"Modern fluorescence imaging technologies, including deep-tissue imaging and super-resolution microscopies, require novel fluorescent labeling tags possessing non-conventional optical features, among which most desired ones are high brightness in the far-red/near-infrared (NIR) region and turn-on/off control in a spatiotemporal manner. Previously, we demonstrated the ability of fine tuning the absorption spectra of a protein-bound natural chromophore over an unprecedented range (474 6030364 nm). The goal of this PhD research is to exploit protein-ligand interactions for the development of protein-based pigments as NIR fluorescent tags for background-free live cell imaging. In the past half century, tremendous efforts have been invested in the optimization and derivatization of GFP-like fluorescent proteins (FPs). More recently, growing attention on phytochrome-based FPs has even upsized the repertoire of available FPs with many enhanced optical features. Giving this advancement, certain pitfalls are still limiting their uses in modern fluorescence imaging. In this context, synthetic dyes provide a broader chemical space for tailoring desired optic features including spectral wavelengths, brightness, stability, and many more photophysical and/or photochemical functionalities. To achieve high contrast imaging with minimal background interference, three different strategies have been applied here. 1) NIR emission is approached by utilizing a dye capable of specific complexation with a target protein via imine bond formation. Upon protonation of the imine, the complex experiences a large bathochromic shift as a result of a strong intramolecular charge transfer (ICT) process. A light-triggered imine isomerization is further incorporated to furnish a photoswitchable tag and negate the routine wash steps in live cell experiments. Rational protein engineering affords a faster variant that allows unprecedented spatiotemporal control of this no-wash bright NIR imaging. (2) A rare organic super photobase is identified, exhibiting a 14-unit change in pKa upon light excitation. Steady-state and ultrafast spectroscopic measurements ascribe this event to an excited-state proton transfer (ESPT) process. This ESPT feature is recapitulated in a protein-ligand micro-environment, yielding protein-dye complexes with extremely high fluorescence quantum yields (up to 92%) and large pseudo-Stokes shifts (> 200 nm). Our optimal mutant bound to the dye boasts millisecond binding rate and enables live cell imaging with negligible background. (3) A general approach to fluorogenicity, i.e., the ability to turn on fluorescence, is designed by coupling a quenching moiety capable of photoinduced electron transfer (PeT) to our dyes. The fluorescence is negligible before the Michael addition of engineered cysteine residue (the trigger) with the quencher moiety. A 30-fold fluorescence enhancement is achieved in vitro with an electronically tuned quencher group. Currently, further modifications are in progress to optimize the quenched system for in vivo applications."--Pages ii-iii.
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