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

• Published in: Analytical chemistry (Washington) Vol. 91; no. 17; pp. 11129 - 11137
• Main Authors: Krmpot, Aleksandar J, Nikolić, Stanko N, Oasa, Sho, Papadopoulos, Dimitrios K, Vitali, Marco, Oura, Makoto, Mikuni, Shintaro, Thyberg, Per, Tisa, Simone, Kinjo, Masataka, Nilsson, Lennart, Terenius, Lars, Rigler, Rudolf, Vukojević, Vladana
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 03-09-2019
• Subjects: Avalanche diodes; Chemistry; Correlation analysis; Data acquisition; Diffractive optical elements; Diffusion; Excitation spectra; Fluorescence; Fluorescence microscopy; Fluorescence spectroscopy; Focal plane; Fruit flies; Glucocorticoids; Graphics processing units; Green fluorescent protein; Imaging; Mapping; Medicin och hälsovetenskap; Microscopy; Nuclear transport; Photodiodes; Photon avalanches; Pixels; Salivary gland; Salivary glands; Scanning; Signal processing; Transcription factors; Translocation; Transport processes
• ISSN: 0003-2700; 1520-6882; 1520-6882
• DOI: 10.1021/acs.analchem.9b01813

Functional fluorescence microscopy imaging (fFMI), a time-resolved (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 cross-correlation between first- and 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.