Scanning-Free functional Fluorescence Microscopy Imaging Toward Spatial Mapping of Biomolecular Information in Live Cell.

Live cells form signaling network and control it via molecular interactions and transporting processes. Through molecular interactions and transporting processes, they precisely regulate spatio-temporally the concentration and mobility of biomolecules. To understand the dynamic integration of molecular interactions and how cells control them spatio-temporally, quantitative methods with high sensitivity and spatio-temporal resolution are needed. Fluorescence microscopy- and correlation spectroscopy-based methods have been proven to be indispensable for this purpose, and advanced methods such as Single Plane Illumination Microscopy (SPIM)/Total Internal Reflection Microscopy (TIR)-based Fluorescence Correlation Spectroscopy (SPIM-FCS/TIR-FCS) have been developed. However, both methods also have limitations. In SPIM-FCS, fluorescence signal is simultaneously read-out; but light sheet propagation may be affected by obstacles in the sample, giving rise to an uneven fluorescence excitation across the sample. TIR-FCS is limited to study at the basal plasma membrane due to an evanescence illumination. In this situation, we have developed scanning-free massively parallel FCS (mpFCS) with high spatio-temporal resolution (~240 nm and ~10 µs) and single-molecule sensitivity, which allows us to quantitatively characterize fast dynamic processes and precisely measure the concentration of fluorescently labeled biomolecules in live cells [Krmpot A.J. et.al., Anal. Chem. 2019; Oasa S. et.al., Anal.Chem. 2021]. mpFCS records fluorescence signals from independent 256/1024 excitation spots generated by the diffractive optical elements (DOEs) using single-photon avalanche diode (SPAD) camera. Time series of fluorescence intensity are transformed to autocorrelation curves to determine the concentration and diffusion time. Our instrument also allows Fluorescence Lifetime Imaging Microscopy (FLIM), to characterize the immediate surroundings via fluorescence lifetime, and FLIM-based Förster Resonance Energy Transfer (FLIM-FRET), to assess the biomolecular interaction via donor-acceptor interaction. We have applied mpFCS/FLIM to spatially map in live cells the local concentration, diffusion and local environment of a transcription factor in the cell nucleus and have characterized the effect of allosteric inhibitor of transcription factor dimerization [Oasa S., et.al., PNAS 2020]. I will also discuss another potential of the instrument in spatial cross-correlation analysis toward the molecular transporting in live cells.