The focus of this dissertation research project was to study the effect of near-IR and IR femtosecond pulses on a living organism via nonlinear optical microscopy (NLOM) of biological specimens. Laser parameters — wavelength, pulse duration, pulse energy and the spatial quality of the beam — were optimized to maximize nonlinear optical signals with minimal photodamage during deep (down to 0.3 mm) tissue microscopy imaging of non-transparent biological specimens. The photodamage in living tissues was assessed. Experimental techniques performed include the following: femtosecond pulse characterization; adaptive spatial and temporal pulse shaping; in vivo and in vitro depth-resolved multimodal nonlinear optical microscopy imaging; spectrally resolved one- and two-photon excitation fluorescence lifetime measurements; and the scoring of a photodamage on living D. melanogaster. The experimental results are presented in four chapters. Chapter 2 describes three major topics: (1) the technical aspects of delivering compressed femtosecond laser pulses with optimal spatial properties into deep portions of a biological specimen; (2) pulse shaping for selective excitation; and (3) the simultaneous detection of different color two-photon excitation fluorescence (TPEF) optical signals using a single PMT detector. Chapter 3 describes the use of advanced laser sources, such as ultrabroadband Ti:Sapphire and Yb-fiber femtosecond lasers, in depth-resolved multimodal NLOM of biological specimens. A second-harmonic generation (SHG) microscopy of a fixed human skin demonstrated an 80% increase in attenuation length for a sub-40 fs, 1060 nm Yb-fiber oscillator in comparison to a >100 fs, 800 nm Ti:Sapphire laser. Chapter 4 describes the investigation of human red blood cells (RBC) using TPEF, third harmonic generation (THG) and three-photon excitation fluorescence (3PEF) microscopy imaging. Hemoglobin was the primary source of the TPEF signal in RBCs and their membranes, as determined by a comparison of spectrally resolved fluorescence lifetime decays of reagent-grade endogenous fluorophores, as well as linear and transient absorption measurements. THG imaging was performed on RBCs through a blood storage bag to demonstrate THG’s potential application in the non-invasive assessment of stored RBCs. Chapter 5 summarizes the experimental results and in vivo assessment of laser-induced damage during NLOM imaging in living D. melanogaster. Short-term photodamage occurred via a nonlinear process and was estimated by scoring the mechanical damage and fluorescence increase. In contrast, long-term photodamage exhibited a linear relationship with laser intensity and was scored by lethality, apoptosis and necrosis in irradiated D. melanogaster population. The study reveals the benefit of utilizing shorter pulses — 37 fs and shorter — for NLOM imaging of highly pigmented tissues wherein linear (thermal) damage is the greatest contributor to the overall photodamage of the living organism.My research successfully demonstrates the potential use of femtosecond fiber-based and ultrabroadband solid state laser systems to overcome a key performance limitation of NLOM technology, while providing low barrier-to-access alternatives to current Ti:Sapphire sources. These results should accelerate the movement of noninvasive NLOM into clinical practice.
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