Shedding new light on intracellular signaling pathways-establishing live-cell fluorescence imaging techniques using genetically encoded fluorescent biosensors

Official URL: https://risc01.sabanciuniv.edu/record=b2486437_(Table of contents)

Genetically encoded biosensors are indispensable tools in cell biology and biotechnology. However, utilizing these powerful tools often require sophisticated and expensive optical imaging devices. New generations of genetically encoded biosensors are engineered as single FP based intensiometric probes that permit imaging on a single excitation and emission wavelength. In this study, we tested whether commonly used single FP based biosensors can be utilized on a simple widefield fluorescence microscope. For this purpose, we exploited a conventional and affordable epifluorescence microscope equipped with only three standard filter-sets and a three-color LED light source. We tested three differently colored biosensors including GECO’s for Ca2+ imaging, geNOps for NO imaging, and HyPer7 for H2O2 imaging. Our results demonstrate that even a low-resolution and simple microscope yields the same results as a sophisticated imaging device in terms of spatial and temporal resolution. In the second aim in these studies, we established more complex FRET imaging approaches for quantification of intracellular Ca2+ signals using FRET-based genetically encoded biosensors such as D3-cpV. Besides, we applied and established FRET analysis techniques with biosensors - termed as Youvan’s algorithm – to increase the spatial resolution of FRET occurrence. In parallel to the FRET studies, we in silico designed and generated a novel FRET-based Acetyl-CoA sensor based on a bi-molecular construct differentially targeted to the cytosol and mitochondria. Overall, in these studies, we demonstrate strategies and establish (i) live-cell fluorescence imaging on a simple conventional microscope (ii) the application of FRET localization algorithms using genetically encoded FRET biosensors (iii) and design and develop FRET-based genetically encoded Acetyl-CoA sensors