Functional Fluorescence Microscopy Imaging: Quantitative Scanning-Free Confocal Fluorescence Microscopy for the Characterization of Fast Dynamic Processes in Live Cells

Abstract

Functional fluorescence microscopy imaging (fFMI), a timeresolved (21 μs/frame) confocal fluorescence microscopy imaging technique without scanning, is developed for quantitative characterization of fast reaction-transport processes in solution and in live cells. The method is based on massively parallel fluorescence correlation spectroscopy (FCS). Simultaneous excitation of fluorescent molecules in multiple spots in the focal plane is achieved using a diffractive optical element (DOE). Fluorescence from the DOE-generated 1024 illuminated spots is detected in a confocal arrangement by a matching matrix detector comprising 32 × 32 single-photon avalanche photodiodes (SPADs). Software for data acquisition and fast auto-and cross-correlation analysis by parallel signal processing using a graphic processing unit (GPU) allows temporal autocorrelation across all pixels in the image frame in 4 s and crosscorrelation between firstand second-order neighbor pixels in 45 s. We present here this quantitative, time-resolved imaging method with single-molecule sensitivity and demonstrate its usefulness for mapping in live cell location-specific differences in the concentration and translational diffusion of molecules in different subcellular compartments. In particular, we show that molecules without a specific biological function, e.g., the enhanced green fluorescent protein (eGFP), exhibit uniform diffusion. In contrast, molecules that perform specialized biological functions and bind specifically to their molecular targets show location-specific differences in their concentration and diffusion, exemplified here for two transcription factor molecules, the glucocorticoid receptor (GR) before and after nuclear translocation and the Sex combs reduced (Scr) transcription factor in the salivary gland of Drosophila ex vivo.

Imaging FCS delineates subtle heterogeneity in plasma membranes of resting mast cells

“…Valuable as a general approach for many cell types, with ImFCS we found that the membrane organization of resting RBL mast cells is predominantly Lo-like, as sensed by lipid probes, that protein modules in lipid-anchored probes modulate partitioning, and that diffusion of TM probes is selectively influenced by protein-based interactions. The simple analytical framework we describe to evaluate D distributions can be adapted readily to other spatially resolved fluorescence fluctuation methods (Ries et al, 2012;Wiseman, 2012;Digman et al, 2013;Bag and Wohland, 2014;Moens et al, 2015;Hendrix et al, 2016;Krmpot et al, 2019). In the context of mast cell activation, future studies will build on the foundation established here to examine stimulated signaling as mediated by the participating proteins within the environment of the responding membrane.…”

Imaging FCS Delineates Subtle Heterogeneity in Plasma Membranes of Resting Mast Cells

“…proven valuable for discerning weak interactions underlying plasma membrane heterogeneity in the poised, steady-state, as sensed by distinctive probes and as modulated by the actin cytoskeleton. This simple analytical framework can be readily adopted in other spatially resolved fluorescence fluctuation methods (48,49,(93)(94)(95)(96)(97). In the context of mast cell activation, future studies will build on the foundation established here to examine stimulated signaling as mediated by the participating proteins within the environment of the responding membrane.…”

Correlative nanophotonic approaches to enlighten the nanoscale dynamics of living cell membranes

“…In this aspect, machine-learning approaches are promising to not only automatize data analysis but also to remove human bias. Besides, machine learning has been employed to create tailor-made optimized designs for nanoantenna designs [ 130 ].…”