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Functional Fluorescence Microscopy Imaging: Quantitative Scanning-Free Confocal Fluorescence Microscopy for the Characterization of Fast Dynamic Processes in Live Cells.
Krmpot, Aleksandar J; Nikolic, 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; Vukojevic, Vladana.
Afiliación
  • Krmpot AJ; Department of Clinical Neuroscience (CNS), Center for Molecular Medicine (CMM) , Karolinska Institutet , Stockholm 17176 , Sweden.
  • Nikolic SN; Institute of Physics Belgrade , University of Belgrade , Belgrade 11080 , Serbia.
  • Oasa S; Department of Clinical Neuroscience (CNS), Center for Molecular Medicine (CMM) , Karolinska Institutet , Stockholm 17176 , Sweden.
  • Papadopoulos DK; Institute of Physics Belgrade , University of Belgrade , Belgrade 11080 , Serbia.
  • Vitali M; Department of Clinical Neuroscience (CNS), Center for Molecular Medicine (CMM) , Karolinska Institutet , Stockholm 17176 , Sweden.
  • Oura M; Max-Planck Institute for Molecular Cell Biology and Genetics , Dresden 01307 , Germany.
  • Mikuni S; Sicoya GmbH , Berlin 12489 , Germany.
  • Thyberg P; Laboratory of Molecular Cell Dynamics, Faculty of Advanced Life Science , Hokkaido University , Sapporo , Hokkaido 001-0021 , Japan.
  • Tisa S; Laboratory of Molecular Cell Dynamics, Faculty of Advanced Life Science , Hokkaido University , Sapporo , Hokkaido 001-0021 , Japan.
  • Kinjo M; Department of Applied Physics , AlbaNova University Center, Royal Institute of Technology , Stockholm 10691 , Sweden.
  • Nilsson L; Micro Photon Devices (MPD) , Bolzano 39100 , Italy.
  • Terenius L; Laboratory of Molecular Cell Dynamics, Faculty of Advanced Life Science , Hokkaido University , Sapporo , Hokkaido 001-0021 , Japan.
  • Rigler R; Department of Biosciences and Nutrition , Karolinska Institutet , Huddinge 14183 , Sweden.
  • Vukojevic V; Department of Clinical Neuroscience (CNS), Center for Molecular Medicine (CMM) , Karolinska Institutet , Stockholm 17176 , Sweden.
Anal Chem ; 91(17): 11129-11137, 2019 09 03.
Article en En | MEDLINE | ID: mdl-31364842
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.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Factores de Transcripción / Receptores Opioides mu / Microscopía Confocal / Proteínas de Drosophila / Microscopía Fluorescente Límite: Animals / Humans Idioma: En Revista: Anal Chem Año: 2019 Tipo del documento: Article País de afiliación: Suecia Pais de publicación: Estados Unidos
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Factores de Transcripción / Receptores Opioides mu / Microscopía Confocal / Proteínas de Drosophila / Microscopía Fluorescente Límite: Animals / Humans Idioma: En Revista: Anal Chem Año: 2019 Tipo del documento: Article País de afiliación: Suecia Pais de publicación: Estados Unidos