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Functional Fluorescence Microscopy Imaging: Quantitative Scanning-Free Confocal Fluorescence Microscopy for the Characterization of Fast Dynamic Processes in Live Cells
Karolinska Inst, CMM, Dept Clin Neurosci CNS, S-17176 Stockholm, Sweden.;Univ Belgrade, Inst Phys Belgrade, Belgrade 11080, Serbia..ORCID iD: 0000-0003-2751-7395
Karolinska Inst, CMM, Dept Clin Neurosci CNS, S-17176 Stockholm, Sweden.;Univ Belgrade, Inst Phys Belgrade, Belgrade 11080, Serbia..
Karolinska Inst, CMM, Dept Clin Neurosci CNS, S-17176 Stockholm, Sweden..
Max Planck Inst Mol Cell Biol & Genet, D-01307 Dresden, Germany.;Univ Edinburgh, Inst Genet & Mol Med, MRC Human Genet Unit, Edinburgh EH4 2XU, Midlothian, Scotland..
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2019 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 91, no 17, p. 11129-11137Article in journal (Refereed) Published
Abstract [en]

Functional fluorescence microscopy imaging (fFMI), a time-resolved (21 mu s/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 x 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.

Place, publisher, year, edition, pages
AMER CHEMICAL SOC , 2019. Vol. 91, no 17, p. 11129-11137
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Analytical Chemistry
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URN: urn:nbn:se:kth:diva-261359DOI: 10.1021/acs.analchem.9b01813ISI: 000484644800031PubMedID: 31364842Scopus ID: 2-s2.0-85071631165OAI: oai:DiVA.org:kth-261359DiVA, id: diva2:1357744
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QC 20191004

Available from: 2019-10-04 Created: 2019-10-04 Last updated: 2019-10-04Bibliographically approved

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