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Methods. 2018 May 1;140-141:62-73. doi: 10.1016/j.ymeth.2017.09.010. Epub 2017 Sep 28.

Optimized processing and analysis of conventional confocal microscopy generated scanning FCS data.

Author information

1
Wolfson Imaging Centre, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK. Electronic address: dominic.waithe@imm.ox.ac.uk.
2
MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK.
3
MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK; Department of Physics, King's College London, London WC2R 2LS, UK.
4
MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK; Wolfson Imaging Centre, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK.

Abstract

Scanning Fluorescence Correlation Spectroscopy (scanning FCS) is a variant of conventional point FCS that allows molecular diffusion at multiple locations to be measured simultaneously. It enables disclosure of potential spatial heterogeneity in molecular diffusion dynamics and also the acquisition of a large amount of FCS data at the same time, providing large statistical accuracy. Here, we optimize the processing and analysis of these large-scale acquired sets of FCS data. On one hand we present FoCuS-scan, scanning FCS software that provides an end-to-end solution for processing and analysing scanning data acquired on commercial turnkey confocal systems. On the other hand, we provide a thorough characterisation of large-scale scanning FCS data over its intended time-scales and applications and propose a unique solution for the bias and variance observed when studying slowly diffusing species. Our manuscript enables researchers to straightforwardly utilise scanning FCS as a powerful technique for measuring diffusion across a broad range of physiologically relevant length scales without specialised hardware or expensive software.

KEYWORDS:

Confocal; Correlation; Diffusion; STED; Scanning; Spectroscopy

PMID:
28963070
PMCID:
PMC6026296
DOI:
10.1016/j.ymeth.2017.09.010
[Indexed for MEDLINE]
Free PMC Article

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