14 July 2015 Quantitative confocal fluorescence microscopy of dynamic processes by multifocal fluorescence correlation spectroscopy
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Quantitative confocal fluorescence microscopy imaging without scanning is developed for the study of fast dynamical processes. The method relies on the use of massively parallel Fluorescence Correlation Spectroscopy (mpFCS). Simultaneous excitation of fluorescent molecules across the specimen is achieved by passing a single laser beam through a Diffractive Optical Element (DOE) to generate a quadratic illumination matrix of 32×32 light sources. Fluorescence from 1024 illuminated spots is detected in a confocal arrangement by a matching matrix detector consisting of the same number of single-photon avalanche photodiodes (SPADs). Software was developed for data acquisition and fast autoand cross-correlation analysis by parallel signal processing using a Graphic Processing Unit (GPU). Instrumental performance was assessed using a conventional single-beam FCS instrument as a reference. Versatility of the approach for application in biomedical research was evaluated using ex vivo salivary glands from Drosophila third instar larvae expressing a fluorescently-tagged transcription factor Sex Combs Reduced (Scr) and live PC12 cells stably expressing the fluorescently tagged mu-opioid receptor (MOPeGFP). We show that quantitative mapping of local concentration and mobility of transcription factor molecules across the specimen can be achieved using this approach, which paves the way for future quantitative characterization of dynamical reaction-diffusion landscapes across live cells/tissue with a submillisecond temporal resolution (presently 21 μs/frame) and single-molecule sensitivity.
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Aleksandar J. Krmpot, Aleksandar J. Krmpot, Stanko N. Nikolić, Stanko N. Nikolić, Marco Vitali, Marco Vitali, Dimitrios K. Papadopoulos, Dimitrios K. Papadopoulos, Sho Oasa, Sho Oasa, Per Thyberg, Per Thyberg, Simone Tisa, Simone Tisa, Masataka Kinjo, Masataka Kinjo, Lennart Nilsson, Lennart Nilsson, Walter J. Gehring, Walter J. Gehring, Lars Terenius, Lars Terenius, Rudolf Rigler, Rudolf Rigler, Vladana Vukojevic, Vladana Vukojevic, } "Quantitative confocal fluorescence microscopy of dynamic processes by multifocal fluorescence correlation spectroscopy", Proc. SPIE 9536, Advanced Microscopy Techniques IV; and Neurophotonics II, 95360O (14 July 2015); doi: 10.1117/12.2183935; https://doi.org/10.1117/12.2183935

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