier shutter is closed. The subroutine STNDBY is
contained in the file STNDBY.FOR.

The system user can get help when using the subroutine HELPME. This
subroutine gives information on each of the menu selections in the
LASCON program. The subroutine HELPME is contained in the file
HELPME.FOR.

The initial set up of the laser is completed by using the LASET
subroutine of LASCON. LASET allows for focusing the beam, centering
the beam, verifying intensity choices, checking shutter<unmfitions,
setting PMT controls, and checking strip chart recorder settings. This

subroutine is contained on the file LASET.FOR.

39

The execution of a FRAP experiment is performed by the subroutine
EXPRUN. This subroutine controls all the system hardware and collects
the recovery intensities using the parameters set by the user or the
default values. EXPRUN also performs the necessary housekeeping chores
of opening files, closing files, and writing data into files.

The equipment control and data acquisition in EXPRUN is performed by
two interrupt service routines (ISRs). Further information on ISRs can
be found in (21-24). The two ISRs used in EXPRUN are LTIME and ATDC13.

The ISR LTIME controls both the bleach time and the run time of the
FRAP experiments. The other, ISR ATDC13, controls sampling of data and
mirror positioning. ATDCIB uses A/D channel 13 to take data. This ISR
also uses the BIA channel of the computer to position the scanning
mirror.

The subroutine EXPRUN continuously checks for a (done) flag while
the ISRs are functioning. Once the run time is completed and the flag
is detected, EXPRUN completes the file management. It then offers the
user the choice of running another experiment with the same parameters
or returning to the main menu of LASCON.

Both operating systems, programs and subroutines, may be reviewed in

Appendix B.

3.4 Operational Characteristics

.In this section characteristics and response of the hardware and
software of the laser photobleaching system are presented.

The response of the energy generation system and the other systems
hardware is dependent on the operational settings. The following

operational settings remain the same throughout the characterization of

40

the laser photobleaching system. The power supply is in the current
contixxl mode. The current is set to 30A. The magnetic field control is

set to its maximum. The laser is set to 514.5 nm in the single line
mode. The heat exchanger provides a minimum flow rate of 2.5 gal/min
at 40 psi to the power supply and the laser. At these settings the
laser produces 500 mW of power when operating at peak output. See
Appendix A for peaking laser output.

The first component of the beam manipulation system which is
characterized is the attenuation by the neutral density filters (NDFs).
With the laser producing 500 mW of power, the NDFs provide intensities
ranging from 25 pH to 500 mW. For each intensity level within the range
of a calorimeter type power meter (Scientech 1736-0001), the predicted
intensities are compared with experimental intensities. The results of
these comparisons are listed in Table 3.2. The intensities for level 8
and 9 have asterisks because estimated errors reflect fluctuations of

the power meter.

41

TABLE 3.2: INTENSITY COMPARISONS

INTENSITY LEVEL PREDICTED INTENSITY (W) EXPERIMENTAL INTENSITY (W)

 

 

 

 

 

 

 

8 0.50 mW 0.5 i 0.5 mW*
9 2.50 mW 2.26 t 0.1 mW*
10 5.00 mW 4.95 i 0.05 mW
11 25.00 mW 26.0 i 0.15 mW
12 50.00 mW 51.5 i 0.5 mW
13 250.00 mW 263.0 f 1.5 mW
14 500.00 mW 505. i 5 mW
0.9“
. 487714
0.7‘
0.5 - BP 515—560 LP 590
0.3 -*
0.1 . i )
t

 

 

 

I I
1. (nm) 400

Fig. 3.5: Fluorescence response curve for green excitation, reproduced
from (41).

42

The mechanical response of the neutral density filter positioning
\nrit is measured using the photomultiplier system and an oscilloscope.
The mechanical response time for removing a filter from the beam path is
found to be 4 i l msec. Replacing a filter yields a slightly slower
response time, 6 i 1 msec.

The filtration system is not characterized experimentally. The
fluorescence response curve reproduced from (41) for the green ex-
citation filter set (Ziess, #487714) used in most of the applications of
this laser photobleaching system can be viewed in Figure 3.5.

Theonly characterized response of the protection system is the
response time of the shutters. The laser and PMT shutter opening and
closing times are measured with a photo-diode system and an oscil-
loscope. The opening and closing times are found to be 1.5 i .2 msec.

The results of the beam positioning are qualitative in some cases
and quantitative in others. Qualitative results come from the alignment
of the beam optical path with the optical path of the microscope. Any
misalignment of optical paths results in multiple reflecthnusand a
non-symmetrical emission spot at the sample. Only whentflmeoptical
paths of the beam and of the microscope match is a symmetrical (assumed
Gaussian) spot seen. Viewing the beam with only a dichroic mirror gives
symmetric interference patterns. A single secondary reflection is seen
when aligned properly.

Quantitative results come from the position of the scanning mirror.
Using a video tape recording of the emission spot from a fluorescently

coated microscope slide, a total beam excursion limit of 11.0 t 0.5 um

43

is measured. This excursion limit corresponds to r 5.6 pm movement from
a centered position. Eleven locations are used to define the beam
position over 10 pm of the sample. The point where the beam is centered
in the microscope is defined as position six. Each change in position
corresponds to a 1 pm position change on the sample.

The results from the beam reduction are also determined with a
video tape recording of the emission spot of a fluorescently coated
microscope slide using a color camera. Table 3.3 shows the measured
emissixni spot diameters for the objectives available in our laboratory.
The errors reflect the resolution possible from the television monitor

for each objective.

TABLE 3.3 BEAM REDUCTION RESULTS

 

 

OBJECTIVE EMISSION SPOT DIAMETER
Ziess Neopluar 10X 3.0 t 0.5 pm
Ziess Neofluar 25X 1.5 t 0.4 pm
Ziess Neofluar 63X 1 t 0.2 pm
Leitz Hoffman Modulation 20X 2.0 i 0.5 pm

It should also be noted that a faint symmetric emission pattern was seen
surrounding the emission spots except at intensities below level 6. A
higher intensity or a shorter effective focal length of the objective
increases the intensity of this symmetrical emission pattern.

The operational characteristics for each operating system are
presented separately. The control only operating system (run image

LOPERA.SAV) is characterized first. Then the operational response of

44

the data acquisition and control operating system (run image LASCON.SAV)
is presented.

The operational characteristics of the control only system software
are dependent on the laser photobleaching system's hardware and the
computer system hardware. The response of the control only operating
system interaction with the hardware has already been presented as part
of the hardware response. Only operational characteristics of the
control-only system software with the computer system hardware are
addressed here.

The subroutine FPLACE controls the neutral density filters and
shutters (FPLACE) of the control only system. It uses the parallel
line unit (DRVll). This subroutine selects which TTL channels are to be
activated. The channel is activated by setting one of 16 bits of the
DRVll output buffer. The bit numbers used and their corresponding

functions are listed in Table 3.

TABLE 3 DRVll OUTPUT BUFFER CONFIGURATION

 

BIT # FUNCTION
4 PMT shutter closed when set
5 Laser shutter closed when set
12 Filter #1 (Optical Density 0.3) removed from path when set
13 Filter #2 (Optical Density 1.0) removed from path when set
14 Filter #3 (Optical Density 2.0) removed from path when set
15 Filter #4 (optical Density 3.0) removed from path when set

45

The subroutine MPOSIT controls the mirror positioning for the
control only operating system. It uses the digital to analog (D/A)
converter. The first positioning option increases or decreases the
digital value supplied to the D/A converter moving the mirrors about a
center of deflection. This center of deflection is the digital value of
307110 (57778) corresponding to + 2.53 V. The minimum deflection of the
mirror is at a digital value of 409510 (77778) corresponding to 0V. Its
maximum is at the digital value 010(08) or +5.0V. The reason for these
odd values is that the scanning mirror responds to an input range of
-5.0 to 5.0 V and the D/A converter operates over a range from O to 10V.
The D/A output signals are also inverted, so that the digital value
409510 (77778) supplies 0V and 010(08) supplies +10V.

The scanning option of the subroutine MPOSIT of the control only
system uses the real-time clock for the initiation.of the interrupt
service routine DTACHA. The real-time clock is limited by software to
two selectable frequencies, 60 Hz and 100 Hz. The DTACHA ISR positions
the mirror using the D/A converter within the user selectable range of 0
- 5.12V DC. The number of cycles run is left to the operator dis-
cretion.

Some of the operational characteristics of the data acquisition and
control system software are identical to those of the control only
system software. Other characteristics display only minor differences
between the two operating systems. Still other responses are unique to

the acquisition and control operating system.

46

Identical operating characteristics are found in controlling the
neutral density filters and the shutters. These characteristics are
identical because the subroutine INSHUT is in fact the subroutine FPLACE
organized somewhat differently.

Minor differences between the two operating systems characteristics
are seen in the positioning of the mirror. In the control only opera-
ting system the mirror position in the manual mode is user selectable.
The acquisition and control operating system manual positioning in the
subroutine LASET restricts the mirror to one of only eleven positions.
These eleven positions correspond to a diffraction limited scanning
range of :5 pm from the center position at the sample membrane cor-
responding to [digital values from 304610 (57468) to 309610 (60308)]. In
both cases positioning of the mirror is defined by sending a single
digital value to the D/A converter and applying voltage to the scanning
mirror positioning control unit.

In both operating systems the scanning portion of the mirror
positioning is accomplished using the D/A converter channel A in
interrupt service routines. Again the acquisition and control operating
system uses the same eleven defined points while the control only system
scans the entire range set by the operator. The final minor difference
stems from the scanning rate. It is dependent on the data collection
rate in the acquisition and control operating system, while being user
selectable in the control only system.

The unique operating responses of the acquisition and control
Operating system are in timing and data acquistion. The fastest rate

that data could be collected is once every 5 msec. Attempts to collect

47

at faster rates cause a computer system failure. Other data sampling
rates are user selectable: 8 msec, 10 msec, 100 msec and 1 sec. All
rates are compatible with 1 kHz real-time clock frequency.

Other unique operational characteristics of the acquisithnnand
control operating system involve timing of controlled events. The
bleach times are user selectable : 250 msec, 500 msec, 1 sec, and 2 sec.
These bleaching times corresponded to those used by other researchers
(1) (2) (15) (18) (19). The experimental run times are calculated from
the maximum allowable space in the program temporary storage arrays and
the software selectable sampling rate for data.

Once the operational characteristics of the hardware and software
for this laser photobleaching system had been determined, application of

the system began.

48

49

4.0 Applications of the System

The laser photobleaching system was applied to four microscopy
techniques. Two of these techniques employed the system to quantize the
number of lamellae (bilayers) in liposomes. The other two techniques
were used to verify the FRAP capabilities of the system. In the
following sections the quantization techniques, the FRAP techniques, and

the results of these applications are presented.

4.1 Quantization Techniques

It was proposed to use the laser photobleaching system in developing
a fluorescence microscopy technique for the quantization of bilayers in
liposomes "in silnf‘. Two microscopy techniques were evaluated. One
metluni was a fluorescence version of a phase contrast technique (27,38)
for the determination of lamellae in liposomes. The other method
compared fluorescently labeled liposomes to fluorescently labeled single
bilayer standards.

The fluorescence contrast technique was attempted at the suggestion
of John J. McGrath, Ph.D. This study began by evaluating a phase
contrast technique (27) to quantize the number of bilayers in liposomes.
The work continued by fluorescently labeling liposome membranes and then
observing the emission differences at low intensitytnufltation. These
observations were to form the basis for the proposed fluorescence
contrast method.

The fluorescence contrast technique to quantize the number of
bilayers varied slightly from the phase contrast technique (27) in

liposome composition and detection response. The liposomes used in the

phase contrast technique were composed entirely of EPC and hydrated in
water. The liposomes of the fluorescence contrast technique were
prepared from EPC (99.9% pure egg phosphatidylcholine, Leon Labs), diI
(3.3'-dioctaderylindotricarbocynanine, Molecular Probes) and hydrated in
distilled water. The fluorescent probe, diI, was in a 1:1000 mole ratio
with EPC to avoid possible variations in membrane properties.

The difference in the intensity response of the two contrast
techniques is that they are inverted with respect to each other. The
fluorescence contrast technique has a response that is above the back-
ground intensity level within the liposome membrane and at the greatest
intensity at the membrane. The phase contrast response is at the
background intensity within the liposome membrane and has the lowest
intensity at the membrane. Refer to Figure 4.1 to compare the detec-
tion response of these two techniques from typical experimental records.
'There is cany a minor difference in procedure between the fluorescence
contrast and phase contrast techniques. This difference :hsin the
movement of the liposome past the detection point. THuefduorescence
contrast technique allows the liposome to float through the detection
point (laser beam). Both responses in Figure 4.1 are fnmnljrmsomes
floating through the detection point. The phase contrast method (27)
moved the liposomes on a step motor controlled microscope stage, with
0.5 pm resolution, through the detection point.

The analytic method for the fluorescence contrast technnmmefor
quantization of lamellea is based on that used by Servuss and Boroske

(27,38). Their analysis method takes the difference in intensity at the

50

*membrane, AI, and normalizes it with respect to the maximum intensity,
Io. These intensities are depicted for both techniques in Figure 4.1.

The normalized intensity (IN I AI/Io) is then plotted versus liposome

 

 

 

 

 

 

 

 

 

   

Fig. 4.1 Intensity detection of a) phase contrast b) fluorescent
contrast from float passed method

51

radius. This plot of normalized intensities shows groupings correspond-
ing to different numbers of lamellea Figure 1.2, reproduced from (27)
shows this data.

The development of the comparison technique for quantization of
lamellea in liposomes was a joint effort. The possibility of such a
comparison technique was conceived by John J. McGrath, Ph.D., associate
professor of the Mechanical Engineering Department at M.S.U. The
experimental equipment, parameters and procedures used to realize this
concept were developed as part of this study. The experiments for this
comparison technique were conducted by Serge Weiss, an undergraduate
student completing an independent study in the field.

The development of this comparison technique required first that
experimental parameters and procedures be defined. The first experi-
mental parameters defined the criterion of the comparison method. A
major requirement for the method was its ability to be used "in situ"
prior to quantitative determination of membrane characteristics. The
method also required flexibility for use with a diffusion chamber (50),
a computer controlled freezing stage (51), and a microscope slide.
Further the comparison technique needed to be easily applied, quick, yet
accurate.

The next parameters to be determined were the standards to be used
in the comparison technique. The two standards used in the comparison
technique were a known single bilayer standard and a normalizing
standard. The known single bilayer standard had to consist of a

verifiable single bilayer. The normalizing standard was to allow for

52

intensity comparisons between the liposomes and the known single bilayer
standard.

The known standard selected was the bilayer lipid membrane (BLM).
This selection was made because of its widespread use (31, 32, 34, 35,
36). BLMs were also chosen because a technique already existed to
verify that they consisted of a single bilayer. This technique,
described in (31), stated that a BLM observed to thin to "black"
(non-passage of light through a membrane due to diffraction) can be
shown to consist of a single bilayer.

The normalizing standard chosen was fluorescent polystyrene micro-
spheres (Duke Scientific). The microspheres were considered initially
because they were small (9.5 pm diameter) and said by the manufacturer
to be inertu ILt was two other'manufacturers' claims that determined
factor the choice to use these microspheres as the normalizing standard.
The claim that the microspheres would provide a constant fluorescent
intensity when excited. They also claimed that the intensity from
microsphere to microsphere would be uniform.

The last group of experimental parameters dealt with the composition
of tin: liposomes and BLMs. The composition of each system had to have
the same molecular ratio of constituents. The major constituent of the
liposomes and BLMs was EPC (99.9% pure Leon Labs). Cholesterol was
included as a component of each system since it helped form more stable
BLMs. The last constituent was the fluorescent probe diI (Molecular
Probes). This probe was chosen because of its use by others in both

liposomes and BLMs (1, 2, 6, 8, 19, 32).

53

The actual compositions used in the comparison technique were
dependent on the BLM preparation solution. The "recipe" for the BLM
preparation solution is based on one regularly used in Tien's laboratory
from (31). This BLM preparation solution consisted of 6.7% EPC, 1.1%
cholesterol, and 0.0067% diI (percentages from weight of solute/ weight
of solvent). The solvents used in the BLM preparation solution were
hexane and octane. The diI percentage was kept small (1:1000, diI:EOC
molecular ratio) to prevent possible membrane property changes due to
its presence. The liposome membrane reflected these component ratios
for similitude.

The comparison technique for quantizing the number of bilayers in
liposomes consists of three procedures. The first of these procedures
is the forming and transferring of BLMs. The next procedure is the
collection of normalized intensities of BLMs as the known single bilayer
standard. The final procedure is the acquisition of normalized intensi-
ties of liposomes.

The formation and transferring procedure of BLMs was derived from
methods used in Tien's laboratory (31). The BLM formation apparatus
used in this procedure is pictured in Figure 4.2. The formation
apparatus consists of a formation tank, a light source, a 2.5x binoc-
ulars, a BLM formation stage, ring stands, and supports. The use and
description of each component is described as the procedure to form BLMs
is presented.

BLMs require an aqueous environment to be formed. The construction
of an 8" x 6" x 3" formation tank meets this requirement. This tank is

large enough to provide easy access to the BLM formation stage.

54

 

Fig. 4.2 BLM formation apparatus.

 

Fig. 4.3 BLM formation stages.

55

Tien’s procedure calls for the BLM prepartion solution to be applied
across an opening in a teflon cup. For the purposes of this study the
cup has been replaced by a teflon disk with a central opening. These
teflon disks are housed in the BLM formation stage, designed as part of
this work. Figure (4.3). The formation of BLMs requires the ability
to seal off the BLM for its removal from the tank and placement on the
Inicroscope stage. The formation stage could be sealed by sliding 22mm
square coverslips over the teflon disks.

Allowing the membrane to thin to "black" on the teflon disk verifies
that the BLM is a single bilayer for use as the known single bilayer
standard. For further description of this portion of the experimental
procedure refer'tna (31) and (39). Once the BLMs thinned to "black",
coverslips were slid into place sealing off the BLM in the formation
stage.

The final step in the formation and transferring procedure is to
transfer the BLM held in a vertical orientation in the tank to the
microscope stage. The sealed formation stage has to be raised from the
tank. The stage must then be released and placed in a horizontal
orientation. Once in the horizontal orientation procedure it is
transported to the microscope stage.

The next procedure of the comparison technique is the collection of
normalized intensities of BLMs. The formed and transferred BLM is
supported in one side of the BLM formation chamber. In the other end of
the chamber fluorescent microspheres are sealed hnxathe opening of
another teflon disk with coverslips. The microspheres, the normalizing
standards, are sealed in this location to assure the microspheres and

BLM were at the same focal plane.

56

The collection of normalized BLM intensities follows these steps.
The laser's excitation intensity is adjusted until the microspheres
produced a voltage of 9.0V on the photomultiplier display for a pre-
selected gain. Once adjusted to this voltage the formation stage is
moved to the BLM portion of the chamber. Several emission "normalized"
intensity readings of the BLM are then taken using the photomultiplier
excited at the same intensity of the laser beam. This portion of the
procedure is repeated until a statistically significant number of the
BLM emission intensities are recorded. It is from this statistical
sample that a mean intensity value for a single bilayer standard can be
determined.

The final procedure in the comparison technique is to apply the
laser photobleaching system and find normalized intensities for lipo-
somes. The laser intensity is attenuated in the same manner using the
fluorescent microspheres as the normalizing intensity standards to get
9.0V on the photomultiplier display at a preselected gain. The lipo—
somes, sealed into a teflon disk, replace a BLM in one end of the BLM
formation stage. This replacement assures that no stage dependence has
been introduced in the procedure. Once again the formation stage is
moved from the microspheres (the normalizing standard) end to the
liposome end where "normalized" emission intensities can be taken.

The intensity readings are taken from multilamellar and unilamellar
liposomes (distinguished by qualitative visual selection). The intensity
readings for liposomes normalized by the intensity of the microspheres
(9.0V) can then be compared to the mean intensity of the similarly
normalized single bilayer standard. Those intensities within two

standard deviations (95% confidence level) are classified as a single

57

bilayer. Intensities greater than this mean single bilayer intensity
are classified as multi-layered. Hopefully further information on the
number of layers in these multi-layered liposomes can also be produced

by the comparison technique.

4.2 FRAP Techniques

The laser photobleaching system has multiple application capa-
bilities. Besides its quantization capabilities, the system can
perform both single point and multi point FRAP techniques. 131this
section, the experiments conducted to verify the laser photobleaching
FRAP capabilities are presented.

The experiments verifying the single point technique follow experi-
ments presented by Axelrod et al. (1). The laser photobleaching system
tests its single point FRAP capabilities on the diffusional recovery of
aqueous solutions of rhodamine 6G (Sigma Chemical).

The parameters defined for the verification experiments follow
those presented by Axelrod et al. (1). The aqueous solutions of
rhodamine 6G for the experiments have a concentration 4.6 x 10—6M. The
solvents are distilled water and glycerol : cfistilled water<1sl by
volume). These aqueous solutions are sealed into thin layers on
microscope slides using (24 mm x 50 mm) coverslips and silicon grease
instead of the rectangular sealed container 100 pm thick used by Axelrod
(1).

The next parameters needed for the single-point FRAP verification

experiments were settings for the laser photobleaching system. The

diameters for the beam used in verification experiments are 1.5 - 20 um.

58

These diameters are function of the distance between the beam focusing
lens (+ 40 mm focal length) and the microscope objective (25 x magnifi-
cation, 7.1 mm effective focal length). The bleaching intensities need
in these experiments are 5 mW and 500uw (bleaching intensity level 10
and 8 respectively). The photomultiplier gain selectors were adjusted
to display 5.0 volts for the fluorescence emission intensity of a sample
with the laser attenuated to 5 uW or 0.5uw (observing intensity level 4
or 2).

The procedure for these experiments followed earlier FRAP theory.
The sealed aqueous solutions of rhodamine 6G were focused on the
microscope stage using phase contrast. The beam attenuated to an
observing intensity level was focused to the desired spot diameter of
2.0 pm. The pinhole stop was then selected to be slightly larger than
the emission spot diameter. The photomultiplier measuring amplifier
gains were then selected to produce 5.0V on the display. The FRAP
experiment was then run with the desired time intervals. The aqueous
solution of rhodamine 66 was bleached using a bleaching level (levellO).
The recovery due to the diffusion of fluorescently labeled molecules
into the bleached spot was monitored by the photomultiplier system. The
time history of the recovery was recorded by the data acquisition
portion of the program. The diffusion coefficient was obtained from
these collected time histories

The results of the verification of the spot FRAP technique are
presented in the next section.

The ability of the laser photobleaching system to perform the
multi-point FRAP technique (37) was briefly examined. Verificathmm

experiments were designed to give preliminary information on the system

59

characteristics using the multi-point technique. The system character-
istics were gained from applying the multi-point FRAP technique to the
aqueous solutions of rhodamine 66 from the spot technique.

The experimental parameters used to examine the characteristics of
the multi-point FRAP technique closely followed those of the spot
technique described in (l). The same aqueous solutions of rhodamine 66
(4.6 x 10“-6 M) were used, as was the same system of sealed microscope
slides. A bleaching intensity of 5 mW (level 10) for 1 second was used
in these experiments. The observation intensity was 5 11W (level 4)
Data was collected at 5 msec intervals, implying the intensity for each
jpoint was measured every 55 msec. The wavelength used in these experi-
ments was 514.5 pm. The emission spot of the beam was focused to 2.0 pm.

'The experimental procedure for the multi-point FRAP experiments was
nearly identical to the spot technique used by Axelrod (1). The laser
photobleaching system was preset with the experimental parameters given

above. The sealed sample was placed on the microscope and focused.
The sample was then bleached with a high intensity beam (5 mW, level 10)
for 1 second. The response at all eleven points was monitored by the
system using 5 pW (level 4). These monitored values weretuflJected
using A/D channel 13 and written into data files. It was from these
recovery intensity data files that the operational characteristics of

the multi-point technique were determined.

4.3 Application Results
Both qualitative and quantitative results were determined for the
four microscopy techniques to which the laser photobleaching system was

applied. The results of the two techniques used to quantize the number

60

of lamellea in liposomes are presented first. The findings of the
application of the two FRAP techniques follows.

The development of the fluorescence contrast technique required the
verification of the published data using the phase contrast technique
(27). The normalized intensities as afunction of radius for the phase
contrast experiments are plotted in Figure 4.4. Figure 4.4 was repro-
duced fromn(27) and the results from this study were superimposed on to
this figure. The normalized intensities for the phase contrast verifi-
cation experiments are plotted with zerosn Figure 4.4. 2&Mflxumlti-
lamellar and unilamellar liposomes were chosen for these experiments by
a visual selection technique. The results agreed with those measured by

Servuss and Boroske (27).

 

 

 

 

In ck '
0.5— - ' , ° .
o z . X . ,
0.4- . , :° . 3 ° .
,1 . . .. , . .
03- ,° ‘ ° 0 ,
° r' $117: ". .°, .
. gs é: ; . )1. , x % : h
02 . . 1a r. 3 .12» i. 7.; fxfif a. . 5 :0
.; ~ .01 3“ T " '1 - x '
. <.o x~1?' . . o 0.
01— o’.¢;%a 0.80 x
. :' :1
0 1 l l l l l l
0 I. 6 8 1o 12 R/um

Fig. 4.4 Phase contrast and fluorescent contrast results: refluor-
escent contrast data; o-phase contrast data; --Servuss &
Boroske phase contrast data; background reproduced from (27).

61

The normalized intensities as a function of radius for the fluores-
cent contrast were plotted with X's on Figure 4.4: Again both multi-
lamellar and unilamellar liposomes were selected by a visual selection
technique under phase contrast. Although fluorescent contrast intensi-
ties cannot be compared closely to the phase contrast intensities,
similar trends were seen.

The comparison technique to quantize the number of bilayers in
liposomes was a two-stage project. The results of the stages of the
development of the technique are presented here in both qualitative and
quantitative form.

The initial stage of the comparison technique was the determination
of procedures to form BLMs. Using a formation stage designed by R.A.
Callow and a hexane-based BLM preparation solution BLMs were able to be
"formed" (observed to thin to a "black" state). These BLMs seemed
stable for approximately 10-15 minutes in a vertical orientation. All
attempts to tranfer the thinned to "black" BLMs to a horizontal orienta-
tion on the microscope failed. In each attempt the presence of the
fluorescently labeled BLM was determined by exciting the opening in the
teflon disks with a mercury lamp source. At this time it was believed
that the BLM ruptured due to the shearing forces of the water during
removal. The formation stage was redesigned to seal the BLM from the
rest of the external water.

The final stage of the comparison technique experiments were
designated as an undergraduate independent study project conducted by
Serge Weiss. Under direct supervision he conducted formation and

transportation experiments, single bilayer standard characterization

62

experiments, normalizing standard characterization experiments, and
liposome characterization experiments. The major results of this
independent study project are reproduced and presented as part of this
study. For complete results of this independent study refer to (39).

The formation and transportation experiments conducted in (39) can
be summarized by the following results. BLMs formed using the hexane
based solution in conjunction with the redesigned stage could be formed
in 10-15 minutes. These BLMs were stable in the vertical position for
10-15 minutes. The formation stage could be sealed after the BLMs
thinned without rupture. Attempts at removing the BLM from the forma-
tion tank and placing in a horizontal orientation were not successful.

Further formation and transportation experiments were conducted in
(39) after the BLM formation chamber was redesigned, the preparation
solution was modified, and the formation tank was replaced. BLMs formed
using the octane based solution with cholesterol added could be formed 4
in 10-20 minutes. These BLMs were stable in the vertical position for
up to 30 minutes. Sealing, removing, and placing a thinned BLM in a
horizontal orientation was successful no more than 5% of the time.
Attempts at transporting the thinned BLMs to the microscope to measure
their intensities failed (39).

Other methods were tried to form BLMs so that their intensities
could be measured. One method sealed the BLM formation chamber before
thinning was complete. This method failed since the membrane could not
be observed to thin to black. Forming a BLM in a horizontal orienta-
tion on the microscope was also attempted. These attempts failed

because proper orientation of the light source and binoculars needed to

63

visualize the diffraction pattern were unobtainable on the microscope.
The last method tried was varying the diameter of the aperture over
‘which the BLMs were formed. Six different apertures diameters were
investigated (0.125-0.203 inches). Results showed that stability
increased with decreasing diameter of the aperture. The time required
to form the BLM also increased when aperture diameter decreases. The
optimal aperture diameter for BLM formation in 10-20 minutes with
increased stability was found to be 0.156 inches (39).

The inability to successfully transport a.thinned BLM forced the
abandonment of the single bilayer standard characterizatflnrexperi-
ments. The independent study project continued with the normalizing
standard characterization experiments.

The findings from the normalized standard characterization eXperi-
ments were different than what was expected. The first group of
experiments were to determine the reproducibility of a mean intensity

for the microspheres. Results of these experiments are reproduced from

(39) in Table 4.1.

TABLE 4.1. MEAN FLUORESCENT INTENSITIES OF MICROSPHERES

Group I Group II
RUN INTENSITY RUN INTENSITY

1 2.65 t 0.25 V l 3.10 1 0.50 V
2 2.30 t 0.20 V 2 5.50 I 0.25 V
3 1.65 t 0.20 V 3 4.75 I 0.75 V
4 1.90 t 0.20 V 4 2.95 i 0.20 V
5 2.20 t 0.20 V 5 4.05 t 0.25 V
6 0.60 x 0.10 V 6 4.75 r 0.25 V
7 1.20 i 0.15 V

8 0.50 r 0.20 V

9 0.25 t 0.10 V

64

These experiments showed the inability of the microspheres to reproduce
a constant mean intensity value to be used as a normalizing standard.
The microspheres were exposed to laser excitation 5-10 seconds before
intensity readings were taken for these experiments. The voltages
listed represent the emission intensities detected by the photomulti-
plier. The errors presented represent the fluctuation of the analog
output from the photomultiplier measuring amplifierlxnn Values in
Table 4.1 were determined by exciting the microspheres with l6.hflJ
(obtained at intensity level 6 with an additional neutral density
filter). The two groups of values represent 2 different samples«of
microspheres at different photomultiplier gains.

Further characterizations of the normalizing standards were deter-
mined in long exposure experiments. In these experiments the time
histories of the intensities of the microspheres were recorded by the
strip chart recorder. A typical response reproduced from data supplied
by (39) can be seen in Figure 4.5. These experiments were approximately
10 minutes in length. The microspheres were excited by 16.7 pW at 514.5
tun.‘The microsphere intensities decreased by approximately 50% during
the experiments as shown in Figure 4.5. Also evident from these long
exposure experiments records were noise to signal ratios on the order of
1:5 in all.<uases (39). The noise in all cases was of a high frequency

nature.

65

. . “at.
fitéi‘f“
.1 ‘ :23“- ,_z"i;";“‘,
.!,7.'~z,,.332’-'
:1”; {$.33
. 3114'
f” 1r" ‘ "'"—""’T

Fig. 4.5 Long term exposure response of fluorescent microspheres
reproduced from data supplied by (39).

Still other characterizations of the "normalizing standard" (micro-
spheres) helped determine the laser photobleaching system dependence on
the focusing of samples. In the first focusing experiments many
tdifferent microspheres were taken in and out of focus. Their intensi-
ties deviated only 10% from a mean in focus intensity (39)when an
excitation intensity of 16.7 pH was used. In other focusing experiments
intensity readings were taken at equal distances above and below a
microsphere focal plane. These experiments showed less than a 5%
difference in mean intensity levels for above and below the sample
focal plane (39).

The final experiments conducted as part of the independent study
project were liposome characterization experiments. Long exposure time
characterizations of liposomes also showed a decrease of intensity.

These decreases of intensity were typically 10—Nfiéfbr the Nlndnute

66

length of the experiment (39). Even more evident were the large noise
to signal ratios, 1:4 in most cases (39). Typical long term liposome
response can be seen in Figure 4.6, reproduced from (39). Another point
to be made about Figure 4.6 is how erratic the high frequency noise of

the signal was.

 

 

 

MT

 

Fig. 4.6 Long term exposure response of liposomes reproduced from data
supplied by (39).

The major results of this study came from the remaining two appli-
cations of the laser photobleaching system. These two applications were
the spot FRAP technique and the multi-point FRAP technique. The results
from the multi-point technique provided only preliminary information.
IT“; spot technique confirmed FRAP capabilities of the the laser photo-

bleaching system.

67

The preliminary information gained by the multi-point technique
application covered many areas. The laser system with a 55 m sec
cycling time was unable to monitor the diffusion of rhodamine solution
with calculated 8 msec characteristic time for diffusion for 2 pm
diameter focused beam . In the quantitative multi-point experiments it
was found that the emission intensities for the eleven locations (:5 pm
from the center point) decreased the farther the beam was from the
optical center. In the qualitative multi-point experiments it was found
that the emission "spot" became more asymmetrical the farther the beam
was from the optical center. These multi-point experiments also
revealed that an average of many initial intensities should be taken
instead of only one initial intensity for a sample.

With the experience and information gained from the multi-point
experiments some successful applications of the spot FRAP technique were
obtained. 0f the sixteen spot-technique experiments conducted, twelve
produced some type of recovery. The remaining four experiments showed
no change in detected intensities. The unchanging intensity was due to
not supplying enough energy to bleach the sample (intensity level 7).
Of the twelve experiments displaying some type of recovery seven
displayed an "expected" fluorescence versus time exponential recoveries.
A typical "expected" recovery for an experiment can be seen in Figure
4.7. The other five experiments surpassed the initial intensities
measured for the sample then in time returned to a value slightly less
than the initial intensity. A typical response of an "abnormal"

recovery can be seen in Figure 4.7.

68

 

Fluorescence (arbitrary units)

250‘; v Abnormal
I Normal

0. VVVVVVVVV V—rV‘TYjYTT“? VYT' 1" IVY1VI"

0.00 0.05 0.10013 0.20 0.25 0.50 0.535 0.40 0.45' 10.150
Time (seconds)

Fig. 4.7 Typical recovery responses for spot - FRAP technique
The diffusion coefficient for 4.6 x 10-6 M rhodamine in water was
calculated by two methods from the spot technique recovery data. The
first method was a three point fitting method used by (1). This three
point method predicted a mean diffusion coefficient for the seven
exponential recoveries to be (1.22 t .68) x 10‘6 cmzlsec. The error of
the calculated mean diffusion coefficient reflects the mean of standard
deviation from the seven recoveries. The diffusional coefficients
calculated using the three point method can be seen in Table 4.2. The
errors listed reflect the uncertainty in beam diameter (:0.5 pm) and in

the recovery time (:2.5 msec).

69

Table 4.2 Diffusion Coefficients

 

 

Exp; Three Point Method Least Squares Fit
(1x10.6 cmzlsec) (1x10.6 cmzlsec)

41 1.11 i 0.56 0.20 (-0.99)

42 1.30 i 0.71 0.44 (-0.98)

49 1.13 i 0.76 0.79 (-0.98)

50 1.15 i 0.78 0.79 (-0.98)

51 1.13 t 0.76 0.79 (-0.98)

52 1.36 i 0.59 0.67 (-0.97)

53 1.36 i 0.59 0.67 (-0.97)

A technique using a least squares fit of the data predicted lower

diffusion coefficients for (4.6x10-6

M) rhodamine solutions. The mean
diffusion coefficient of the least squares fits was 0.62 x 10.6 cm2/sec.
The mean of the standard deviations of these seven diffusion coefficient
values was i 0.22 x 10m6 cmzlsec. The diffusional coefficients calcu-
lated using a least squares fit can be seen in Table 4L2. The cor-

relation coefficients for each of the least squares fits are listed in

parentheses in Table 4.2.

70

The five "abnormal" recoveries could not be explained. Yet they did
provide substantive information about the laser photobleaching system.
These recoveries revealed that a mismatch between the A/D converter and
PMT output existed. They showed that shorter experimental run times
could be used for rhodamine in water experiments. Finally, these
abnormal recoveries showed that the emission intensities were constant
for periods much greater than the diffusional characteristic time for

the (4.6 x 10.6 M) rhodamine solutions.

71

72

5.0 Discussion

The results from the applications of the laser photobleaching
system revealed the system is functional within certain limits. These
limits were due to problems and situations faced throughout the develop-
ment and applications of the laser system. The solutions, explanations,
or evasions of these problems and situations is presented in this
section. Then data reduction methods, and application results are
discussed.

The laser photobleaching system hardware posed many problems. The
energy generation group, beam manipulation group, and light detection
group of the hardware each provided major difficulties.

The energy generation group problems began before the laser and
power supply arrived. The laser and power supply initially proposed was
an air cooled 300 mW Lexel Argon Laser. Instead a water cooled 4W
Spectrui Physics Argon Laser was purchased this required the building of
a heat exchanger. Constructing the heat exchanger, even though it was
simple to design, added extra time to the development of the system.

The arrival of the laser brought further problems. The laser
.arrived in a damaged.shipping crate. It was unknown whether the laser
was seriously damaged or not. Also, since this was a demonstration
model, ru) replacement was available. The shipping company refused to
cover the loss of the laser until it was shown to be damaged. Many
weeks were wasted because there was no service contract or technical
support supplied by Spectra Physics for setting up and testing the

laser. Ron Bass a technician from M.S.U.'s chemistry department,

finally was able to help show that the laser "worked".

Indeed, the laser "worked" but not to the manufacturer's specifi-
cations. The problem apparently stemmed from the mishandling of the
laser during shipping. The laser system was brought\u)to specifi-
cations by re-aligning the laser tube within the field magnet and
centering the beam in the laser tube. Again,<hmato the lack of a
service contract, Ron Hass provided instruction on centering the tube in
the field magnet, centering the beam in the tube, and cleaning the laser
optics. Many weeks were spent optimizing the lasers output to meet the
manufacturer's specifications. Complete details for these power
optimizations methods are presented in Appendix A.

More extensive usage of the laser disclosed one last major problem
with the energy generation group. The laser field magnet required a
large current to handle its enhancement fOr ultra violet work. This
large current requirement kept blowing fuses in the power supply when
run above 30 amps. It was found ultimately that Spectra Physics had
mistakenly supplied and installed the fuses for a non-ultra violet
enhanced laser. Ron Hass and Spectra Physics spent manyrmue'weeks
trying to isolate this problem.

After the laser was operating properly, difficulties were discovered
in the beam manipulation group. The problems were in the areas of
attenuation, direction, filtration, fixation, position, and reduction.

The problem with attenuation of the laser bemnvms thatrmfltiple
reflections of the beam seen at the sample were introduced by the
neutral density filters. The multiple reflections were caused by the

beam reflecting off the surface of a neutral density filter and

73

striking another filter. The mistaken belief that the filters had to
be critically aligned to avoid these reflections wasted time unneces-
sarily. The multiple reflections were in fact eliminahuitw'arbi-
trarily positioning the neutral density filters to avoid having the beam
reflect on any other filters.

IMultiple reflections were also a problem with the filter sets for
the laser system. These reflections occurred where the beam reflected
off the surfaces of the excitation, barrier and dichroic mirrors of the
filter sets. Some of the reflections were removed by eliminating the
excitation filters from the beam path. The excitation filters were
deemed unnecessary because the laser was being operated in single
wavelength mode at 514.5 nm. Other reflections of the beam were
eliminated by aligning the barrier filters properly in the epi-
illuminator housing of the microscope.

The last multiple reflection was caused by the dichroic mirror. The
dichroic mirror had been improperly installed. This caused the beam to
be reflected down to the sample by the front (substrate) surface and
then the (reflective) coated surface. The proper orientation of the
dichroic mirror was obtained by placing the coated surface nearest to
the beam's source. This re-orientation of the dichroic mirror elimi-
nated the final multiple reflection.

The next difficulty which arose in the beam manipulation group was
the alignment of the optical axis of the laser beam with the optical
axis of tfluelnicroscope. The first step was to affix all the equipment
to the optical table. This provided a frame reference for the optical

paths of the laser and microscope. The next step was to direct the beam

74

to the back of the microscope keeping it parallel or orthogonal to the
optical table (reference frame) at all times. The final step was to use
the multiple degree of freedom microscope mounting plate to align the
optical path of the microscope to the beam. Attempts at aligning the
beam optical path to the microscope optical path failed. Complete
alignment techniques are presented in Appendix A.

The most time consuming problem with the beam manipulation group was
the reduction of the beam. Three methods were applied to reduce the
beam from 1.25 mm at the source to the 1-2 pm diameter desired at the
sample focal plane. Two of these methods proved unsatisfactory. The
first unsatisfactory method came directly from optical physics.
Parallel light (source) passed through two convex lenses with the
correct ratio of focal lengths (magnification) produces parallel light
at a reduced diameter an "inverted telescope". The problem with this
"inverted telescope" method was that the incoming beam did not fill the
entire lens. That caused an interference pattern to also be visible at
the sample focal plane. This interference pattern appeared as concen-
tric rings with the desired spot diameter in the center point.

The second unsuccessful beam reduction method was a variation of the
"inverted telescope" method. This method tried to eliminate the
concentric interference rings with pinhole stops. fHuepdnhole stops
seemed to work, but, there was no way to mount and position them
accurately. If these stops were positioned improperly other inter-
ference patterns were introduced.

Finally, a method suggested by Jack Holland, Ph.D., a professor for
the Biochemistry Department at M.S.U. solved the beam reduction problem.

He suggested placing a convex lens in the beam path and matching its

75

focal point to the effective focal length of the microscope focusing
objective. Tflrfs method worked for observing intensities but a diffuse
emission pattern could be seen around the 2 pm emission spot of the beam
for bleaching intensities greater than level 7. This diffuse emission
pattern occurred because the beam did not diverge enough thus exciting
any fluorescent probe in the area surrounding the beam. This reduction
‘method was used even though the fluorescent probe surrounding emission
spot was being excited during bleaching. It was assumed that the
fluorophore in this diffuse region would not receive enough energy to be
bleached thus not affecting the recovery. The spot FRAP experiments
showed that this assumption was valid.

Two final problems of the beam manipulation group did not become
apparent until the system was being used for the FRAP application
experiments. The first problem was that the beam was only able to scan
11 pm at tine sample focal plane. This limited scanning length was due
to the diffraction of the beam as it was moved off the optical center of
the reducing lens and the microscope objective. This problem was evaded
by limiting the scanning length to 10 pm (:5 pm from a center point).

The other problem found while trying to apply the system to was FRAP
experiments was the low reflectivity of the scanning mirror. When at
bleaching or observing intensity the scanning mirror permitted an
undeterndJuui portion of the beam to be transmitted through its reflec-
tive surface. This problem had no effect on the detenmhmujon of the
diffusion coefficient since normalized intensities were used. However,
the theoretical fluorescence could not be calculated since attenuated
power supplied to the sample was unknown because of this low reflect-

ivity{

76

Difficulties with the light detection group appeared during the
quantization application experiments using the laser photobleaching
system. The erratic output of the photometer system during character-
ization experiments was a major concern. A typical erratic emission
response from a 2 pm spot of a fluorescently labeled liposome can be
seen in Figure 4.6 in chapter 4. The erratic outputs did not permit the
determination of anything but trends from liposomes and microspheres.
This erratic output was corrected by eliminating 60 cycle noise and high
frequency noise. The addition of a 470 pF capacitor to the reference
voltage circuit of the photomultiplier tube eliminated the high fre-
quency noise. The 60 cycle noise was eliminated by converting the PMT
measuring amplifier ground from an earth ground to a floating ground .

Another problem with the light detection group which was found
during characterization experiments was a low frequency drift of the
photometer output. The period of this drift has been measured to be
11-13 minutes. The magnitude of the drift was approximately 15-20°/o of
the total output. This amplitude drift has not been solved. A "period"
of the low frequency drift (oscillating output) of the photometer can be

seen in Figure 5.1.

' . '_ altos»! -'

 

Fig. 5.1 Low frequency drift of the photometer output.

77

The noise present in Figure 5.1 was due to the expanded scale used (5 V
fullscale). This low frequency drift was avoided by using rhodamine in
water with an 8 msec characteristic time for diffusion.

The final difficulty with the light detection group was a mismatch
of the PMT analog output and the A/D range of the computer. This problem
resulted from not understanding that a bipolar 10.24 V A/D board meant
:5.12 V not 110.24 V. The mismatch went unnoticed until the final
stages of this study. This mismatch has not been corrected.

As the system development progressed problems with the software also
became apparent. The first software difficulty to arise was the
inability to change the output filename interactively using FORTRAN IV.
The software was rewritten to allow the changing of tflmerun number
after each experiment. The output filename now has the form of FTN--.
DAT, where the spaces represent a number between 10 and 99.

Another software problem was the limited speed in which the inter-
rupt service routines (ISRs) could be operated. The ISRs could only be
operated at 200 Hz (5 msec) response without causing a computer
failure. It is believed this rate may be improved by using machine
language programs. Data collected at 5 msec rate supplied 5—6 points
to calculate the diffusion coefficient of rhodamine solution using the
least squares fit.

.Application of the system revealed another fault with the software.
The software only acquired one intensity reading for the initial
concentration intensity of the sample before bleaching. Taking just one
reading did not allow for the possibility of an extraneous intensity

reading. Extraneous intensities were compensated by taking ten readings

78

and then using the mean of these as the initial concentration intensity.
Further use of the system showed that a larger sample should be taken,
but this change was not incorporated into the software.

Still another difficulty with the laser photobleaching system
software was it did not allow the operator to plot results. This
graphics capability has not been incorporated into the operating system
because of'tflua incompatibility of an ADM-3A terminal with the graphics
'package PLOT 10 (TEKTRONICS). The ability to see the "recovery" for an
experiment would provide the operator with an immediate check for the
proper functioning of the system.

The final problem with the software evolved from the conversion from
the multi-point-technique to the spot technique. The conversion was
made because the recovery of the 2 pm bleached spot was too fast for the
55 m sec cycle of the multi-point technique. The only software change
to the multi-point operation was not to move the scanning mirror. In
making this change the system was able to collect data at 5 msec
intervals, required to be moved from one position to the next in the
multi-point technique. The 5 msec interval was fast enough to obtain at
least 5-6 points before the bleached spot of the rhodamine solution had
recovered. It was from these 5-6 points that the diffusion coefficients
were determined. Separation of the two techniques should improve on the
5 msec sampling rate presently obtainable for the spot technique.

The application and data reduction of the FRAP experiments disclosed
(“finer difficulties. The 5 msec cycling time of the spot technique did
not permit the measurement of the fluorescence intensity immediately
after bleaching. The intensity at zero time was required for Axelrod's

three poiJH: fit method (1) to determine the diffusion coefficient. In

79

this study, the zero time intensity had to be approximated. The
--t/'co
approximation was made assuming f(t) - 1 - e fbr the fluorescence

recovery. Using a least squares fit of the recovery data in the

following form

(F(W) - F(t))v a _ 42 (F(@) - F(o)v
Ln ( 10 v ) ( 2) t + &n ( 10v ) (5.1)

W

 

 

provided the slope (-4D/w2) and the fluorescence at t=0. This ap-
proximation was the basis for'the least squares method of determining
the diffusion coefficient D. The diffusion coefficient was determined
from the slope of equation 5.1. A complete derivation of this equation
can be seen in Appendix G.
Diffusion coefficients determined using the three point fit method
(1) were compared to the values determined from the slope of the least
squares fit method. The values can be seen in Table 4.2. The mean
values of the diffusion coefficients for each method generally varied by
a factor of two. The largest difference predicted by,the two methods
varied by a factor of five. Determination of which method was pre-
dicting the more accurate value for the diffusion coefficient was not
made. It should be noted that Axelrod (1) uses onLytfluee points to
determine the diffusion coefficient. The least squares fit method used
all the recovery intensities.
The possible differences in the calculated diffusion coefficients of
this study originated from a few sources. One source was the assumption
made in developing the least squares fit using f(t)=='1-'ent/To instead

-t/t
of f(t) ' 1 + Be or the infinite series solution Equation 2.6. Bv

.r

assuming B = l.():it was assumed that complete recovery occurred. This

80

assumption was supported by the mobile fractions of greater than 95%
found by (1) for rhodamine solution. Another source of the differences
in the predicted values was that one Axelrod's method used only three

points F(0), F(m), and F(t1 2) to predict D, while the least squares fit

/
used all the data points.

The results obtained during this study by using the three point fit
method (1) for determining the diffusion coefficients were compared to
those obtained by Axelrod et al (1). They agreed within stated error
ranges. Axelrod predicted the diffusion coefficients to be (1.2 t .3) x
10m6 cm2/sec for solutions of rhodamine in water (1) in 12 samples. The
laser photobleaching system predicted a mean diffusion coefficient to be
(1.22 t .68) x 10‘.6 cm2/ sec for a sample size of 7.

The larger error for the diffusion coefficient prediction was due to
the sample size of only 7, a 25% uncertainty in the effective beam
diameter, the uncertainty of the fluorescence immediately after bleach-
ing (F(0)) and the uncertainty of the actual time when the normalized
fluorescence recovery was equal to one half (f1:(t) = 0.5). The
effective beam diameter as stated in Table 3.3 was (2.0 t 0.5) um for
the 20x objectdxne. The time uncertainty of i 2.5 msec for the normal-
ized fluorescence intensity reflected 5 msec sampling rate.

The quantization application results raised questions of intensity
requirements, probe selectitnh single bilayer standard formation, and
normalizimu; standard selection. Concerns about the intensity require-
ments appeared during the characterizations of the fluorescently labeled
microspheres and the liposomes. The long term exposures shown Figure
4.5 and 4.6 both displayed a definite decrease in intensity over ten

minutes using a 16.7 uW observing intensities. It was initially

81

believed that this observing intensity had too high an energy density.
This high density caused "bleaching" of the fluorescent probe in the
sample region. However, when the energy densities used by others (18,
19, 40) were compared to the energy densities supplied bylflmelaser
photobleaching system the density level was not too high. In Table 5.1
the energy densities used by others for bleaching the diI fluorescent

probe are presented.

Table 5.1 - Bleaching Energy Densities

 

 

System energy density w/um2 Length of Exposure
-3 2

Axelrod (40) 6.5 x 10 w/um 1.0 sec

Schlessinger (18) 9.1 x 10‘3 w/umz 0.5 sec

Wolf (19) 9.1 x 10"3 w/umz 0.15 sec

The energy density used in this study (2.6 x 10-6 w/pmz) was three
orders of magnitude less than the bleaching energy density recommended
by (18, 19, 40) for observation. Some decrease of'fluorescence*was
expected for long exposures, but 30-50% decrease in 10 minutes was
unacceptable.

The diI probe had been selected because of its widespread use. No
characterization of the probe was attempted before its use. Therefore
no further explanation of the decrease in emission intensimvcflfthe
probes in the 10 minute exposure to the 16.7 uW intensity can be made at

this time.

82

The results from the single bilayer formation and transferring
attempt showed that the microbiology background and experience in
successfully forming a single bilayer standard was absent. Axbasic
understanding of what BLMS were and how they were formed was obtained.
However, a physical understanding of why the BLM formation and trans-
portation failed has yet to be ascertained.

The results from the characterization experiments of normalizing
standards showed that the microspheres were of no use when using diI as
the fluorescent probe in liposomes. The emission intensities of the
fluorescently labeled microspheres and liposomes could not be taken with
the same PMT gain settings. Further, the emission from the microspheres
decreased twice as much as fluorescently labeled liposomes for 10 minute

exposures.

83

84

6.0 Conclusions

Based upon the results of this study the following conclusions can

be made:

1)

2)

3)

A laser photobleaching system for use with unique microscopy

techniques has been developed.

The laser photobleaching system has been shown to be functional
although limitations exist. These limitations include: a) low
frequency drift in photometer output, b) low reflectivity of the
scanning mirrory <2) confined beam scanning length on a sample
focal plane, and d) mismatched analog to digital input range

(35.12 V) photometer output range (0 - 10 V).

The laser photobleaching system using a spot Fluorescence
Recovery After Photobleaching technique has predicted lateral
diffusion coefficients. The predicted lateral diffusion
coefficients of rhodamine (4.6 x 10-611) in water were within

the same order of magnitude as values published by Axelrod (1).

The laser photobleaching system was unable to monitor the
recovery of rhodamine in water using a multi-point Fluorescence

Recovery After Photobleaching technique. The 55 111 sec cycling

5)

6)

time for the multi-point technique was too slow for the cal-
culated 8 10 sec characteristic time for diffusion of rhodamine

in water.

The development of a microscopy technique using the laser
photobleaching system to quantitatively determine the number of
bilayers present in liposomes produced inconclusive results.
Both the fluorescence contrast and comparison methods of
quantization of the number of bilayers require further study to

be classified as valid quantization techniques.

To use the laser photobleaching system to its maximum potential

will require more collaboration with researchersxflmahave an

extensive background in biochemistry or microbiology.

85

86

7.0 Suggestions for future work

The results of this study displayed only some of the capabilities
and limitations of the laser photobleaching system. During this study
several components of the laser system required corrections. However,
some applications of this laser system worthy of future investigations
were disclosed.

M components of the laser photobleaching system hardware need to
be replaced, repaired, or redesigned before further applications are
conducted. Of the items requiring replacement the scanning mirror with
its poor reflectivity should have the top priority. Uncertainty about
its reflectivity prevented precise energy density calculations. Measure-
ment of the energy lost due to this mirror should be made. Other system
components that should be considered for replacement are the stand for
the fifth neutral density filter and the convex lens which reduces the
beam diameter. The fifth neutral density filter, which allows ad-
ditional attenuation of the beam, is held in place with masking tape.
The convex reducing lens produces a diffuse emission pattern around the
focused beam for bleaching intensities and should be replaced.

Some of the computer system components should also be considered for
replacement. The printer should be replaced as soon as possible. It is
needed for both printed hardcopy and graphics. Another component which
should be replaced is the graphics terminal because its resolution is
too low. The last computer component that should be replaced is the
parallel line unit (transistor logic input-output board DRVll, D.E.C.).
On the DRVll board presently in use 4 of the 16 channels are not

functioning.

The only laser photobleaching system component which required repair
was the photometer. The existing low frequency drift of the photometer
output will effect the results of FRAP experiments with larger charac-
teristic times for diffusion. The problem has been isolated to the
photomultiplier tube or the electronics in the photomultiplier tube
housing. The electronics of the measuring amplifier box have been
thoroughly examined and eliminated as the source of the low frequency
drift of the output of the photometer.

Components that should be considered for redesigning are the beam
directing and reducing groups and the beam scanning system. The beam
directing and reducing groups presently direct the beam in the rear of
the microscope to be reduced. If replacement of the reducing convex
lens does not eliminate the emission pattern around the focused beam, an
alternate beam path to the sample should be considered. The beam
scanning system must be repositioned to allow for a longer scanning
range at the sample focal plane. The scanning range is presently only
10 um at the sample focal plane.

Some components of the laser photobleaching system software need to
be modified or added to the operating systems before further appli-
cations are conducted. The highest priority for operating system
software should be given to separating the spot and multi-point FRAP
techniques into different subroutines. Separation of the two techniques
should elicit a quicker data collection cycle for the spot FRAP tech-
nique than the present rate of 5 msec. Compensation for the A/D board
and photometer output in the operating system software should be made
until the hardware mismatch can be eliminated. The interrupt services

routines also need to be changed. The cycling times of the spot and

87

multi-point techniques should be reduced by converting the interrupt
service routines from FORTRAN IV to MACRO-11. A final software component
that needs to be added to the operating system is graphics. Graphics
capability would allow immediate recognition of problems with fluores-
cence recoveries. Before graphics capability can be added the present
graphics software needs to be made compatible with the present terminal.

After making the necessary corrections to the laser system compo-
nents, proposed applications of the system should include further
verification of application experiments conducted in this study as well
as some new applications. The spot FRAP technique should be the first
application to be verified. This verification is needed to understand
and explain why both "expected" and "abnormal" recoveries were detected
in this study. Next the multi-point FRAP technique should be examined.
The present study failed to verify the FRAP multi-point technique
capability. Both techniques of quantization using laser photobleaching
require further investigation. The fluorescent contrast method of
quantization showed some promise as a technique for determining single
bilayer liposomes. However no solid conclusions could be drawn from the
small sample size of fluorescence contrast information. The comparison
method of quantization method is based on a sound hypothesis, but an
easily formed single bilayer standard and a stable normalizing standard
are needed. Finding two workable standards would allow evaluation of
this technique as a method of quantization.

The new applications that should be investigated are the next
logical step in the progression of the laser photobleaching system's
capabilities. The most interesting application would be the integration

of image analysis with the present System. With such an integration

88

researchers cxnxhi eliminate the photometer system. This elimination
would be possible because the image analysis system has the capability
to resolve intensity differences. The image analysis system would also
allow for data collection over an increased area. This increased area
would be equal to the viewing area of the low light video camera. The
image analysis system would not reduce the 5 m sec cycling thmacflfthe
interrupt service routines (ISRs) for the multi-point system. The image
analysis is limited by the 33 m sec per frame state of vhhx>tapes.
Intensities at multiple points could be monitored simultaneously by the
image analysis system.

Even without image analysis integration, there are other new
applications which would expand the capabilities of the laser photo-
bleaching system. The first two applications that should be conducted
involve using the laser photobleaching system on a membrane sysman.
These studies would be important for further verification of the spot
and multi-point FRAP technique capabilities of the system.

Another interesting application for the system would be determining

the effect of temperature on the diffusion of molecules in a membrane.
This study; if used in conjunction with image analysis, could disclose
some of the mechanisms which cause damage during freezing.

The present study has demonstrated that the laseryflmmobleaching
system is operational. The system has some limitations, but once these
are resolved a very powerful experimental tool will exist. It will then
be up to other researchers to expand and refine the laser photo-

bleaching system and its applications.

89

APPENDICES

 

APPENDIX A: LASER OPERATION AND CARE

Laser Operation and Care

A.1:

A.2:

Laser Start Up

10.

11.

12.

Turn on tap water to heat exchanger (5-6 gal/min)
Open valve for flow and pressure bleed off downstream pump.
Turn on pump for recirculating cooling water.

.Adjust water pressure going to laser to be 40 psi (30-50
psi limits for laser) this will provide a 2.5-3.0 gal/min.
Replace water filter if under 2.2 gal/min.

Turn on the main wall circuit breaker box.

Turn power supply key on.

Set the control settings to the following:

+ meter - 30 A

+ current - 50% of fullscale

+ mode - current

+ field - maximum

Check for water indication light on the power supply

Turn on power supply circuit breakers

Wait 30 seconds (start light will glow) and press start

If fails refer to operating manual.

One half hour is required for stabilized operation.

Laser Shut Down

1.

2.

Turn off laser at power supply circuit breakers

Turn off key of power supply

90

3. Turn off main circuit breaker

4. Continue running heat exchanger until water returning from
laser is 20°C

5. Open pressure valve, pressure should drop to zero.

6. Turn off recirculating pump, and then close pressure
valve.

7. Turn off tap water.

A.3: Mode - Selection

The laser has two operating modes, the current mode and the
light mode. The current mode regulates the power output by
regulating the current supplied to the laser tube. The light
mode regulates the power output by sampling beam and adjusting
the current accordingly. The current mode is claimed to have a
stability of i 3% after 30 minute warm up and only 1% rms noise
(at 514.5 nm) (49). The light mode is claimed to have a
stability of 1': 0.5% after 2 hour warm up and 0.2% rms noise
(49). The current mode is safer to use since any time the beam
is blocked the light mode supplies maximum current to counter

the drop in the sampling intensity.
The selection of modes should be done after start-up. The

settings used before changing modes should be at start-up

values. Then to select mode flip mode selection switch.

91

A.4:

 

Cleaning and Peaking Output

In lasers losses due to unclean or misaligned<unjcs, which
might be negligible in other optical systems, can cause enough
loss to disable a laser. Dust, films, and atmosphericcunv-
taminants on any of the optical surfaces can cause loss of
output power or operational failure. Even with clean optical
surfaces the output power may be limited by misalignment of the
optics. The required materials and procedures for general
cleaning and peaking are listed below. If the laser output
still does not meet specifications refer to advanced techniques

listed in the operating manual (49).

Materials -

1. Filtered, Nitrogen (25 um limit on filter mesh)
2. Lens cleaning tissue

3. Cotton swabs

4. Hemostat (non-metallic preferred)

5. Acetone (CH COCH spectroscopic grade

3 3
6. Methanol (CHBOH) spectroscopic grade
7. Power meter
Procedure - If specified power output is achieved prior to the

completion of all the steps, the remaining steps

are optional.

92

I.

Peaking Output - Complete peaking only required once every 2 months

or before laser changed to single-line mode.
Before any cleaning step the laser should be peaked
for maximum output using multi-line lasing. Note:
when peaking output of laser work at one end only.
In the case of losing lasing capabilities it will
be easy to get lasing again. If lasing is lost
refer to operation manuals trouble shooting section

(49).

Before laser start-up remove cover and insert cover interlock

defeat plug.

Start-up laser. Allow a half hour for warm-up.

.Always start at rear end plate (anode end, where cooling water

‘is supplied) adjust vertical adjustment thumbwheel until power

is peaked. Next adjust horizontal adjustment thumbwheel until

power is peaked. If lasing is lost do not touch any other

 

adjustment. Using adjustment thumbwheel used when lasing was
lost turn back slowly until lasing re-established. Finish
peaking using that thumbwheel. Repeat cycle at‘fluarear end

plate until no more output power is gained.

93

 

.After peaking power at rear end peak output power at the front
end plate (cathode end, where laser beam is emitted). Ik1not
touch any other adjustments until peaking at the front plate is
completed. Again start with vertical adjustment peak then go to
horizontal. If lasing lost use that adjustment set screw
without touching another to get lasing to return. Repeat cycle

at the front end plate until no more output power is gained.

Repeat steps 3 & 4 until no more output power is gained.

If specified power is not achieved yet start cleaning procedure.

II. Cleaning - Clean only one optical component at a time. If lasing

jhost after cleaning and replacement refer to operation

manual (49).

Always start with rear end mirror,tflmn1front end, then front

Brewster window, and then rear Brewster window.

Repeat steps 1-4 for each optical component.

1. Blow away dust with filtered air.

2. Use folded (1/2" square) lens tissue held in hemostat when

applying methanol or acetone.

94

Start wiping with methanol. Wet tissue rid excess by
shaking. Wipe with even pressure only once over surface.
Replace lens tissue. Repeat but wipe in a direction 90
degrees to previous wipe. Repeat two more times.

Replace optical component.

If specified power still is not achieved then repeat above

cleaning steps 2-4 but adding wiping with acetone
before wiping with methanol.

'If specified power still is not achieved try peaking power
again.

If still does not achieve specified power levels go to

advanced cleaning steps outlined in operation manual (49).

NOTE: Cleaning prism for single-line operation DO NOT TAKE APART. Use

 

cotton swabs with methanol or acetone to wipe surfaces clean.

A.5:

Lasing -- Multi-Line

Multi-line operation is used for complete peaking laser power

(required every 2 months or before laser changed to single-line

operation). lhilti-line operation achieved by just inserting

planar rear end mirror into rear end plate. Peak power only at

rear end plate after insertion. Other end should not have

changed.

95

A.6:

Lasing - Single-Line

Laser must be completely peaked before inserting prism for
single-line operation. The following procedure is then
required to peak the power for single line operation. Ac-
cording to Spectra-Physics the prism for single-line operation
and the planar mirror for multi-line operation can be inter-
changed without realignment. But for this laser realign-
ment is required to peak power of the single line. If the
prism has not been jarred the laser should lase when the prism
replaces the planar mirror after complete peaking (APPENDIX

A.4).

Peaking single-line power is required if laser output at the
desired wavelength is less than 25% of the "ideal" specified
power. (49). Always peak prism initially at the 514.51m1

(bright green) wavelength.

Warning: the procedure in the operation manual is the only way

to peak the power for single line operation.

The procedure sounds easy in theory but is very difficult in

practice. Listed below are a few hints that will help.

1. After each adjustment to the prism replaces the planar

mirror (multi-line). Repeak using rear end plate only

then replace prism to see if further adjustment is needed.

96

If lasing is lost use "rocking procedure" discussed in
manual (49) to find which direction the prism must be

moved.

To see where prism is deflecting beam insert piece of paper
in path of beam. Center reflection about laser tube using
adjustments in step 3 to get it to lase if it was not
lasing before. If the laser was lasing the rocking
procedure discussed in manual (49) will indicate which

adjustments in step three will be needed.

97

APPENDIX B: LASER PHOTOBLEACHING SYSTEM PROGRAMS

B.1 CONTROL ONLY OPERATING SYSTEM

PROGRAN LOPERA

 

THIS IS A PROGRAN THAT CONTROLS THE NEUTRAL DENSITY FILTERS,
AND SCANNING NIRROR FOR BASIC LASER OPERATION (LOPERA). THE
PROGRAH CONBINES HANY OF THE SAHE SODROUTINES USED IN THE
LASCON PROGRAN (F.R.A.P. CONTROL). THE PROGRAH HOST BE LINKED
IITH THE FDLLDHING SDDROOTINES NPOSIT,FPLACE,DTACHA,AND CLOCIN

 

 

finnnnnnnnnn

DECLARATIONS

 

CONNON IDTOAIIVLTSI,IVLTSZ,IVLTSS,ICODNT,ICYCLE,IVD,ISLOPE,

+IDONE,IDACHA

CONNOR ICLOCK/ ICLBPR,ICLCSR,12CONP,ICLKST,ICLKGD
CONNDN [SHUT] IDRV11,ILSHDT, IPSWTJDSIRIT, ISHUT,ISHDTN
CONNON IFILTER/ ODSERV,ODSVAL,DLECHV,BLEVAL,TRNSO,TRNSD
CONNOR IFILE/ FNANE

INTEGER ODSVAL,OBSERV,DLEVAL,BLECHV

REAL RESP

REALtO FNANE

DINENSIDN IDLISTIZI)

EXTERNAL DTACHA

 

nnnn

IDLIST FDR RESETTING REGISTERS TO ORIGINAL STATUS

 

101151111s-10777o 109011 0.5.9.
1011511212190091-1071701 100910915 09911 0.5.9.
10115113121107772 ' 109911 001901 000009
101151141=19009111077721 100910915 09011 0.0.
1011511512-170420 19.1. 01009 0.5.9.
101151101=190091-17o4201 100910915 9.1. 01009 0.5.9.
101151171a-110122 19.1. 01009 0.9.
101151101=19009111704221 100910915 9.1. 01009 9.9.
muwuuunmo mmcsm.
1011511101:190091-177ooo1 100910915 910 0.5.9.
1011511111=1177002 1910 0.9.
1011511121=1900x111770o21 100910915 A/D 0.9.
1011511131=-17044o 1019 0.5.9. A
1011511111:190091-1704401 100910915 019 0.5.9. 9
1011511151=-170442 !DIA 0.5.9. 0
1011511101=190091-1104421 100910915 0.5.9.0
1011511111=117o444. 1019 0.5.9. 0
1011511101:190091-1704441 100910915 0.5.9. 0
IDLIST119)='170446 1019 0.5.9. 0
1011511201:190091'1704401 100910915 0.5.9. 0
1011511211=o 1090 00 0151 0105

 

CALL DEVICE1IDLIST1 !UPDN EXIT RESETS REGISTERS IN IDLIST

 

953

 

 

 

 

C INITIAL VALUES
C
IDRV118'167772 EDRVII OUTPUT BUFFER ADDRESS
ILSNUT='20 EOCTAL VALUE TO SHUT LASER SHUTTER
IPSHUT3'40 !OCTAL VALUE TO SHUT PNT SHUTTER
IBSHUT='60 !DCTAL VALUE TO SHUT BOTH SHUTTERS
ISHUTN=3 ESHUTTER FLAG BOTH CLOSED
ISHUT=IBSHUT !OCTAL VALUE FOR BOTH CLOSED
OBSERV=I !INTENSITY LEVEL 1
OBSVAL3'0 EOCTAL VALUE FOR LEVEL 1
TRNSO=.000050 €PERCENTAGE TRANSNIT DBS.NDDE
BLECHV=7 fINTENSITY LEVEL 7
BLEVAL8'60000 EOCTAL VALUE FOR LEVEL 7
TRNSB'.050 EPERCENTAGE TRANSNIT BL. HODE
C
CALL IPONEIIDRVII,IBSHUTT ECLDSES BOTH SNUTTERS
10 CONTINUE
NRITE17,8)
IRITE(7,81’ LASER OPERATION HENU-ENTER E-TO EXIT PROGRAN,’
NRITE(7,8)’ I-TO SELECT BEAN INTENSITY AND SELECT SHUTTER’
NRITE(7,8)’ CONDITIONS,P-TO POSITION BEAN’
READ(7,201 RESP
20 FORHATIAI)
IF(RESP.ED.’E’)GO TO 999
IFIRESP.EO.’I’)GO TO 40
IFTRESP.EO.’P’TGO TO 30
NRITE17,!)’ UNABLE TO DETERNINE RESPONSE’
GO TO 10
30 CONTINUE
CALL NPDSIT
GO TO 10
40 CONTINUE
CALL FPLACE
GO TO 10
999 CONTINUE
CALL IPOKEIIDRV11,'0) !SHUTS DFF EVERYTHING
HRITE17,11' NORHAL PROGRAN EXIT’
C
CALL EXIT !RESETS REGISTERS TO INITIAL VALUES OF IDLIST
C
END

599

SUBROUTINE DTACHA

 

 

 

 

 

C
C THIS SUBROUTINE IS AN I.S.R. FOR THE SCANNING NIRROR CONTROL
C PROGRAN OR SUBROUTINE. THIS I.S.R. POKES A DIGITAL EQUIVALENT
C DE A VOLTAGE TO D/A CHANNEL 1. IT ALSO KEEPS TRACK OF THE
C NUHBER OF COHPLETED CYCLES. NUST BE LINKED NITH NCNTRL AND
C MPOSIT SUBROUTINES, NNICH ARE PART OF THE PROGRAHS LASCON
C AND LOPERA RESPECTIVELY.
C
C
C
C DECLARATIONS
C
COHNON /DTOAIIVLTSI,IVLT52,IVLTS3,ICOUNT,ICYCLE,IVB,ISLOPE,
+IDONE,IDACHA
CONNON lCLOCK/ ICLBPR,ICLCSR,12CONP,ICLKST,ICLKGO
COHNON ISHUT/ IDRV11,ILSHUT,IPSHUT,IBSHUT,ISHUT,ISHUTN
CONNON /FILTER/ DBSERV,OBSVAL,BLECHV,BLEVAL,TRNSO,TRNSB
CONNON /FILE/ FNANE
INTEGER OBSVAL,OBSERV,BLEVAL,BLECHV
REAL RESP
REALIB FNANE
C
CALL IPOKEIICLCSR,ICLKGO) !RESETS CLOCK PDKES GO BIT
IFIISLOPE) 10,20,20 fCHECKS POS. OR NEG SLOPE
10 CONTINUE
IVLTSI=IVLTSI+I
IF(IVLTSI.GT.IVB) GO TO 110
CALL IPOKEIIDACHA,IVLTSII !POKES NEXT VOLTAGE INCREHENT
GO TO 190
20 CONTINUE
ISLOPE=0 !RESETS SLOPE FLAG
IVLTSI=IVLTSI~I
IFIIVLTSI.LT.IVLT52) GO TO 100
CALL IPOKEIIDACHA,IVLTSI) !POKES NEXT VOLTAGE INCREHENT
GO TO 190
100 CONTINUE
ISLOPE=-I FREDIRECTS SLOPE FLAG
GO TO 190

110 CONTINUE
ICOUNT=ICOUNT+1

IVLTSI=IVB ERESETS INITIAL VOLTAGE
ISLOPE=1 !REDIRECTS SLOPE FLAG
IFIICOUNT.LT.ICYCLE1 GO TO 190
CALL IPOKEIICLCSR,'0) !POKES CLOCK OFF
IDONE=I 1SETS DONE FLAG

190 RETURN
END

1130

SUBROUTINE FPLACE

THIS SUBROUTINE IS USED TO CONTROL THE NEUTRAL DENSITY FILTER
BOX AND TO CONTROL THE SELECTION OF SHUTTER CONDITIONS. THE
FILTER BOX CAN ATTENUATE THE LASER BEAN BY PLACING ANY
CONBINATION OF THE FOUR NEUTRAL DENSITY FILTERS IN THE
LASER’S PATH. THERE ARE FOURTEEN DIFFERENT LEVELS OF INTENSITY
POSSIBLE. THIS SUBROUTINE IS PART OF THE PROGRAN LOPERA.

THIS FILE ALSO CONTAINS THE SUBROUTINES TRANSV AND SHUTTR
NHICH ARE CALLED BY FPLACE

 

DECLARATIONS

finnmnfinnnnnnm

 

009909 101091191151,191152,191155,100091,101010,190,151000,
+10090,109099

009909 1010091 101009.101059,120099,101951,101950

009909 159011 109911,115901,105901,105901,15901.159019

009909 10111091 005099,005991,010099,010991,19950.19950

009909 101101 09990

1910509 005991,00509v,010991,01009v

9091 9050

909110 09990

FOR LATER USE NITN DATA ACQUISITION PORTION FDR LASER SYSTEN

HRITEI7,!)’ ENTER DATA FILE NAHE’
READ(7,101 FNAHE

FORHATIAB)

NRITEIT,201 FNANE

FORHATIIX,AB)

nrfinnnrfinnc—x
F.) H
o o

01-.)

00911900
9911017,:1
9911017,:1
9911017,1oo) 005099,19950
100 00999111 005. 109011.13,1:1,01o.7,1 0090091 00 009911
9911017,:1
9911017,:10) 010099.19950
110 00999111 010909 10901',13,1:1,012.7,1 0090091 00 009911
9911011,:1
101159019.00.11 9911011,:11 19509-0009,091—0105001
101159019.00.21 9911011,:11 19509-010500,091-00091
101159019.00.31 9911017,:11 19509-010500,091-0105001
101159019.00.41 9911017,:11 19509-0009,091-00091
9911017,:1
9911011,:1
9911017,:11 09109 9 90500950 0909 190 001109195 909011
9911017,:11 00-10 099950 00509991109 1910951111
9911017,:11 00-10 099950 010909 1910951111
9911017,:11 05-10 099950 5901109 501119551

1(11

30

40

45

SD

55

9911017,:11 01-10 0111 191095111 9000101

9911017,:11 01-10 591150100 9119 109015 990 501119551
909011,271 9050

0099911921

1019050.00.10011 50 10 5o

1019050.00.10011 50 10 1o

1019050.00.10511 50 10 45

1019050.00.10111 50 10 199

1019059.00.10911 50 10 so

9911017,:11 099010 10 001099190 905009501

50 10 25

00911900

9911017,:1

9911017,:11 09109 005099195 191095111,1 109051-5 91590511
909011.11 005099

101005099.51.01 50 10 50

101005099.11.11 50 10 so

0911 0019001005099,0059911

0911 1999591005099,199501

19950=100119950

50 10 25

00911900

9911017,:1 .
9911017,:11 09109 010909195 191095111,7 109051-11 91590511
909011,:1 010099

101010099.11.71 5010 40
101010099.51.141 50 10 40

0911 0019001010099,0109911

0911 1999591010099,199501
19950=1995011oo

50 10 25

00911900

0911 590119

50 10 25

00911900

9911017,:)

9911017.:11 09109 090100 0909 001091
9911017,:11 0-099950 501119551
9911017,:11 0-00509991109 90001
9911017,:11 0-010909 90001
9911017,:11 0-0111 191095111 9000101
909017.5519050

0099911911

1019050.00.101150 10 25
1019050.00.101150 10 50
1019050.00.101150 10 10
1019059.00.101150 10 199

9911017,:11 099010 10 001099190 905009501
50 10 50

00911900

191091=15901.09.005991

102

CALL IPOKEIIDRV11,INTENT)
GO TO 25
TO CONTINUE

C
C NEED TO INSERT AN INTERRUPT SERVICE ROUTINE TD TINE BLEACH AND DATA
C ACOUISTION TO BE ADDED LATER
NRITET7,X)’ BLEACH HODE’
INTENT=ISHUT.OR.BLEVAL
CALL IPOKEIIDRVII,INTENT1
DO 90 I=I,1000
II=1
CONTINUE
NRITE17,X)’ OBSERVATION NODE’
INTENT=ISHUT.OR.OBSVAL
CALL IPONE(IDRVII,INTENT)
GO TO 25
199 CONTINUE
RETURN
END

nnnrocvn
-O
C-

SUBROUTINE TRANSV(IANS,TVAL1

THIS SUBROUTINE NATCHES THE AHOUNT OF LASER OUTPUT ALLONED THROUGH
TO NICROSCOPE TO THE INTENSITY VALUE ENTERED.

“fit-Dr)

IF(IANS.EO.11 TVAL=.00000050

(“’1 n (‘1 (‘1 (”A

IFTIANS.EO.2)
IFTIANS.ED.3)
IFTIANS.EO.41
IFIIANS.ED.5)
IFIIANS.ED.6)
IFIIANS.ED.7)
IFTIANS.ED.B)
IFIIANS.ED.9)

TVAL=.0000010
TVAL=.0000050
TVAL=.000010
TVAL=.000050
TVAL=.00010
TVAL=.00050
TVAL=.0010
TVAL=.0050

IFIIANS.ED.IO) TVAL=.DIO
IFTIANS.ED.II) TVAL=.050
IFTIANS.ED.12) TVAL=.10
IFIIANS.ED.13) TVAL=.50
IFIIANS.ED.14) TVAL=I.00

RETURN
END

SUBROUTINE SHUTTR

SUBROUTINE THAT CONTROLS THE LASER SHUTTER AND P.H.T. SHUTTER.
THIS IS PART OF FPLACE SUBROUTINE NHICH IS PART OF THE LOPERA

OR LASCON PROGRAHS

1113

 

fin":

DECLARATIONS

 

COHHON IDTOA/IVLTSI,IVLTSZ,IVLTS$,ICOUNT,ICYCLE,IVB,ISLOPE,IDONE,

+IDACHA

009909 1010091 101009,101059,120090,101951,101950
009909 159011 109911.115901,105901.105901,15901.159019
009909 10111091 005099,005991,010099,010991,19950,19950
009909 101101 09990

1910509 005991.005099,010991.010099

9091 9050

 

ID

40

SC

CONTINUE

NRITE17,81’ LC-LASER SHUTTER CLOSED / PHT SHUTTER OPEN’
NRITEI7,!I’ PC-PHT SHUTTER CLOSED l LASER SHUTTER OPEN’
NRITE17,1)’ BC-BDTH SHUTTERS CLOSED’

HRITEIT,X)’ BO-BOTH SHUTTERS OPEN’

READ17,101 RESP

FORNATIAZ)

IFIRESP.EO.’LC’IGO TO 20

IFIRESP.EO.’PC’)GO TO 30

IFIRESP.EO.’BC’IGO TO 40

IFIRESP.ED.’BO’)GO TO 50

NRITE17,l)’ UNABLE TO DETERNINE REEPONSE’

GO TO 5

CONTINUE

ISHUT=ILSHUT ‘OCTAL VALUE TO CLOSE LASER

,ISHUTN=2 fFLAG FOR LASER CLOSED

GO TO 99

CONTINUE

TSHUT=IPSHUT 'OCTAL VALUE TO CLOSE FHT
ISHUTN=I IFLAG FOR PHT CLOSED

GO TO 99

CONTINUE

ISHUT=IBSHUT 'OCTAL VALUE TO CLOSE BOTH
ISHUTN=3 ‘FLAG FOR BOTH CLOSED

GO TO 99

CONTINUE

ISHUT=”O ‘JCTAL VALUE TO OPEN BOTH
ISHDTN=4 'FLAE FOR BOTH OPEN
CONTINUE

RETURN

END

SUBROUTINE OCTREFIINFCT,IOUTI

SUBROUTINE TAKES AN INTEGER INPUT ~90 RETURNS OCTAL
REPRESENTATION SF DESIRED INTENSITY

1134

IFIINPUT.ED.1)
IFIINPUT.EO.2)
IFTTNPUT.ED.31
IFIINPUT.ED.41
IFIINPUT.EO.S)
IFIINPUT.EO.6)
IFIINPUT.ED.71
IFIINPUT.E0.0I
IFIINPUT.ED.9)

IOUT=‘D
IOUT='IODDDO
IOUT='40000
IOUT='140000
IOUT='20000
IOUT='120000
IOUT='60000
IOUT='160000
IOUT='50000

IF(INPUT.EO.IO) IDUT='150000
IFIINPUT.EB.III IOUT='30000
IFIINPUT.ED.12) IOUT='130000
IFIINPUT.EO.13) IOUT='70000
IFIINPUT.EO.IA) IOUT='170000

RETURN
END

1115

SUBROUTINE HPOSIT

THIS SUBROUTINE IS TO BE PART OF THE LASER OPERATION PROGRAH
(LOPERA) IT ALLONS FOR SELECTION OF THE OPERATIONAL NODE,

TD SCAN HIRROR OR TO POSITION NIRROR. IT HILL ALSO BE THE
BASIS OF THE SUBROUTINE TO BE USED BY LASCON PROGRAN (F.R.A.P.
LASER CONTROL PROGRAN). THIS SUBROUTINE HOST ALSO BE LINKED
NITH THE SUBROUTINE DTACHA AND CLOCIN

 

DECLARATIONS

 

nnnnnnnnnnn

009909 101091191151,191152,191153,100091,101010,190,151000,
+1m,109019

009909 1010011 101099,101059,120099, 101151.101900

009909-159011 109911,115901,1951911,1051911,151911,159019

009909 10111091 005099,005991,010099,010991,19950,19950

009909 101101 09990

009909 190111 9191,9019,9991

1910509 005991,005099,010991,010099

9091 9059

909110 0119110

019095109 1011511211

01109991 019099

 

n

INITIAL VALUES

 

IVEC3'440 ICLOCK OVERFLON VECTOR

ID=I IISR ID NUNBER

IPRTY=7 IPRIORITY NUNBER

ICOUNT80 ICOUNT OF NUHBER OF CYCLES

IZER03‘7777 IDIGITAL VALUE FOR 0.0 D/A OUTPUT

ISLOPE=I ISETS INITIAL SLOPE FOR VOLTAGE INCRENENTS
ICLBPR8'170422 IREAL-TIHE CLOCK BPR

ICLCSR='170420 IREAL-TIHE CLOCK CSR

IDACHA8'170440 ID TO A CH. A (NON-D.E.C. STANDARD REG.)

 

roman

400 CONTINUE
NRITEI7,2402) ‘
2402 FORHATI’O’,’IN NPOSIT HODULE, NANT TO CONTINUE?(Y,N)’)
READ(7,24041 RESP
2404 FORHATIAI)
IFIRESP.EO.’Y’T GO TO 2406
IFTRESP.ED.’N’1 GO TO 2499
NRITEI7,81’ UNABLE TO DETERNINE RESPONSE’
GO TO 2400
406 1 CONTINUE

 

OPERATING HODE CHOICE

1136

 

9911017,11
9911017,24001

2400 0099911101,199109 9000 15 0051900? 15-5099,9-90511109,

+09 0-10 0111 9000.011)

9090(7,241019059

2410 0099911911
1019059.00.15') 00 10 2412
1019059.00.1911 00 10 2450
1019059.00.1011 50 10 2499
991100.111 099010 10 001099190 9105909501
50 10 2400

 

 

C
C FOLLDNING ALLONS THE PROGRAN TO BE REPEATED
C
2

 

 

 

 

412 CONTINUE

ICOUNT=0 IRESETS COUNTER FOR REPEATED CYCLES
IDDNEID IRESETS DONE FLAG

C

C SETTING D/A CHANNEL ONE TO ZERO

C
CALL IPOKEIIDACHA,IZERO) IDIAIREG.NONSTANDARD)TO 0.0 V

C

C SETTING UP I.S.R.

C
CALL CLOCIN ISUBROUTINE ALLONING NULTIPLE CLOCK SETTINGS
CALL IPOKEIICLBPR,12CONP1 ESETS BPR 2’S CONP INT.ICYC.
CALL IPOKEIICLCSR,ICLKST) ISETS CSR TO STANDBY

C
IERR=INTSETIIVEC,IPRTY,ID,DTACHAT ISETS ISR PARAHETERS

 

 

C
C DELAY FOR I.S.R. TO BE INSTALLED
C
2

414 CONTINUE
NRITE17,!)’ ARE YOU READY TO CONTINUE? (Y,N)’
READ(7,24I6) RESP
2416 FORHATIAI)
IFIRESP.ED.’Y’) GO TO 2418
IFIRESP.ED.’N’) GO TO 2414
NRITEI7,41’ UNABLE TO DETERNINE RESPONSE’
GO TO 2414

 

no

PROGRAN INPUTS 4 OF CYCLES 0 VOLTAGE RANGE INPUTS

 

2419 CONTINUE
NRITE17,!)’ ENTER INITIAL CYCLE VOLTAGE, RANGE 0.D--5.12 V’
READ(7,!) VINT
IFIVINT.LT.0.0.0R.VINT.GT.10.241 GO TO 2418

2420 CONTINUE
HRITEIT,X)’ ENTER FINAL CYCLE VOLTAGE, RANGE VINT--S.12V’
READI7.I) VFIN

1(17

1019019.11.9191.09.9019.51.10.241 50 10 2420
9991=5.12 19911909 9011950 9110900
0911 009991

9911017,11

9911011,111 51991195 919909 90511109: 1,191151
9911017,111 019159195 919909 90511109: 1,191152
9911017,11

9911017,111 09109 190 909009 00 010105 10 00 909.1
909017,11 101010

 

rant":

STARTING CLOCK AND END OF PROGRAN CHECK

 

to
.h
r.)
to

2424

2450

0911 190101109099,1911511 190905 1911. 9011950
0911 190101101059,1011501 100105 01001 50 011
9911017,111 9911195 009 9905999 10 010101

1911017,11

104151090.00.11 9911047,111 009910100 01010 11.100091
10110090.00.01 50 10 2422

9911017,111 9905999 009910100 01011951

00911900

9911017,11

9911017,111 09109 090 00 190 001109195 0901005-- 5-90501 50991
9911017,111 9999901095, 0-901099 10 919909 9000 090100. 091
9911017,111 0-10 0111 990511 9000101

909017,24251 9059

0099911911

1019050.00.1011 50 10 2400

1019059.00.1511 50 10 2412

1019050.00.1011 50 10 2499

9911017,111 099010 10 001099190 905909501

50 10 2424

00911900

9911017,111 90511109195 90001

9191=0.0

9019=2.55

9991=5.12

0911 009991

0911 190101109099,1911521 190105 009109195 9011950
9911017,11

9911017,:11 109051 919909 00511109 11911: 1.191151
9911011,111 9159051 919909 90511109 11911: 1.191153
9911017,11

9911017,:110905091 919909 90511109: 1,191152

0911 90909

00911900

9911017,11

9911017,111 09109 090 00 190 001109195 0901005-- 9-90905111091
9911011,111 919909, 0-901099 10 919909 9000 090100. 09 0-101
9911017,1)1 0111 900511 9000101

909017,245419059

009991191)

1118

IFIRESP.EO.’C’I GO TO 2400
IFIRESP.E9.’P’1 GO TO 2450
IFIRESP.ED.’E’I GO TO 2499
HRITEIT,4)’ UNABLE TO DETERNINE RESPONSE’

 

 

 

 

 

00 10 2452

2499 00911900
901099
090
5009001190 009991

0

0

0 1915 5009001190 00990915 9 9091 90119505 1910 1910509

0 00019910915. 1915 5009001190 15 0500 01 990511 5009001190,

0 99109 15 9150 9991 00 190 9905999 109099

0

0

0 000199911095

0
009909 101091191151,191152.191155,100091,101010,190,151090,

+10090,109099

009909 190111 9191,9019,9991

0
919099=10.24110114-11 1909009 00 9011950 1909090915
9011s1=1919119190991 19091 1911191 9011950 900.
901152=1901919190991 19091 01991 9011950 909.
901151=1999119190991 19091 01991 9011950 900.

-191151=901151 11910509 9011. 909.

191152=901152 11910009 9011. 900.
191153=901155 11910509 9011. 900.
0001:901151-191151
1010001.51.0.51 191151=191151+1 100990015 009 9099190095
191151=17771-191151 100990015 009 19909100 019
0002:901152-191152
1010002.51.0.51 191152=191152+1 100990015 009 9099190095
191152=17777~191152 100990015 009 19909100 019
0003:901155-191153
1010005.51.5.51 191155=191155+1 100990015 009 9099190095
191155=17771-191153 100990015 009 19909100 019
190:191151 11911 9011950 0500 19 019099
901099
090
5009001190 90909

0

0 1915 5009001190 1909090915 09 0009090915 019 901950 001901

TO THE NIRROR CONTROL BOX. ROUTINE KEEPS LODPING UNTIL USER

1119

 

 

 

0 15 591150100 9119 19509 0099 10091109. 1915 5009001190 15
0 0500 09 190 990511 5009001190, 99109 15 9991 00 190 9905999
0 109099
0
0
0
0 000199911095
0
009909 /0109/191151,191152,191153,100091,101010,190,151090,
+10090,109099
9091 9059
2470 00911900
9911017,:1
9911017,:1' 919909 009950 9010519091--09109 0-10 9090’
9911017,:1' 919909 09,0-10 9090 919909 0099,0-10 50 10'
9911017,:1' 0190 9010519091,1-10 10990 90511109195 1009’
909017.24721 9059
2472 0099911911
1019059.00.'0'100 10 2450
1019059.00.'0 150 10 2474
1019059.00.'0'100 10 2474
1019059.00.'1’150 10 2459
9911017,:1' 099010 10 001099190 90590950’
50 10 2470
2479 00911900
190909=o 1905015 99100
9911017,:1’09109 190 909009 1909090915 00519001
909017.24751 190909
2475 0099911141
1019059.00.'0'11912:1911525190909
1019059.00.'0'119122191152—190909
1011912.51.191151.09.1912.11.191155150 10 2473
191152=1912
0911 190901109099,1911521
9911017,:1 19905091 10091109: ',191152
50 10 2470
2475 00911900
9911017,:1’ 190 0910900 909009 995 001 00 99950’
9911017,1119905091 10091109: 1.191152
50 10 2470
2450 00911900
9911017,:1
9911017,:1’ 09109 0-10 9090 919909 09, 0-10 9090 919909 0099.1
9911017,11’ 0-10 50 10 009950 9010519091,09 1-10 10990 1009’
909017.245219059
2452 0099911911

IF‘IRESP.EO.’L’)BO TO 2469
IF(RESP.EO.’C’)GO TO 2470
IFIIRESP.EO.’U’!SO TO 2463
IFéRESP.E9.’O’TGO TO 2464
GO TO 21160

110

2464

2465

2467

2469

CONTINUE

IVLT52=IVLT52-l

GO TO 2465

CONTINUE

IVLT52=IULT52+I

CONTINUE

IFIIVLT52.SE.IVLTSI.OR.IVLT52.LE.IVLT53) GO TO 2467
CALL IPOKEIIDACHA,IVLT52)

NRITE47,t)’PRESENT LOCATION: ’,IVLT52

SO TO 2460

CONTINUE

NRITEI7,!)’ OUT OF RANGE NUST NOVE IN OTHER OIRECTION’
NRITEIT,!)’PRESENT LOCATION: ’,IVLT52

SO TO 2460

CONTINUE

RETURN

END

IJLl

SUBROUTINE CLOCIN

 

 

 

 

 

0
0 1915 5009001190 15 0500 9119 190 50999195 919909 00919119091911
0 9905999/50090091190. 11 911095 009 190 501001109 00 9091-1190
0 01009 09000090105 990 9000 00 090991109. 19050 501001109 990
0 1909 90900 1910 190 059 990 099, 905900119011. 9051 00 119900
0 9119 50090011905 909191 09 990511, 99109 990 9991 00 190
0 99059995 195009 990 109099 905900119011.
0
0
0
0 000199911095
0
009909 /0109/191151,191152,191153,100091.101010,190,151090.
+10090,109099
009909 101009/ 101099.101059,120099,101151,101950
009909 159011 109911,115901,195901,105901,15901,159019
009909 10111091 005099,005991,010099.010991.19950.19950
009909 10110/ 09990
1910509 005991,005099,010991,010099
9091 9059
909110 09990
0
0
0
10 00911900
9911017,:1' 190 90551010 09000090105 00 190 9091-1190 010001
9911017,:1' 099 00 501 10 999 00 190 001109195:'
.9911017,11
9911017,:1' 100 92 - 09109 2 001091
9911017,:1' 5o 91 - 09109 1 001091
9911017,:1
9911017.:1' 09109 190 909009 90090 0099059090195 10 190’
9911017,:1’ 0051900 9091-1190 01009 0900009091
909017,20,099=1o1 10900
20 0099911121
10110900.11.1.09.10900.51.21 00 10 1o
10110900.00.21 50 10 70
10 110900.00.11 50 10 00
50 10 10
70 00911900
101951=-152 15015 059 10 5199009 190 91
101900=1153 15015 059 50 011
010=-1.002 1905. 00 909009 00 0101051500
50 10 90
00 00911900
101951=1172 15015 059 10 5199009 50 91
101950=-175 15015 059 50 511
090=-5.001 1905. 00 909009 00 0101051500
50 00911900

9RITE17,X)’ THE OEFAULT VALUE FOR Z'S CONFLINENT FOR THE’

JLLZ

95

100

I40

HRITE17,t)’ NUNBER OF INTERRUPTS PER CYCLE IS SET TO ONE’
HRITEI7,t)’ DO YOU HANT THE DEFAULT VALUE? (Y,N)’
READI7,9SI RESP

FORHATiAl)

IFIRESP.EO.’N’I GO TO 100

IFIRESP.EO.’Y’) GO TO 120

HRITEI7,l)’ UNABLE TO DETERNINE RESPONSE’

GO TO 90

CONTINUE

HRITEI7,!)’ ENTER THE NUNBER OF COUNTS BEFORE AN INTERRUPT’
NRITEI7,t)’ IS TO OCCUR. THE NUNBER OF COUNTS CANNOT BE’
NRITEI7,3)’ GREATER THAN THE NUHBER OF CYCLES ’,CYC,’OR’
NRITEI7,t)’ LESS THAN ONE.’

READI7,4) COUNTS

R2CONP=ICYCICOUNTSI ICYC IS NEG. SO GIVES 2’5 COHP.
IF(R2COHP.LT.-I.OR.R2CONP.GT.CYC) GO TO 125

NRITET7,I)’ 2'5 CONPLIHENT IS OUT OF RANGE’

GO TO 100

CONTINUE

NRITEI7,!)’ IF 2'5 CONPLIHENT IS GREATER THAN 32,000 RESET TO’
NRITE(7,t)’ A SNALLER CLOCK FREDUENCY’
IFIRZCONP.ST.32000) GO TO 10

IZCONP=INTIR2CONPI

GO TO 130

CONTINUE

I2COHP=(-I)

CONTINUE

NRITEI7,!)’ THE NEH VALUES ARE:’

HRITE(7,3)

NRITEI7,I)’CLOCK FREOUENCY’,CYC.’CYCLES!SEC’
HRITE(7,!)’2'S COHPLINENT IS’,I2COHP

NRITEI7,!)

NRITEI7,!)’ DO YOU NANT TO CHANGE EITHER OF THESE VALUESOIY/N)’
READ47,140) RESP

FORHATIAI)

IF(RESP.EO.’Y’) GO TO 10

IFIRESP.E9.’N’) GO TO 199

HRITE17,!)’ UNABLE TO DETERHINE RESPONSE’

GO TO 130

CONTINUE

RETURN

END

I513

3.2 DATA ACQUISITION AND CONTROL OPERATING SYSTEM

runnnr‘ar‘)

(fit—’1“:

(“I (”I I.) (”J ('7!

(“'1 r1 r“. r) n n ('1

PROGRAH LASCON

THIS PROGRAN CONTROLS ALL HAJOR FUNCTIONS OF FLUORESCENCE RECOVERY
AFTER PHOTOBLEACHING SYSTEN FOR THE BTP LABORATORY. IT FOLLONS A
PROGRAM GIVEN TO US BY JOHN HOLLAND,PH.D., N.S.U. NITH PERHISSION
OF HERIDIAN INSTRUHENTS, OKENOS, HICHIGAN.

 

DECLARATIONS

 

009909 191001 190000,190059,19110,190901
009909 1099111 109911,191090,191090

009909 101091 109099,19091,19051

009909 1019551 199191,11990

009909 101101 09990.10911

009909 10111091 010099.010991,005099,005991,19950.19950
009909 115951 10.19911,1900,10,19911,1900

009909 11010091 01190,00091,101590,109901,110059,91190,1190
009909 19010091 01190,101099,101059,101950.101951,120099
009909 159011 100909,1059u1,115901,195901.15901,159019
009909 1991001 101099,1090,19001,11090

009909 1111091 1001,11109,1109

019095109 19001111,2001

019095109 101151171

019095109 190511111

01109991 910015.11190

1910509 010099,010991,005099,005991

9091 9059

909110 09990

 

 

SETTING UP IDLIST FOR RESETTING REGISTERS TO ORIGINAL STATUS
IELISTTI)="167772 TDRVII OUTPUT BUFFER
IDLISTTZ)=IPEEK('I67772) !CONTENTS DRVII O.B.
IDLIST13)='170420 TR.T. CLOCK C.S.R.
IDLISTT4)=IPEEKT'1704201 !CONTENTS R.T. CLOCK I.S.R.
IDLISTTSI='177546 FLTC C.S.R.
IDLISTT6)=IPEEKT' 77546) ECONTENTS LTC C.S.R.
IBLIST17)='O FEND OF LIST FLAG

 

0911 00910011011511 10909 0111 905015 905151095 19 101151

 

SUBROUTINE THAT SETS INITIAL VALUES FOR PROGRAN AND SUBROUTINES

 

CALL VA UES

 

ILLA

99:1017,11

’ PLEASE TYPE IN THE THO LETTER CODE FRON LIST BELOH’

 

 

 

 

9911017,11 1 009 190 091109 100 9159 10 00991010.1
9911017,11
9911017,11 1 09 - 099950 99999010951
9911017,11 1 01 - 0111 99059991
9911017,11 1 09 - 099950 0110 99901
9911017,11 1 90 - 90191
9911017,:1 1 19 - 1151 99999010951
9911017,:1 1 90 - 909 010909 01909190911
9911017,:1 1 91 - 909 19509 501 091
9911017,11 1 50 - 51990 011
9911017,11
909017,2o1 995

20 0099911921

0

0

0 091109 00015109 01009

0

0
101995.00.10911 50 10 40
101995.00.10111 50 10 5o
101995.00.10911 50 10 00
101995.00.19011 50 10 100
101995.00.11911 50 10 120
101995.00.19011 50 10 150
101995.00.19111 50 10 100
101995.00.15011 50 10 200
9911017,:1 1 099010 10 001099190 905909501
50 10 30

0

0 5009001190 09115

0

0

40 00911900
0911 099999
50 10 30

0

50 00911900
0911 99900
00 10 30

0

:00 00911900
0911 901990
00 10 :0

120 00911900
0911 199999
00 10 :0

150 00911900

115

CALL EXPRUN

 

 

 

 

 

 

GO TO 30
C
180 CONTINUE
CALL LASET
GO TO 30
C
200 CONTINUE
CALL STNDBY
GO TO 30
C
60 CONTINUE
NRITET7,I)’ NORNAL END OF LASCON PROGRAN’
CALL IPOKEILTCCSR,LDISAB) ETURNS OFF LTC
CALL IPOKEIICLCSR,LDISAB) ETURNS OFF RTC
CALL IPOKEIIDRV11,IZERO) ECLEARS DRVII OUTPUT BUFFER
C
C
C
C CALL EXIT !ACTIVATE5 DEVICE SUBROUTINE, RESETS REGISTERS
C
C
C
END
SUBROUTINE VALUES
C
C THIS SUBROUTINE SETS DEFAULT AND INITIAL VALUES USED BY LASCON
C PROGRAN AND ITS SUBROUTINES.
C
C
C DECLARATIONS
L

009909 191001 190000,190059,19110.190901

009909 1099111 109911,191090,191090

009909 101091 109099,19091,19051

009909 1019551 199191,11990

009909 101101 09990.10911

009909 10111091 010099,010991,005099.005991.19950,19900
009909 115951 10.19911,1900.10,19911,1900

009909 11010091 01190,00091.101590,109901.110059,91190,1190
009909 19010091 01190,101099,101059,101950.101951,120099
009909 159011 100909,105901,115901,195901,15901,159019
009909 1991001 101099,1090,19001,11090

009909 1111091 1001;11109.1109

019095109 19001111,:001

319095109 190511111

01109991 910015.11190

1910509 010099,010991,005099,005991

9091 9059

909110 09990

];L6

 

c..-

C’IC—J

INITIAL VALUES

 

(‘JnnnC'JL-JC-Jfinfifi

BLECHV=7
BLEVAL='60000
BTIHE=0.5360.
DIIHE=1.00
FNANE=’FRAP.DAT’
IADBUF='177002
IADCSR='IIIDOO
IBOPEH='O
IBSHUT=‘50
ICLBPR='170422
ICLCSR='170420
ICLKSO='I43
ICLRSI=‘I42
ICOL=I

ID=2
IDACHA='170440
IDRVII='167772
IELEVN=II
ILSHUT=‘20
INITD='6401

INTENO=OBSVAL.OR.IBOPEN
INIENB=BLEVAL.OR.IPSHUT

IONE=1

IPCNI=6
IPOSTIII=3046
'IPOSTI2)=0051
IPOSTI3I=3056
IPOSTIAI=306I
IPOSTISI=3066
IPOSTIBI=0071
IPOSII7I=3076
IPOSTIBI=3081
IPOSII9I=3086
IPOST(10)=7091
IPOSIIIII=3096
DO 333 I=I,Il
IPOSIiII=307I
CONTINUE
IPRTY=6
IPSHUI='40
IREADY:‘6600
ISHUT=‘B0
ISHUTN=4
ITICK=0
IUNIT=IO
IVEC='44D
IZERO=0
I2COHP=(-1)t100

SBLEACH INTNESITY CHOICES 17-14)
!VALUE FOR BLEACH LEVEL 7

!INIESER REP. OF BLEACH IINE 0.55EC
STIHE BETHEEN RIC ISR INIERRUPTS
!DEFAULT NANE PUT INTO FILE

!AID BPR ADDRESS

9ATD CSR ADDRESS

EVALUE TO OPEN LASER AND PM? SHUTTERS
!VALUE TO CLOSE LASER AND PHI SHUTTERS
YRTC BPR ADDRESS

FRTC CSR ADDRESS

!SEIS RIC CSR NOOEI,I RHZ,CLK BO
fSEIS RIC CSR NODEI,I KHZ,CLK OFF
!INITIAL VOUT COLUNN POINTER

!RTC ISR ID NUHBER

‘OIA CHANNEL A CSR

9DRV11 OUTPUT BUFFER

!UPPER POSITION LIHIT FOR NIRROR
IVALUE TO CLOSE LASER SHUTTER

!VALUE INITIATES DATA CONVERSION CHIS
!OBS. INT 1 AND SHUT COND BO

IBLEACH INT 7 AND SHUT COND PC

!LONER POSITION LINII FOR NIRROR
!POINTER HIRROR POSITION ARRAY

!INI REP. HIRROR POSITION ONE

fEACH POSITION IS APPROIINAIELY

!2 NICRONETERS DISTANT FRON

!POSITION ABOVE OR BELOH CURRENT
!POSIIION

!INT. REP. NIRROR CENTER PT.

IALLONS FOR PLUSININUS 5 NICRONS
!FRON CENTER POSITION

IINI. REP. NIRROR POSITION ll

!RTC ISR PRIORITY NUNBER

9VALUE TO CLOSE PHI SHUTTER

!VALUE DATA CONVERSION DONE CHIS
!VALUE SET FRON ITSHUI VALUES

!FLA6 FOR SHUTTER COND. BOTH OPEN
!INIERRUPT COUNTER

FDEFAULT OUTPUT LOGICAL UNIT

!RTC INTERRUPI OVERFLON VECTOR
!VALUE OF ZERO

IS’COHP 100 COUNISIINTERRUPI RIC SPR

1QL7

LD=I
LDISAB='0
LEHABL='100
LPRTY=7
LTCCSR='177546
LVEC='100
OBSERV=I
OBSVAL='0
RTIHE=5.!60.
IICK=0.0
TIHE=0.
TRNSB=0.05
TRNSO=0.00005
RETURN

END

!LTC ISR ID NUHBER

!VALUE TO DISABLE LTC

!VALUE IO ENABLE INTERRUPI BII b
!LTC ISR PRIORITY NUNBER

ELTC CSR ADDRESS

!LIC INTERRUPI OVERFLOH VECTOR

IOBS. INTENSITY CHOICES (1'6)

!VALUE FOR OBS. INT LEVEL I

!INTESER REP. OF RUNNING IIHE 5 SEC
!TINE KEEPER

ECOUNTER FOR TINE FRON RIC
!PERCENIA6E OF BEAN TRANSNITTED BLEACH
IPERCENTASE OF BEAN IRANSHITIED OBS.

JQL8

SUBROUTINE CPARAH

THIS SUBROUTINE IS PART OF THE LASER CONTROL PROGRAN (LASCON).
ITS FUNCTION IS TO ALLON THE USER TO CHANGE THE PROGRANS
PARANETERS OR USE DEFAULT PARANETERS.

 

nnnnnnrar-n

DECLARATIONS

 

COHNON IATDDI IADBUF,IADCSR,INITD,IREADY

COHNON IDRVII/ IDRVII,INTENB,INTENO

COHNON IDTOA/ IDACHA,IPCHT,IPOST

COHNON /FLAGS/ LPRINT,LTAKE

CONNON IFILE/ FNANE,IUNIT

COHNON [FILTER] BLECHV,BLEVAL,OBSERV,OBSVAL,TRNSO,TRNSB
CONNON /ISRS/ ID,IPRIY,IVEC,LD,LPRTY,LVEC

CDNNON lLCLOCK/ BTIHE,COUNT,LDISAB,LENABL,LTCCSR,RTIHE,TIHE
CONHON IRCLOCKI DTINE,ICLBPR,ICLCSR,ICLKGO,ICLKST,12CONP
COHHON ISHUI/ IBOPEN,IBSHUT,ILSHUT,IPSHUT,ISHUT,ISHUTN
CONNON IVALUE/ IELEVN,IONE,IVOUI,IZERO

CONNON IZTICK/ ICOL,IIICK,TICK

DINENSION IVOUTIII,ZBOI

OIHENSION IPOSTI?)

INTEGER BLECHV,BLEVAL,OBSERV,OBSVAL

REAL RESP

REALtB FNANE

 

nr'an

403
406

0011017,:1

0011017,:11 001001 0110 0900 15 10 0000 010__.091, 100 0000001
0011011,:1' 10 010-_.091 15 1000000100 01 000 000 0900 000'
0011017,:1' 0009011 0900 0011100 1010 0110 010__.091 15’
0011017,:1' 0090.091, 0500 091 009000 10 0051000.'
0011017,:1

0011011,11' 011 001000 10 00011000’

009011,4031 0050

0000911911

00011000

0911 109090

 

J-t-J ("I 0")

00011000

0011017,:1

0011011,:11 00100 0000 000 100 090900100 100 0150 009000?
0011011,:1

0011017,:1’ 00-01100900 000 0919 0010011

0011011,:1' 15-101005111 100015 900 00500.5001100 00001110001
0011017,:1’ 51-501 1100: 010900, 0919, 000’

0011011,:1’ 10-1151 0909001005'

];l9

410

NRIIEI7,I)’ OK-SATISFIED NITH PARANETERS’
READ(7,410) RESP

FORNAIIAZ)

IFIRESP.ED.’IS’)GO TO 412
IFIRESP.EO.’ST’)GO TO 4I6
IFIRESP.EO.’LP’)GD TO 406
IFIRESP.ED.'FN’IGO TO 424
IF(RESP.EB.’OK’)GO TO 499

HRITEI7,!)’ UNABLE TO DETERNINE RESPONSE’
GO TO 408

 

CONTINUE
CALL INSHUT
GO TO 406

 

CONTINUE
CALL SETIHE
GO TO 406

 

CONTINUE
CALL NAHEF
GO TO 406

 

CONTINUE .

NRITE(7,!)’RETURNING TO LASCON NENU’
RETURN

END

IJZO

SUBROUTINE INSHUT

THIS SUBROUTINE IS USED TO CONTROL THE NEUTRAL DENSITY FILTER
BOX AND TO CONTROL THE SELECTION OF SHUTTER CONDITIONS. THE
FILTER BOX CAN ATTENUATE THE LASER BEAN BY PLACING ANY
CONBINATION OF THE FOUR NEUTRAL DENSITY FILTERS IN THE
LASER’S PATH. THERE ARE FOURTEEN DIFFERENT LEVELS OF INTENSITY
POSSIBLE. THIS SUBROUTINE IS PART OF THE PROGRAN LOPERA.

THIS FILE ALSO CONTAINS THE SUBROUTINES TRANSV AND SHUTTR
HHICH ARE CALLED BY INSHUT

 

DECLARATIONS

 

nnnnnnnnnfinnc—I

100

110

CONNON lDRVIIl IDRVII,INTENB,INTENO

CONNON IFILTER/ BLECHV,BLEVAL,OBSERV,OBSVAL,TRNSO,IRNSB
CONNON ISHUT/ IBOPEN,IBSHUT,ILSHUT,IPSHUT,ISHUT,ISHUTN
INTEGER BLECHV,BLEVAL,OBSERV,OBSVAL

REAL RESP

00011000

0011017,:1

0011017,:1

0011017,1oo1 005000,10000

00009111 005. 10001’,13,':’,01o.7,' 0000001 00 0090')
0011011,:1

0011017,1101 010000,100sn

0000911’ 010900 10001',13,':',012.7,' 0000001 00 0090')
0011017,:) ‘
0011017,:1'----00500091100 5001100 0000111005 ----- ’
101100010.00.11 0011017,:1' 19000-0000,001-010500’
101100010.00.21 0011017,:1' 19500-010000,001-0000’
101150010.09.31 0011011,:1' 19500-010000,001-010500'
101100010.00.41 0011017,:1' 19500-0000,001-0000'
0011017,:1

0011017,:1

0011017,:1' 00100 9 00500050 0000 100 001100100 0000:’
0011011,:1' 00-10 009000 00000091100 101005111'
0011017,:1' 00-10 009000 010900 101000111'
0011017.:1' 00-10 009000 0001100 50111000’
0011017,:1' 01-10 0111 101005111 0000101

0011011,:1' 00-10 591150100 0110 100010 900 00111000’
009017,271 0050

0000911921

1010000.00.’00'1 00 10 so

1010050.00.’00'1 00 10 4o

1010000.00.’05’1 00 10 45

1010050.00.'01'1 00 10 so

1010050.00.100'1 00 10 so

0011011,:1' 009010 10 001000100 00500000?

00 10 25

1221

30

40

45

I‘ll-1n“)

00011000

0011017,:1

0011017,:1’ 00100 005000100 101000110,1 100051-0 0100051’
009017.11 005000

101005000.01.01 00 10 so

101005000.11.11 00 10 10

0911 0010001005000,0050911

0911 1090001005000,100001

10050=1oot10050

00 10 25

00011000

0011017,:1

0011011,:1' 00100 010900100 101005110,7 100051-14 01000511
009017.11 010000

101010000.11.71 0010 40

101010000.01.141 00 10 40

0911 0010001010000,010091)

0911 1090501010000,100501

100502100501100

00 10 25

00011000

0911 000110

00 10 25

00011000 ‘

5015 091005 10 00 00000 1010 00011 050
101000=15001.00.005091 109100 000 005. 900 5001 0000.
101000=105001.00.010091 109100 000 010900 900 001 5001
001000

000

5000001100 10905011905,10911

THIS SUBROUTINE NAICHES THE ANOUNI OF LASER OUTPUT ALLOHED THROUGH
TO NICRDSCOPE TO THE INTENSITY VALUE ENTERED.

IFIIANS.EB.II TVAL=.00000050
IFIIANS.ED.2) TVAL=.0000010
IFiIANS.EO.3) VAL=.0000050
IFIIANS.EO.4I TVAL=-000010
IFiIANS.EO.SI TVAL=.000050
IFIIANS.EO.6) TVAL=.ODOIO
IFIIANS.EO.7) TVAL=.00050
IFIIANS.EO.B) TVAL=.OOIO
IFiIANS.EO.9I TVAL=.0050
IFIIANS.EO.IO) TVAL=.OIO
IFIIANS.EO.III TVAL=.050
IFIIANS.ED.12) TVAL=.10
IFIIANS.ED.IS) IVAL=.50
IFIIANS.EO.I4) TVAL=I.OO

1222

RETURN
END

SUBROUTINE SHUTTR

SUBROUTINE THAT CONTROLS THE LASER SHUTTER AND P.N.T. SHUTTER.

THIS IS PART OF FPLACE SUBROUTINE NHICH IS PART OF THE LOPERA
OR LASCON PROGRANS

 

nnnnnnnn

DECLARATIONS

 

CONNON ISHUT/ IBOPEN,IBSHUT,ILSHUT,IPSHUT,ISHUT,ISHUTN
REAL RESP

 

10

30

4o

99

CONTINUE

NRITEI7,3)’ LC-LASER SHUTTER CLOSED I PNT SHUTTER OPEN’
NRITEI7,II’ PC-PNT SHUTTER CLOSED / LASER SHUTTER OPEN’
NRITEI7,I)’ BC-BOTH SHUTTERS CLOSED’

NRITEI7,II’ BO-BOTH SHUTTERS OPEN’

READI7,10) RESP

FORNAT(A2)

IFIRESP.EO.’LC’)GO TO 20

IFIRESP.EO.’PC’)GO TD 30

IFIRESP.EO.’BC’)GO TO 40

IFIRESP.EO.’BO’)SO TO 50

NRIIEI7,I)’ UNABLE TO DETERNINE RESPONSE’

GO TO 5

CONTINUE

ISHUI=ILSHUT SOCIAL VALUE TO'CLOSE LASER
ISHUTN=2 EFLAG FOR LASER CLOSED

GO TO 99

CONTINUE

ISHUI=IPSHUT POCTAL VALUE TO CLOSE PNT
ISHUTN=I !FLAG FOR PNT CLOSED

GO TO 99

CONTINUE

ISHUI=IBSHUI !OCTAL VALUE TO CLOSE BOTH
ISHUTN=3 IFLAG FOR BOTH CLOSED

GO TO 99

CONTINUE

ISHUI=IBOPEN FOCTAL VALUE TO OPEN BOTH
ISHUTN=4 FFLAG FOR BOTH OPEN
CONTINUE

RETURN

END

SUBROUTINE OCTREPIINPUT.IOUT)

1123

C10") ("J

SUBROUTINE TAKES AN INTEGER INPUT AND RETURNS OCTAL
REPRESENTATION OF DESIRED INTENSITY

IFIINPUT.EO.II IOUT='0
IFIINPUI.EO.2) IOUT='100000
IFIINPUT.ED.3I IOUT='40000
IFIINPUT.ED.4I IOUT='I40000
IFIINPUT.EO.5) IOUT='20000
IFIINPUT.EO.6I IOUT='120000
IFIINPUT.EB.7I IOUT='60000
IFIINPUT.EO.BI IOUT='160000
IFIINPUT.ED.9) IOUT='50000
IFIINPUT.E9.10) IOUT='150000
IFIINPUT.ED.III IOUT='30000
IFIINPUI.ED.IZ) IOUT='130000
IFIINPUT.ED.13) IOUT3'70000
IFIINPUT.EB.I4I IOUT='I70000
RETURN

END

1224

SUBROUTINE SEIINE

 

THIS SUBROUTINE IS PART OF THE LASCON PROGRAN. IT ALLONS
USER TO SELECT LENGTH OF BLEACHING, LENGTH OF RUN (DATA
COLLECTION, AND TINE INTERVAL BETNEEN PNT READINGS.

 

nnr'annnn

DECLARATIONS

 

2500

2502

2504

2506

2510

2520

nenq
bJ‘L

000000 11010001 01100,00001,101590,100901.110050.01100,1100
000000 10010001 01100.101000,101050,101000,101151.120000
0091 0050

00011000

11000201100100.o

11000=01100100.o

0011017,2502111000

0000911'0','100010 00 010900 ',05.3,' 5000005’1
0011017,2504111000

0000911'0','100010 00 000 ',05.1,' 5000005'1
0011017,2500101100

0000911’o’,'101000001 10100091 ',05.3,' 50000051)
0011011,:1

0011017,:1' 010950 00100 000100 0000 1151 00100'
0011017,:1' 01—10 501 010900100 1100’ .

0011017,:1' 01-10 501 0000100 1100 1 101000001 10100091'
0011011,:1' 00-10 591150100 0110 091005'

009017,25101 0050

0000911921

1010050.00.'01'100 10 2520

1010050.00.'01’)00 10 2550

1010050.00.'00’100 10 2590

0011011,:1’ 009010 10 001000100 00500050'

00 10 2500

00011000

0011017,:1' 00100 101100 0000050000100 10 010900 1100 00510001
0011017,:11 9 = 0.25 5000005’

0011011,:1' 0 = 0.50 5000005’

0011017,:1' 0 = 1.00 5000005’

0011017,:1' 0 = 2.00 5000005’

0011017,:1’ 901 01000 00500050 09100 0000900001
009017.25221 0050

0000911911

1010050.00. 9’1 01100=o.25100

1010050.00.’0'1 01100=o.50100

1010050.00.'0’1 01100=1.00100

1010050.00. 0’1 01100=2.oo:00

00 10 2500

00011000

0011017,:1' 00100 101100 0000050000100 10 0051000 101000911
0011017.:1' 9 = 0919 19000 00001 0.005 0000001
0011011,:11 0 0919 19000 00001 0.000 500000’
0011011.:1' 0 0919 19000 00001 0.01 5000001

1225

0011017,:11 0 = 0919 19000 00001 0.1 5000001
0011017,:11 0 = 0919 19000 00001 1.0000 5000001
0011017,:11 900 01000 00500050 09100 0000900001
009017.253210050

2532 0000911911
1010050.00.1911 01100=0.005
1010050.00.1011 01100=0.000
1010050.00.1011 01100=0.01
1010050.00.1011 01100=o.1
1010050.00.1011 01100=1.o

2533 00011000
0011017,:1
0011017,:11 00100 101100 0000050000100 10 0051000 000 11001
0011017,:11 9 = 5 50000051
0011017,:11 0 = 15 50000051
0011017,111 0 = 30 50000051
0011017,:11 0 = 15 50000051
0011017,:11 0 = 00 50000051
0011017,:11 0 = 90 50000051
0011017,:11 0 = 120 50000051
0011017,:11 0 = 150 50000051
0011011,:11 1 = 100 50000051
0011017,:11 1 = 240 50000051
0011017,:11 0 = 300 50000051
009017,25341 0050

2534 000091191)

1010050.00.1911 00 10 2530
1010050.00.1011 00 10 2530
1110050.00.1011 00 10 2530
1010050.00.1011 00 10 2530
1010050.00.1011 00 10 2530
1010050.00.101) 00 10 2530
1010050.00.1011 00 10 2530
1010050.00.1011 00 10 2530
1010050.00.1111 50 10 2531
1010050.00.1111 00 10 2530
1010050.00.1011 00 10 2530
0011017,:11 009010 10 001001000 005000501
00 10 2533

253 00011000
1010050.00.1911 000:5.0101100
1010050.00.1511 000=15.0101100
1010050.00.1011 000=10.0101100
1010050.00.1011 000=45.0101100
1010050.00.1011 000=5o.o101100
1010050.00.1011 000=90.0101100
1010050.00.1511 000=120.0101100
1010050.00.1011 000=150.0101100
1010050.00.1111 000=100.o101100
1010050.00.1111 000=240.0101100
1010050.00.1011 000=3oo.0101100

126

IEIRUN.LE.3ooo) 50 TU 2540
RRITEI7,II
NRITEIT,II' RUN TINE EXCEEDS RAIINUN ALLOHABLE TINE FUR’
URITEI7,:)' INTERRUPT INTERVAL 0F ’,DTIHE,’ SECONDS/INTERRUPT’
50 TU 2533
2540 CONTINUE
IZCDHP=(-l)tDTIHE!1000 !ICDUNTS / INTERRUPI
IFIRESP.ER.'A'I RTIHE=5.0t60.
IFIRESP.EU.'U') RTIUE=15.0:UU.
IF(RE5P.E9.’C’) RTIHE=30.0860.
IF(RE5P.EQ.’D’3 RTINE=45.oTbo.
IEIRESP.ER.’E') RTIHE=60.0!60.
IFiRESP.EQ.’F’) RTINE=Ro.oIbo.
IFIRESP.E9.’6’) RTINE=120.0I60.
IEIRESP.EU.'U’T RTINE=150.0:60.
IF(RESP.ED.’I’) RTInEalao.oIUo.
IF(RESP.E9.’J’) RTINE=24o.o:Uo.
IFIRESP.EU.'K') RTIHE=300.0!60.
80 TU 2500
2599 CONTINUE
RETURN
ENU

1.27

SUBROUTINE NAHEF

THIS SUBROUTINE IS PART OF THE LASER CONTROL PROGRAH (LASCON).
ITS FUNCTION IS TO ALLON THE USER TO CHANGE THE OUTPUT FILE
NAHES NHILE RUNNING THE PROSRAN. IT CAN ALSO BE CALLEO BY
CPARAH SUBROUTINE

 

DECLARATIONS

finnnnnntfir:

 

CONNON IFILE/ FNANE,IUNIT
REAL RESP
REALtO FNAHE

 

canon

00 CONTINUE
NRITEI7,UozT

802 FORNAT(’0’,’ENTER YOUR CHOICE FRON BELOI’)
NRITETT,:T' U-TU USE DEFAULT NAHE PUT INTU FILE’
HRITEI7,!)’ c-TU CHOOSE NANE PUT INTU FILE’
NRITEI7,:T' U-TU CHOOSE OUTPUT FILE UNIT NUNBER’
NRITEI7,xT' E-TU EIIT NANEE HODULE’
READ(7,804) RESP

904 FORHAT(A1)

IEIRESR.EU.'U'T 60 TU 806

IF(RESP.ED.’C’) 60 TU 810

IF(RESP.EO.’U') 60 TU 830

IF!RESP.EO.’E’) 60 To 399

HRITEI7,t)’UNABLE TU UETERNINE RESPONSE’

60 TU 800

 

want”:

06 CSNTINUE
FNAHE='FRAP.DAI’
NRITEI7,UoUT IUNIT,FNAHE
808 FORHATI’U’,’OUTPUT FILE=FTN’.I2,’.DAT:NAHE PUT :NTU ‘ILE=’.R86

 

60 TU 900
c
C.-- ...............
UIU UUUTINUE

JRITEIF.SZUT
3:0 FOR?RT£’U’,’PLEASE ENTER NAHE TU BE PUT :NTU TRE OUTPUT FILE’
: HRITE£7,3)’ IT NUET BE IN THE FORH '---_.DRT“’

EEAOT?.&22) FHAflE
822 FORMATTABI

URITEI?,826} FNAHE
3:: FURNRTT'U’,’NRNE ENTEREU was *.Re.’ , CURREET? IT.R)’)
UERUI7.UEUT RESP

1JZ8

328

330

832

836

838

FURRATIAII

IFIRESP.EO.’Y’) BU TU Boo

IFIPESP.EO.’N’) 60 TU 810

NR!TE(7,I)’ UNABLE TU BETERNINE RESPONSE’

BU TU 510

CONTINUE

NRITEIT,832IIUNIT

FORNATI’O’,’PRESENT OUTPUT FILE=FTN’,IZ,’.DAT’)

NRITEI7,II

NRITEI7,!)’ PRUBRAN INCRENENTS UNIT BY UNE AFTER EXPERIMENTAL’
NRITEI7,II' RUN so FRUBRAN HAY BE RUN REPEATEULT, THE USER’
HRITEI7,I)’ HAY CHANGE UNIT BY ENTERINU A NEN UNIT NUNBER’
NRITEI7,II' BETNEEN 10 ANU 99 BUT IF UNIT NUNBER HAS BEEN USED’
NRITEI7,IT' BEFURE THE EXISTING FILE NILL BE UUERNRITTEN'
REAUI7,IT IUNIT

IFIIUNIT.LT.IUT 60 TU 830

IFIIUNIT.BT.99T so TU 830

NRITEI7,836)IUNIT

FORHATI’O’,’NEH OUTPUT FILE=FTN’,12,’.DAT’)

NRITEI7,:I

NRITEI7,IT' ENTER Y-CORRECT,N-INCORRECT’

READI7,838) RESP

FORHAT(AI)

IFIRESP.EA.’Y’) 60 TU Boo

IFIRESP.EA.’N’) 60 TU aso

HRITE(7,!T’ UNABLE TU DETERNINE RESPONSE’

BO TU 830

 

mnnn

CONTINUE
RETURN
END

1J29

SUBROUTINE HELENE

THIS SUBROUTINE IS PART OF THE LASER CONTROL PROGRAM. ITS
FUNCTION IS TO FURTHER EXPLAIN OPTIONS THAT ARE POSSIBLE.

 

nnfirltfit‘lf‘o

DECLARATIONS

 

REAL RESP

 

”man

000

1040

1080

1082

1042

1044

1046

1048

1050

"-J

1054

1054

1058

CUNTINUE

HRITEI7,1040)

FURNATI'D','INFURNATIUN UN EACH OPTION NILL BE GIVEN’)
URITEI7,:T' ONLY A FER URTIUNS NILL BE LISTED AT UNE TINE’
NRITEI7,IoBoT

FORHATI’O’,’HIT RETURN TU CONTINUE’}

READI7,1082) RESF

FURNATIAIT

NRITEI7,1042T

FORNAT(’I’,’CP - CNANSE PARANETERS’)

NRITEI7,IT' TNIS OPTION ALLUNS USER TU CNANEE PARANETERS. IF’
NRITEI7,:T' NU CHANGES NADE HILL RENAIN As DEFAULT 9ALUES OR’
NRITEI7,:T’ VALUES PREVIOUSLY SET.’

NRITEI7,1044T

FORHATI’O’,’EX - EXIT PROGRAH’)

IRITE(7,1046)

FORHAT(’0’,’FN - ENTER FILE NANE’)

NRITEI7,IT' TRIS OPTION ALLUNS USER TU CHANGE NANE PUT INTU’
NRITEI7,IT’ OUTPUT FILE FTN__.DAT, FTN__.DAT Is INCRENENTED’
HRITEI7,1)’ DY UNE EACN TINE EIRERINENTAL RUN IS CONPLETED’
NRITEI7,IDABT

FORNATI’0’,’HE - HELP’)

BRITEI7,IUSUT

FORHATI’O’,’LP - LIST PARAHETERS’)

URITEI7,8)’ THIS OPTION LISTS TNE PARANETERS AS THEY EIIST’
HRITE(7,!)’ EITHER DEFAULT UR SET DALUES.’

NRITEI7,IUBET

FORHATI’0’,’HIT RETURN TU CONTINUE.’)

READ£7,1064) REEF

FORUATIAII

NRITE(?,1054)

FURNATi’l’,’RD - RUN BLEACN EIFERINENT'I

HR!TEI7,!)’ THIS URTIUN ALLUNS USER ID INITIATE A BLEACH’
HRITEI7,I)’ SEBUENCE. SUBRUUTINES DRTUE FILTERS,5RUTTERS'
NRITE£7,1)’ AND DATA ACQUISTION.’

NRITEI7,IDSBI

FORMATI’O’,’RL - RUN LASER SET UP’)

RRITEI7,I}’ TRIS D TION ALLUNS USER TU RUN SISTER HITHOUT’
NRITEI?.1I’ SLEACN SEQUENCE. USED FUR ADIUSTINU PNT, ETC.’

1L30

1058

1099

RRITEI7,IUSRT

FORRATI’O’,’SB - STAND BY’)

URITEI7,IT' TRIS UPTIUN ALLDNS TNE SYSTEN TU BE PUT IN A’
NRITEI7,IT' SAFE NUDE ANILE SYSTEN IS JUST SITTINS IDLE.’
NRITEI7,IUBDI

FORNATI’O’,’HIT RETURN TU CONTINUE.’)
READIT,IUBUT RESP

FURNATIAIT

CONTINUE

NRITEI7,1070)

FORHAT!’0’,’DO NEED TU SEE UPTIUNS AGAIN? (Y,N)’)
READI7,1072T RESP

FURNATIAI)

IFIRESP.ED.'V'T SU TD 1000

IFIRESP.ED.'N'T 50 TD 1099

UNITEI7,IT' UNABLE TU DETERNINE RESPONSE’

BU TD 1075

CUNTINUE

NRITEI7,IT' LEAVING NELP HODULE’

RETURN

END

1:31

SUBROUTINE LPARAH

 

SUBROUTINE LISTS LASCON PROGRAN PARANETERS ON REQUEST. IT
IS ALSO CALLED DY CPARAN SUBROUTINE

 

nnnnnn

DECLARATIONS

 

CONNON IFILEI FNANE,IUNIT

CONNON IFILTER/ BLECHV,OLEVAL,OBSERV,ODSVAL,TRNSO,TRNSD
CONNON ILCLOCK/ OTIHE,COUNT,LDISAO,LENADL,LTCCSR,RTINE.TINE
CONNON IRCLOCK/ DTINE.ICLDPR,ICLCSR,ICLKGO,ICLKST,I2COHP
CONNON /SHUT/ IBOPEN,IDSHUT,ILSHUT.IPSHUT,ISHUT,ISHUTN
INTEGER BLECHV,DLEVAL,OBSERV,OBSUAL

REAL RESP

REALIO FNANE

 

40

CUNTINUE

NRITEI7,2DT

FORNATI’0’,’THE PRESENT PARANETER VALUES AREz’)
NRITEI7,IT

NRITEI7,IT' ----INTENSITY LEVELSW’

NRITEIT, u ' UBSERVATIUNs' ,UBSERV, ' BLEACIIa' ,BLECNV
NRITEI7,I)' ----UBSERVATIUN SNUTTER CONDITIONS---’
IFIISNUTN.EU.IT NRITEI7,II' LASER-UPEN,PNT-CLUSED'
IFIISNUTN.ED.2T IRITEI7,!)’ LASER-CLOSED,PHI-OPEN’
IFIISHUTN.EA.3) NRITEI7,xT' LASER-CLOSED,PNI-CLOSED’
IFIISNUTN.EU.4T NRITEI7,II' LASER-OPEN,PHT-OPEN’
NRITEI7,IT' -----UUTPUT FILE PARANETERS-----'

'NRITEI7,30) IUNIT,FNANE

FORNATI’ UUTPUT FILE: FTN’,12,’.DAT; NANE PUT INTU FILE=’,A8)
TINED=BTIHEI60.0

TINER=RTINE/Bo.o

BRITEI7,32TTINEB

FORHATI’O’,’LEN6TH UF BLEACH ’,F5.3,’ SECONDS’)
NRITEI7,34TTINER

FORHATI’O’,’LENGTH UF RUN ’,F5.1,’ SECONDS’)
IRITEI7,36)DTINE

FORHATI’O’,’DATA CULLECTED AT ’,F5.3,’ SECUND INTERVALS’)
NRITEIT,:T

NRITEI7,t)’ HIT RETURN TU CONTINUE’

READI7,40T RESP

FURNATIAIT

RETURN

END

1L32

SUBROUTINE EXPRUN

 

THIS SUBROUTINE IS PART OF THE LASER CONTROL PROGRAM FOR THE
F.R.A.P. EXPERINENTS. (LASCON.FOR). THIS SUBROUTINE HHEN
CALLED HILL EXCUTE AN EXPERINENTAL RUN. IT CONTROLS ALL
EOUIPNENT REQUIRED FOR AN EXPERINENTAL RUN AND ALSO TAKES
CHARGE OF THE DATA AOUISITION.

 

finnnnnnnr‘;

DECLARATIONS

 

1600

CUNNUN /ATUD/ IADBUF,IADCSR,INITD,IREADV

CUNNUN IDRVIII IDRVII,INTENB.INTENU

CUNNUN IDTOA/ IDACNA,IPCNT,IPUST

CUNNUN IFLAGS/ LPRINT,LTAKE

CUNNUN IFILE/ FNANE,IUNIT

CUNNUN IFILTER/ BLECNV,BLEVAL,UBSERV,UBSVAL.TRNSU,TRNSB
CUNNUN IISRS/ ID,IPRTT,IVEC,LD,LPRTY,LVEC

CUNNUN ILCLOCKI BTINE,CDUNT,LDISAB,LENABL,LTCCSR,RTINE,TINE
CUNNUN /RCLOCKI DTIHE,ICLBPR,ICLCSR,ICLK60,ICLKST,IZCOHP
CUNNUN ISHUT/ IBUPEN,IBSNUT,ILSNUT.IPSNUT,ISNUT,ISNUTN
CUNNUN /VALUE/ IELEVN,IUNE,IVUUT,IZERU

CUNNUN IZTICK/ ICOL,ITICK,IICX

DINENSIUN IVUUTIII,2BDT

DINENSIUN IPUSTIII)

EXTERNAL ATDC13,LTIHE

INTESER BLECNV,BLEVAL,UBSERV,UBSVAL

REAL RESP

REALIB FNANE

CUNTINUE

NRITEI7,IT

NRITEIT,:T' YOU ARE IN THE EXPERINENTAL RUN NUDE. PLEASE ENTER’
NRITEI7,:I' YOUR CNUICE FRUN TUE FULLUNINS LIST:’
NRITEI7,IT

IRITEI7,t)’ SU - PRUCEED NITN F.R.A.P. EXPERIMENT’
NRITEI7,II' RE - RETURN TU LASCUN HENU’
READ(?,1610) RESP

FURNATIAzI

IFIRESP.EA.’60’) SD TD 1620

IFIRESP.EQ.’RE’) SU TD 1690

HRITEI7,1)’ UNABLE TU DETERNINE RESPONSE’

SD TD 1500

CUNTINUE

 

SETTING INITIAL VALUES,RUN PARANETERS.AND I.S.R.’S

 

CALL IPOKEIIDR911,INTENO) IDRVll TO ODS. A SHUT COND.
COUNT=0. ICOUNTER OF CYCLES TO CALCULATE TINE IN SEC.
ICOL=1 ECOLUNN POINTER

ITICK=1 FINTERRUPT COUNTER

LPRINT=0 !FLAG TO EXIT INIT. DATA COLLECTION

LTAKE=0 9FLAG FOR END OF BLEACH TINE

1:33

TINE=0. PCOUNTER FOR RUNNING TINE
TICK=0. !COUNTER FOR INTERVAL TINE
IPCNT=6 IRESETS NIRROR POSITION POINTER
DO 1300 IR=1,11

DO 1302 IC=1,280

 

 

IVUUTIIR,ICI=U !INITIALIZES VULTASE UUT ARRAY

1302 CUNTINUE

1300 CUNTINUE
OPEN(UNIT=IUNIT,ERR=1629,TYPE=’NEN',INITIALSIZE=3001
CALL IPOKEILTCCSR,LDISAB) SCLEARS LTC CSR FOR I.S.R.
CALL IPUNEIDTACNA,IPUSTIIPCNTTI ESEIS NIRRUR TD IST LUCATIDN
CALL IPOKEIICLBPR,12CONP) !SET5 NUNBER CUUNTS/INTERRUPT
LERR=INTSET(LVEC,LPRTY,LD,LTINE) !SETS ISR FUR LTC
NRITEI7,:T’ DELAY TU SET UP LTC ISR, NIT RETURN TU CONTINUE’
READI7,1621) RESP

1621 FURNATIAIT
IFILERR.NE.DT SD TD 1662 !CHECKS FUR ISR ERRUR
CALL IPOKEIICLCSR,LDISAB) ECLEARS RTC CSR FDR I.S.R.
IERR=INTSETIIVEC,IPRTV,ID,ATDCISI !SETS ISR FUR RTC
NRITEI7,IT' DELAY TU SET UP RTC ISR, NIT RETURN TU CONTINUE’
READ(7,1623) RESP

1623 FORNATIAI)
IFIIERR.NE.DT SD TD 1672 €CHECK5 FUR ISR ERRUR

C

C NRITINS INITIAL VALUES TU UUTPUT FILE

C
NRITEIIUNIT,IB24T FNANE
HRITEI7,1624) FNANE

1624 FURNATIABT
TINEB=BTINE/Bo.
TINER=RTIHE160.
HRITEIIUNIT,1625)TINEB,TINER,DTINE

1625 FURNATIFI .2,' =BTINE ’,F12.1,’ =RTINE ’,F5.3.’ =DT1NE’)
URITEIIUNIT,1626)OBSERV,TRNSO.BLECHV,TRNSB

1525 FORNAT(13,’ =UBS L;TRAN=',F12.B,3x,IT,' =BLE L;IRAN=’,F12.8)
TAKES PREBLEACN,PUSITIUN ZERO READINB FDR RUN
NRITEI7,I)
IRITE(7,t)’ TAKING INITIAL READINGS’

C CALL IPOKEIIADCSR,INITD) !INITIATES DATA CUNVERSIDN

C CNECRS IF INITIAL READINS DATA CUNVERSIUN DUNE

£1628 IFIIIPEENIIADCSRI.AND.IREADVI.EU.ITERUT SU TD 1628

C IVLOUT=IPEEX(IADBUF)

C NRITEIIUNIT,IT

C URITEIIUNIT.1630) IPCNT.TICK,IVLUUT

C NRITEIT,IBTD)IPCNT,TTCK,IVLUUT

C1630 FURNATIIT.2x,F12.3,SI,112)

CALL INTDAT !COLLECTS INITIAL DATA
IFTLPRINT.ED.UT SD TD 7000

TINE=BTINE FSETS LTC ISR SLEACN CNECR
NRITEIT,II

NRITE£7,!)’ BLEACHING’

134

1644

1303

1305

1304

1307

1308
1306

7000

1629

1662

1672

CALL IPOKEILTCCSR,'100) IENAELES INTERRUPI BIT 6 LTC

CALL IP0REIIDRVII,INTENBI !POKE5 B—INTENT AND SNUT CUND.
IFILTARE.EU.01 50 TD 1044 !CHECKS BLEACN DUNE FLAS CNANUE
CALL IPUNEIICLCSR,ICLASUI !POKES SU BIT FUR RTC ISR
NRITEI7,11

NRITEI7,II’ COLLECTING’
CHECKS IF RUN IS DONE,IF DONE TURNS OFF RTC ISR
IFILTAKE.LE.1)GO TO 1656

CALL IPUKEIICLCSR,LDISABI !TURNS RTC ISR UFF
CALL IPUKEILTCCSR,LDISABI !TURNS LTC ISR UFF
NRITEIT,II

NRITEIT,II' NRITINU DATA INTU UUTPUT FILE’
HRITEIIUNIT,1303)IPCNT,TICK,IVOUTIIPCNT,ICOL) !LAST DATA
FURNATIIT,2I,F12.3,SI,1121
NRITEIIUNIT,IIICUL,ITICK,TICN
NRITEIIUNIT,II
NRITINS INTESER VULTASE REPRESENTATIUN INTU UUTPUT FILE
TINTNP=DTINE
DD 1304 IR=6,11
IRITEIIUNIT,1305)IR,TIHTNP,IVOUT(IR,IONE)
FURNATIIT,2I,F12.3,TI,1121
TINTNP=TINTNP+DTINE
CUNTINUE
DD 1300 IC=2,ICUL
DD 1300 TR=I,11
NRITEIIUNIT,13071IR,TINTNP,IVUUTIIR,ICI
FURNATIIT.2I,F12.3,3x.1121
TINTNP=TINTNP+DTINE
CUNTINUE
CUNTINUE
NRITE17,II
NRITEIT,11' NURNAL END UF EIPERINENTAL RUN’
CUNTINUE
CLUSE IUNIT=IUNITI
IUNIT=IUNIT+1 !INCRENENTS UNIT NUNBER
IFIIUNIT.ST.99I IUNIT=0 !DEFINES INPUSSIBLE UNIT
IFIIUNIT.LT.IDI IUNIT=0 !DEFINES INPUSSIBLE UNIT

80 TO 1600

CONTINUE

CALL IPOKEILTCCSR.LDISABI ITURNS LTC ISR OFF
CALL IPOKEIICLCSR,LDISA8) ITURNS RTC ISR OFF
HRITEI?,1)’ ERROR OPENING FILE, RUN ABORTED’

GO TO 1600

CONTINUE

CALL IPOKEILTCCSR,LDISAD) 1TURNS LTC ISR OFF
CALL IPOKEIICLCSR,LDISAG) !TURNS RTC ISR OFF

CLOSEIUNIT=IUNITI

HRITE17,I)’ ERROR SETTING UP LTC ISR. RUN AEORTED’
GO TO 1600

CONTINUE

CALL IPOKEILICCSR.LDISA8) ITURNS LTC ISR CFF

1:35

1690

CALL IPOKEIICLCSR,LDISAO) !TURNS RTC ISR OFF
CLOSEIUNIT=IUNITI

NRITE17,8)’ ERROR SETTING UP RTC ISR, RUN ABORTED’
GO TO 1600

CONTINUE

CALL IPOKEILTCCSR,LDISADI !TURNS LTC ISR OFF
CALL IPOKEIICLCSR,LDISAG) !TURNS RTC ISR OFF
RETURN

END

1:36

SUBROUTINE ATDC13

 

THIS SUBROUTINE IS AN INTERRUPT SERVICE ROUTINE FOR AiD DATA
SAHPLING AND NIRRUR POSITIONING. IT IS PART OF THE EXPRUN
SUBROUTINE NHICH IN TURN PART OF THE LASCON PROGRAH.

 

finnnfinn

DECLARATIONS

 

CONNON lATODl IAODUF,IADCSR,INITD,IREADY

CONNON IDTOAI IDACHA,IPCNT,IPOST

CONNON IFLAGS/ LPRINT,LTAKE

CONNON IRCLOCK/ DTINE,ICLDPR,ICLCSR,ICLKGO,ICLKST,IZCONP
CONNON IVALUE/ IELEVN,IONE,IVOUT,IZERO

CONNON IZTICK/ ICOL,ITICK,TICK

DINENSION IVOUT(11,280)

DINENSIUN IPOSTI11)

 

200

210

CALL IPOKEIICLCSR,ICLKGOI IRESETS CLOCK GO BIT

CALL IPOKEIIADCSR,INITD) IINITIATES DATA CONVERSION
CHECKS IF DATA CONVERSION IS DONE
IFI(IPEEKIIADCSR).ANO.IREADY).EO.IZERO) GO TO 200
IVOUTIIPCNT,ICOLI=IPEEKIIADBUFI IRETRIEVES DATA FRON BUFFER
IPCNT=IPCNT+IONE EINCRENENTS NIRROR POSITION POINTER
TICK=TICK+DTINE !KEEPS TINE

ITICK=ITICK+IONE !COUNTS INTERRUPTS

CHECKS IF INCRENENT RESET IS NEEDED

IFIIPCNT.LE.IELEVN) GO TO 210

IPCNT=IONE

ICOL=ICOL+IONE

CONTINUE

CALL IPOKE(IDACHA,IPOSTIIPCNT)I !NIRROR TO NEXT POSITION
RETURN

END

1:37

SUBROUTINE LTINE

 

THIS SUBROUTINE IS AN INTERRUPT SERVICE ROUTINE - THAT
COUNTS THE NUNBER OF TICKS OF THE LINE TINE CLOCK (LTC)
TO CONTROL THE LENGTH OF BLEACHING AND THE LENGTH OF DATA
COLLECTING FOR AN EXPERINENTAL RUN OF F.R.A.P. SYSTEN.
THE SUBROUTINE IS PART OF THE LASCON PROGRAN, CALLED DY
EXPRUN SUBROUTINE.

 

nnt’lnnnnnr‘xxfl

DECLARATIONS

 

99

CUNNUN IDRVIII IDRV11,INTENB,INTENU
CUNNUN /FLAGSI LPRINT,LTAKE

CUNNUN ILCLDCK/ BTINE,CUUNT,LDISAB,LENABL,LTCCSR.RTINE,TINE
CUNNUN IVALUE/ IELEVN,IUNE,IVUUT,IzERU

DINENSIUN IVUUTIII,2BoI

CALL IPOKEILTCCSR.LENABL) !RESETS ENABLE DIT 6 OF LTC
COUNT=COUNT+IONE

IFICOUNT.LT.TINEI GO TO 99

CALL IPOKEIIDRV11,INTENO) 1DRV11 TO 085. A SHUT COND.
LTAKE=LTAKE+IONE

TINE=RTINE

COUNT=0.0

RETURN

END

1:38

 

SUBROUTINE INTDAT

 

 

C
C TNIS SUBRUUTINE IS PART 0F LASCUN PRUSRAN. IT IS CALLED
C BY TNE SUBRUUTINE EXPRUN. IT’S FUNCTIUN IS TU TAKE TNE
C INITIAL READINSS FUR SIVEN NIRRUR PUSITIUNS AND NRITE
C TNEN INTU TNE UUTPUT FILE AND NRITE TNE AVERACE TU TNE
C TERNINAL SCREEN. TNIS SUBRUUTINE ALSU ALLUNS TNE USER ID
C TU CALIBRATE TNE P.N.T. SU TNAT TNE NAxINUN INTENSITY IS
C NUMNWEUDmICUWULanFmERNL
C
C
C DECLARATIUNS
C
CUNNUN IATOD/ IADDUF,IADCSR,1N1TD,IREADY
CUNNUN /DRVlll IDRV11,INTENB,1NTENU
CUNNUN IDTOA/ IDACNA,IPCNT,IPUST
CUNNUN IFLASS/ LPRINT,LTAKE
CUNNUN IFILE/ FNANE,IUNIT
CUNNUN IFILTER/ BLECNV,BLEVAL,UBSERV,UBSVAL,TRNSU,TRNSB
CUNNUN /ISRS/ ID,IPRTV,IVEC.LD.LPRTI,LVEC
CUNNUN ILCLOCK/ BTINE,CUUNT,LDISAB,LENABL.LTCCSR.RTINE,TINE
CUNNUN IRCLOCK/ DTIHE,ICLBPA,ICLCSR,ICLKSO.ICLKST,IZCOHP
CUNNUN iSHUT/ IBUPEN,1BSNUT,ILSNUT,IPSNUT,ISNUT,ISNUTN
CUNNUN IVALUE/ IELEVN.IUNE,IVUUT,ITERU
DINENSIUN IDLISTIIII
DINENSIUN IPUSTIIII
DINENSIUN AVEIIII
EXTERNAL ATDCII,LTINE
INTEEER BLECNV,BLEVAL,UBSERV,UBSVAL
REAL RESP
REALIB FNANE
6000 CUNTINUE
NRITE17,11
BRITE17,11' ENTER YOUR UPTIUN CNUICE FRUN LIST BELDH:’
NRITEIT,II' CL-TU CALIBRATE P.N.T.’
HRITE17,!)’ RE-TU RETURN TU NAIN NENU’
NRITE17,11' TD-TU TAKE DATA FUR UUTPUT FILE’
'READIT,60101RESP
6010 FURNATIA21
EFTRESP.EA.’CL’) 60 TD 6100
IFIRESP.ED.’TD’) SD TD 6200
IFTRESP.EA.’RE’) BU TD 6300
HRITE(7,!)’ UNABLE TU DETERNINE RESPONSE’
SD TD 6000
6100 CUNTINUE
NRITEIT,11
DD 6110 1:1,11
TVLUUT=0
DD 6120 1:1,10
DD 6130 L=I,1000
5130 CUNTINUE

139

6122

6120

6112
6110

6999

CALL IPUNEIIADCSR.INITD1
IFIIIPEENIIADCSRI.AND.IREADII.EU.IzERUI 60 TD 6122
IVLUUT=IVLUUT+IPEEXIIADBUFI
AVEIII=IVLUUTII
CUNTINUE

NR1TEIT,6112II,AVEIII

FORNAT(’ PUSITIUN :',13,' AVERAGE:’,F8.1)

CUNTINUE

60 TD 6000

CUNTINUE

LPRINT=I !FLA6 FUR CUNTINUINS

URITEIT,11

DD 6210 1:1,11
IVLUUT=0
DD 6220 1:1,10

DD 6230 L=1,Iooo
CUNTINUE
CALL IPOKEIIADCSR,INITD) !INITIATES DATA CULLECTIUN
IFIIIPEENIIADCSRT.AND.IREADTI.EU.12ERUI 60 TD 6222
IVTUUT=1PEENIIADBUFT
IVLOUT=IVLDUT+IPEEKIIADBUF)
AVEIII=IVLUUTIJ
NRITEITUNIT,6224TI,TICX,IVTUUT
FURNAT113,21,F12.1.31,1121

CUNTINUE

NRITEIIUNIT,622611,AVEIII

FURNATI' POSITION:’,13,’ AVERASE: ’,F8.1)

HRITE(7,6228)1,AVEII)

FURNATI' POSITION:’,13,’ AVERAGE: ’,F8.1)

CUNTINUE

BU TD 6999

CUNTINUE

LPRINT=0

CUNTINUE

RETURN

END

141)

SUBROUTINE LASET

 

THIS SUBROUTINE ALLONS FOR SET UP AND INITIALIZATION OF THE
EOUIPNENT USED IN DATA COLLECTION AND CONTROL OF THE LASER
CONTROL PROGRAN (LASCON). IT USES HANY OF THE SANE SUBROUTINES
AS THE EXPRUN SUBROUTINE.

 

rant—Annama-

DECLARATIONS

 

3000

3010

3030

1°?
JUJB

3040

CUNNUN IATOD/ IADBUF,IADCSR,INITD,IREADI

CUNNUN /DRV11/ IDRVII,INTENB,INTENU

CUNNUN /DTUA/ IDACNA,IPCNT,IPUST

CUNNUN IFLAGSI LPRINT,LTAKE

CUNNUN /FILE/ FNANE,IUNIT

CUNNUN IFILTER/ BLECNV,BLEVAL.UBSERV,UBSVAL,TRNSU,TRNSB
CUNNUN /ISRS/ ID,IPRTY,IVEC,LD,LPRTY,LVEC

CUNNUN ILCLOCK/ BTINE.CUUNT,LDISAB,LENABL.LTCCSR,RTINE,TINE
CUNNUN lRCLOCKI DTINE,ICLBPR,ICLCSR,ICLRSU,ICLRST,IICUNP
CUNNUN ISHUT/ IBUPEN,IBSNUT,ILSNUT,IPSNUT,ISNUT,ISNUTN
CUNNUN IVALUE/ IELEVN,IUNE,IVUUT,12ERU

CUNNUN IZTICK/ ICDL,ITICK,TICK

DINENSIUN IVOUT111,280)

DINENSIUN IPUSTIIII

INTESER BLECNV,BLEVAL,UBSERV,UBSVAL

REAL RESP

IP=1PCNT !NIRROR PUSITIUN ELENENT
CUNTINUE

NRITEIT,30101

FORNATI’0’,’IN LASER SET UP NUDULE--PLEASE ENTER CNUICE FRON’I
NRITEIT,II' TNE FOLLOHING:’

HRITEI7,1)’ PU-BEAN PUSITIUN SET UP’

HRITE17,3)’ IN-BEAN INTENSITY SET UP’

uRITE17,11' CP-CNANSE PARAHETERS’

NAITEI7,I)’ LP-LIST PARANETERS’

NRITEI7,!)’ RE-RETURN TU LASCUN HENU’

READIT,:0201RESP

FDRNATIA21

IFIRESP.EA.’CP’) 50 TD 3060

IFIRESP.EA.’IN’) 80 TD 3030

IFIRESP.EQ.’LP’) 00 TD 3030 '

IFIRESP.EQ.’PD’) SD TD 3040

IFIAESP.ED.’RE’) 60 TD 3060

NRITEIT,11’ UNABLE TU DETERNINE RESPONSE’

50 TD 3000

CUNTINUE

ARITE17,30321

FDRHAT(’0’,’BEAH INTENSITY SET UP’)

CALL IPUREIIDRVII,INTENUI !POKES INT AND 5901 2300
GO ID 3000

CUNTINUE

NRITEI7.:UISI

IJAl

3012

3041

3050

3060

 

FORHATI’O’,’BEAN PUSITIUN SET UP’I

NRITEIT,11

CALL IPOKEIIDACHA,IPOSTIIP))

NRITETT,11= PRESENT NIRRUR PUSITIUN Is = ’,IP
NRITE17,11

URITE(7,II’ ENTER NIRRUR PUSITIUN DESIRED, 1-11;6 CENTER:’
NRITE17,11’ NIRRUR RESET BY EIPRUN 50 ND NEED CNANSE SACK’
HRITEI7,!I’ TU URISINAL SETTINS.’

READIT,30441 IP

FURNAT112I

IFIIP.LT.11 SD TD 3040

IFIIP.ST.111 BU TD 3040

CALL IPDKE(IDACHA,IPOST(IP)) !POKES 1N NIRRUR PUSITIUN
60 TD 3000

CUNTINUE

CALL LPARAN

BU TD 3000

CUNTINUE

RETURN

END

IJAZ

SUBROUTINE STNDBY

 

 

 

 

 

P
C TNIS SUBRUUTINE IS PART UF TNE LASER CUNTRUL PRUSRAN (LASCON).
C IT’S FUNCTIUN 15 TU SET ALL NEURTAL DENSITY IN PLACE AND CLUSE
C ALL SNUTTERS ALLURINS FOR TNE USER TU LEAVE SYSTEN UN BUT IN
C A SAFE NUDE.
C
C
C
C DECLARATIUNS
C
CUNNUN /DRVII/ IDRVII,INTENB,INTENU
CUNNUN ILCLOCK/ BTINE,CUUNT,LDISAB,LENABL,LTCCSR,RTINE,TINE
CUNNUN IRCLDCK/ DTINE,ICLBPR,ICLCSR,ICLXSU,ICLKST,12CDNP
CUNNUN ISHUTI IBUPEN,IBSNUT,ILSNUT,IPSNUT,ISNUT,ISNUTN
REAL RESP
C
C
C
2040 CUNTINUE
YRITEIT,11' YUU YISN TU 60 TU TNE STANDBY HODE? IY UR N)’
READ(7,2000) RESP
2000 FURNATIAII
IFIRESP.EA.’Y') 00 TD 2020
IFTRESP.EA.’N’) 60 TD 2099
NRITE17,11'UNABLE TU DETERNINE RESPONSE.’
SD TD 2040
2020 CUNTINUE
C
C SET PARANETERS TU CLUSE BOTH SNUTTERS AND PUT ALL FILTERS IN
C BEAN PATN.
C
CALL IPOKE(IDRV11,IBSHUT) !CLDSES BUTN SNUTTERS
CALL IPUNEIICLCSR,ICLRSTI !TURNS UFF RTC ISR
CALL IPUNEILTCCSR,LDISABI !TUNRS UFF LTC ISA
2060 CUNTINUE
NRITEIT,20501
2050 FORNATI’O’,’SYSTEN IN STANDBY NUDE. ENTER LE TU LEAVE HODE’)
READIT,2UTDI RESP
2090 FURNATIA2I
IFIRESP.EA.’LE’) 50 TD 2099
60 TD 2060
2099 CUNTINUE
YRITEI7,20901
2000 FORNATI’O’,’LEAVIN6 STANDBY HODE’)
RETURN
END

143

 

APPENDIX C:

BEAM ALIGNMENT OPERATIONS

Beam Alignment

Beam alignment must be done only after laser has been
peaked for power output. Peaking the power output of the laser will
assure the beam is along the same optical axis. The procedure that was

developed is as follows:

1. Laser should be set to the lowest observation intenxhnrthat is

visible using the control only program (LOPERA.SAV).

2. Remove first planar mirror from path. Adjust positioning set screws
of the mirror until the 2 plats of mirror are equidistant at each
corner. Removing the mirror also allows for a longer path for the

beam for more precise alignment of the beam.

3. Adjust laser mounting plates until the beam optical axis is parallel
to tapped holes of the optical table (reference frame). Aligning
optical axis of beam is accomplished by affixing two of the extra
mirror mount plates to the optical table parallel (or perpen-
dicular) to a line of tapped holes on the optical table. The
mounting plates are to be placed as near to and far from the laser
as possible. These 2 mounting plates must be equidistant from all
rows of tapped holes, assuring that the edges of the plates are
parallel or perpendicular to all the top holes of the optical table
reference frame. The location of the beam is then marked on a clear

drafting angle (held normal to the optical table by a second

144

drafting angle) as close to the laser as possible. The angle is

then moved to the second mounting plate if it matches a location of

the beam marked the laser is aligned to optical table. If locations

do not match adjust laser mounts. Repeat until both locations

match. (Equidistant from table surface and two mounting plates.)

4.

Replace first mirror such that the front plate of positioning
mount is at a 45 degree angle to the beam without using the

positioning screws.

Remove 2nd mirror, adjust so front and back plate of position-

ing mount are equidistant at each corner.

Again repeat positioning the two extra mirror mounting plates
as near to and as far from the lst mirrors surface. Using the
two angles make the corrections to the beam location using the

positioning screws of the first mirror.

Replace third mirror such that the front plate of positioning

mount is at a 45 degree angle to the beam without using the

positioning screws.

Remove scanning mirror positioning mounts but leaving 1"

diameter post in place.

145

10.

11.

12.

13.

14.

Adjust position of beam using clear drafting angles. One angle
is used to assure second angle is perpendicularIthhe post
(parallel to optical table). The second angle is placed such
that the open center of the angle is around the post holding
the third mirror. The edge of the second angleIuNIthen be
placed against the post of the scanning mirror. The location
of the beam is marked as near to the surface of the third
mirror. The location is again checked at the end of the posts
and the beam is corrected using the positioning screws of the

mirror mount.

The scanning mirror mount is then replaced.

Position scanning mirror at center point of scan locations set

by software.

Affix the two extra mirror mount plates so they will be

perpendicular to the intended beam path.

Remove microscope and three point mounting plates just leaving

the four corner mounting plates affixed to the optical table.

(Warning: the beam will be at eye level when sitting!)

.Adjust the two corner mounting plates so they are equidistant

from all rows and columns of tapped holes.

146

15.

16.

17.

18.

19.

20.

21.

Location of the beam is obtained by using a large pane of glass
and an angle to assure that the pane is perpendicular to the

table.

The mount attaching the scanning mirror to the post is adjusted

so that the beam lines along a line parallel to the post.

The scanning mirror is then raised or lowered or moved to

center the beam on the scanning mirror.

The scanning mirror is then rotated on the shaft to match
locations of beam near and far from scanning mirror. Note
rotating mirror may cause beam to deviate from center so check

after rotating scanning mirror.

Replace microscope and three-point mounting plates. level

mounting plates with level of optical table.

Remove epi-illxmdnator housing and reducing lens from sliding

housing.

Replace sliding housing center beaUIOn fully closed iriSIOf
sliding housing by raising, lowering, or rotating mounting
plates. Using glass plane location marked for scanning mirror

positioning.

147

22.

23.

24.

25.

When centered replace reducing lens, so it is centered in
opening of the sliding housing. Adjust positioning screws of
sliding housing to center beam on location marked for scanning

mirror positioning.

Replace epi-illuminator housing and place fluorescence sample

on microscope.

Focus the beam on the sample by moving sliding housing toward

or away from the epi-illuminator.

If emission spot is not symmetrical open iris of sliding

housing and repeat 22-24 after changing rotation of microscope

about the normal axis of the microscope.

148

APPENDIX D - SOLUTION TO DIFFUSION PROBLEM

The lateral transport equation with no bulk flow (VkO) gave the

diffusion equation (Eq. 25)

s D V C (D.1)

with the boundary condition

C(0,t) I C (D.2)

O
and the initial condition

C(r,0) - C exp[-aTI(r)] (D.3)

0
Equation (D.1) can be written using spherical and assuming no con-

centration dependence on the radius 0 or 41 leaving only those terms

dependent on 9.

 

ac D 1 3 DC
at ' p2 sine 30 (“no 30) (0'3)

The angle 6 defines a circular bleached region, an angle of O radians
denoting a point and an angle of w radians denoting the entire sample
region. Equation (D.3) represents the diffusion of the concentration of
fluorescently labeled molecules only after bleaching. Therefore if 80

describes the circular region of interest immediately after bleaching it

is subject to the following

C I O, O S 9 S 6 (0.4)

9 S 6 S N (D.5)

Applying separation of variables C(8,t) - T(t)R(8) where T(t) represents

time and R(8) represents position.

149

Leaves

2
paT_1 a . 8R._
D St sine 00 Slne 00) A (D'é’

where -X is constant.

Solving for the time portion of the Equation (D.6) leaves

2% . -D/02X (D.7)

The solution to this differential equation is

-XD/02t

T(t) - e (0.8)

Exchanging the radius term with the effective beam diameter w

diameter at 86% intensity of beam at the sample)

-ND/4w2t

T(t) 3 e (D.9)

Solving for the position portion of the Equation (D.6) leaves

 

1 3 . _§
1 - sine 39 (Sine ) (D.10)

Using the substituion u-cose, and the chain rule for 8f/39 - (Bu/86.)
(Bf/Du)

8 -sin8 Bf/Bu leaves

-N 8 2' sin29 £3 (D.11)
Gu 3n

9 0
Using the trigonometry identity sin'8=1-cos 8 and differentiating leaves

9
-2u £3 + (l - u") 25— + A R 8 0 (D.12)
Bu 8u2

which is Legendre equation (16,17). The Legendre equation has a general

solution in the form

a)

R(9) ' E A P (U) (D.13)
n n

n-O

150

The coefficients An depend on neither u or t but instead are only
determined by the boundary condition and initial condition C(u,O). The
Legendre polynomials Pn expressions can be found in many references
(16,17). Combining equation (D.9) and (D.13) leave the solution of the

diffusion equation

2
C(u,t) I :5: An Pn(u) e-X(D/4w ) (D.14)

nIO
Applying the initial and boundary conditions gives the final solution to

the diffusion equation with VOI O as:

O
C/C0 I 1/2 (1+coseo) + :E: égflill [cosO

(n+1) Pn(coseo) -

O
nIl

e-n(n+1)(D/4w2)t

P (cose )] P (u) (0.15)
n-l O n

where (D/4w2) is the characteristic time for diffusion and u I cosG

defines detection region.

151

APPENDIX E - LIPOSOME PREPARATION

Materials - Solvent

10:2 Choloroform - Methonal

Solutes - Egg Phosphatidylcholine*** (EPC)

Oxidized Cholesterol**
diI* (1,1' - dioctadeyl - 3,3,3',3'

tetramethlindocarbocyanine)

* - Fluorescent Probe - Molecular Probes, Inc.

** - Stabilizing Agent to Match BLMs - supplied by Tien's Laboratory

*** - EPC 99.9% pure Leon Labs

Methods - Combine solutes in the solvent to form a stock solution. The

stock solution should contain have molar ratios identical to

BLM preparation solution. For example 1 mole should contain

0.8582 moles of EPC, 0.1409 moles of oxidized cholesterol,

0.0009 moles of diI.

- Stock Solution
+ 25 ml 10:1
+ 0.1371 grams
+ 0.0225 grams

+ 0.0002 grams

- Procedure

chloroform - methanol

of EPC

of oxidized cholesterol

of diI

+ 2-3 ml of stock solution evaporated in rotary evaporator

minimum of 20 minutes at room temperature

152

Solutes were then hydrated in distilled water at 60°C in a

heat bath

H20 level 1-2 mm above solutes on side of rotary flask

Sample left in heated water bath until both cooled to room

temperature
A cloud of liposomes and lipid should be visible in 3 to 4
hours. (Can use liposomes after this 3 to 4 hour period)

Liposomes can be used up to 3 to 4 days after formation

153

APPENDIX F - BLM PREPARATION SOLUTION

The preparation solution used follows prescribed chemical compositions

presented in Bilayer Lipid Membrane (BLM): Theory and Practice by H.T.

 

Tien (31).

Materials - Solvents - Octane and Hexane
Solutes - Egg Phosphatidylcholine*** (EPC)

Oxidized Cholesterol*

diI (1,1' - dioctadeyl - 3,3,3',3' -

tetramethlindocarbocyanine)**.
* - Stabilizing agent for BLMs supplied by Tien's Laboratory.
** - Fluorescent probe from Molecular Probes Inc.
*** - EPC from Leon Labs 99.9% pure
Methods 4 Combine the solutes according to the following:
6.7% W/W of EPC

1.1% W/W of Oxidized Cholesterol

0.0067% W.W of diI

where W/W I weight of solute/weight of solvent.

Hexane: z 0.682 g/ml

Octane: z 0.695 g/ml

Detailed formation technique used is described in Weiss (39).

154

APPENDIX G DETERMINATION OF THE DIFFUSION COEFFICIENT
USING A LEASE SQUARES FIT

The determination of the diffusion coefficient using a least squares
fit was based on the normalized fluorescence recovery. When plotting
the normalized fluorescence intensity versus time that the fluorescence
intensity was measured, the curve obtained can be represented by the

approximation

.(EP.
2.
F(t) - F(o) _ ‘_ Ce w (6.1)

 

m
fk (t) ' 91*) - F(o)

Normalizing both the numerator and denominator which have units of volts

by dividing through by 10 volts gives

(-fl?
2

W

[F(t) - FIoIIv/10.0v . 1 - c. (0.2)

[F(*) - F(o)IV/10.ov

Using I I w2/4D and C I 1 and isolating the exponential term gives

D

[F(t) - F(o)]V/10.0V ‘t/‘D

l ' [F(*) - F(o)]V/10.0V ' e

(0.3)

Taking the natural logarithm of both sides and simplifying leaves

IF(*) - F(t)l/10 . _ 1.
L [F(*) - F(o)}/10] D t (G-A)

Rearranging the equation and substituting in for ID reduces to an

equation of a line (y I ax + b):

An(IFI*) - 01:11/10) - (3%)0 + 1n([F‘*’ I0F(°)1) 10.5)
W

Therefore determining the slope of the least squares fit line through

 

the measured fluorescence recovery intensities versus time give the

diffusion coefficient for a known effective beam diameter w.

' 155

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