Functional Fluorescence Microscopy Imaging: Quantitative Scanning-Free Confocal Fluorescence Microscopy for the Characterization of Fast Dynamic Processes in Live Cells
Autor: | Rudolf Rigler, Masataka Kinjo, Vladana Vukojević, Lars Terenius, Marco Vitali, Lennart Nilsson, Dimitrios K. Papadopoulos, Stanko N. Nikolić, Sho Oasa, Aleksandar J. Krmpot, Simone Tisa, Shintaro Mikuni, Per Thyberg, Makoto Oura |
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Rok vydání: | 2019 |
Předmět: |
Confocal
Green Fluorescent Proteins Receptors Opioid mu Gene Expression Fluorescence correlation spectroscopy 010402 general chemistry PC12 Cells 01 natural sciences Dexamethasone Salivary Glands Analytical Chemistry Green fluorescent protein Genes Reporter Cell Line Tumor Quantum Dots Fluorescence microscope Animals Drosophila Proteins Humans Microscopy Confocal Osteoblasts Pixel Chemistry 010401 analytical chemistry Detector Avalanche photodiode Fluorescence Rats 0104 chemical sciences Protein Transport Drosophila melanogaster Microscopy Fluorescence Biophysics Transcription Factors |
Zdroj: | Analytical Chemistry. 91:11129-11137 |
ISSN: | 1520-6882 0003-2700 |
Popis: | 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. |
Databáze: | OpenAIRE |
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