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Proceedings Paper

Quantitative confocal fluorescence microscopy of dynamic processes by multifocal fluorescence correlation spectroscopy
Author(s): Aleksandar J. Krmpot; Stanko N. Nikolić; Marco Vitali; Dimitrios K. Papadopoulos; Sho Oasa; Per Thyberg; Simone Tisa; Masataka Kinjo; Lennart Nilsson; Walter J. Gehring; Lars Terenius; Rudolf Rigler; Vladana Vukojevic
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Paper Abstract

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.

Paper Details

Date Published: 14 July 2015
PDF: 9 pages
Proc. SPIE 9536, Advanced Microscopy Techniques IV; and Neurophotonics II, 95360O (14 July 2015); doi: 10.1117/12.2183935
Show Author Affiliations
Aleksandar J. Krmpot, Karolinska Institutet (Sweden)
Univ. of Belgrade (Serbia)
Stanko N. Nikolić, Karolinska Institutet (Sweden)
Univ. of Belgrade (Serbia)
Marco Vitali, Berlin Institute of Technology (Germany)
OMICRON Energy Solutions GmbH (Germany)
Dimitrios K. Papadopoulos, Max-Planck Institute for Molecular Cell Biology and Genetics (Germany)
Sho Oasa, Hokkaido Univ. (Japan)
Per Thyberg, KTH Royal Institute of Technology (Sweden)
Simone Tisa, Micro Photon Devices S.r.l. (Italy)
Masataka Kinjo, Hokkaido Univ. (Japan)
Lennart Nilsson, Karolinska Institutet (Sweden)
Walter J. Gehring, Univ. of Basel (Switzerland)
Lars Terenius, Karolinska Institutet (Sweden)
Rudolf Rigler, Karolinska Institutet (Sweden)
Vladana Vukojevic, Karolinska Institutet (Sweden)


Published in SPIE Proceedings Vol. 9536:
Advanced Microscopy Techniques IV; and Neurophotonics II
Emmanuel Beaurepaire; Francesco Pavone; Elizabeth M. Hillman; Peter T. C. So, Editor(s)

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