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Quantitative confocal fluorescence microscopy of dynamic processes by multifocal fluorescence correlation spectroscopy
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2015 (English)In: ADVANCED MICROSCOPY TECHNIQUES IV; AND NEUROPHOTONICS II, 2015, Vol. 9536, 95360OConference paper (Refereed)
Abstract [en]

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 32x32 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 auto- and 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 sub-millisecond temporal resolution (presently 21 mu s/frame) and single-molecule sensitivity.

Place, publisher, year, edition, pages
2015. Vol. 9536, 95360O
, Proceedings of SPIE, ISSN 0277-786X
Keyword [en]
Quantitative confocal microscopy without scanning, Functional fluorescence microscopy imaging (fFMI), Dynamical reaction-diffusion landscapes, Transcription factor, G protein-coupled receptor (GPCR)
National Category
Atom and Molecular Physics and Optics
URN: urn:nbn:se:kth:diva-173291DOI: 10.1117/12.2183935ISI: 000359599300016ScopusID: 2-s2.0-84940205705ISBN: 978-1-62841-701-2OAI: diva2:852567
Conference on Advanced Microscopy Techniques IV; and Neurophotonics II, JUN 24-25, 2015, Munich, GERMANY

QC 20150909

Available from: 2015-09-09 Created: 2015-09-09 Last updated: 2015-09-09Bibliographically approved

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Thyberg, Per
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Experimental Biomolecular Physics
Atom and Molecular Physics and Optics

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