Tailoring optically modulated fluorescent proteins for sensitive fluorescence imaging
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The continuous development and advancement have made fluorescence microscopy one of the most indispensable tools in life science. Fluorescent proteins (FPs) are highly specific and biocompatible. Therefore, they are widely used in fluorescence microscopy, which enables spatio-temporal analysis of complex biological processes. However, cellular metabolites generate autofluorescence background, which blends with the target FP fluorescence and results in reduced sensitivity or even inconsistent results, especially under low FP copy numbers. On the other hand, some FPs transiently reside in µs- to ms-lived dark electronic states after photoexcitation, which can be rapidly depopulated back to the fluorescent states by a lower energy coillumination. Optical modulation modulates the coillumination intensity, which dynamically alters the dark state population, thereby modulating the fluorescence intensity. Those FPs are named optically modulated fluorescent proteins (OMFPs). Synchronously amplified fluorescence image recovery (SAFIRe) developed by the Dickson group utilizes those modulatable fluorescence tags. SAFIRe uses Fourier transform to selectively recover the light signal from only the target of interest. It greatly suppresses the background and significantly improves the imaging contrast. SAFIRe holds great potentials in not only improving cellular imaging sensitivity but also enabling new paradigms of qualitative and quantitative analysis. Currently, there are only a few OMFP variants engineered and reported, albeit their great potentials in a wide array of applications. Herein, we assess the modulatability of various FPs that possess interesting photophysical characteristics. We also worked with the Fahrni lab at Georgia Institute of Technology to perform mutagenesis on several FPs to generate novel modulatable mutants. Notably, we have successfully engineered, identified, and characterized optically modulated yellow fluorescent proteins with unique modulation profiles even with single mutation. Further experimentation and analysis on some OMFPs revealed unique optically activated delayed fluorescence (OADF), which not only elucidates the triplet nature of the short-lived dark state among some OMFPs but also demonstrates great potential in developing expeditious background-free and reference-free imaging methodologies. Our meticulous characterization of photophysics in the YFP family yields highly accurate photophysical and photochemical parameters, which can be used to generate rate matrices based on corresponding Jablonski diagrams. OMFP fluorescence simulation based on excitation and decay rates coupled to the experimental conditions yields simulated modulation and OADF agree strongly to our experimental results. Continuous research on OMFPs since their discovery inspires us to develop novel high-sensitivity fluorescence imaging methodologies. Utilizing those well-engineered and well-characterized OMFPs in cellular imaging will greatly increase the target contrast. Continuous development of new fluorescence microscopic paradigms will enable qualitative and quantitative analysis of targets of interest.
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