Optimized processing and analysis of conventional confocal microscopy generated scanning FCS data
Author(s)
Type
Journal Article
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.
Date Issued
2018-05-01
Date Acceptance
2017-09-24
Citation
Methods, 2018, 140-141, pp.62-73
ISSN
1046-2023
Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
Start Page
62
End Page
73
Journal / Book Title
Methods
Volume
140-141
Copyright Statement
© 2017 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
License URL
Sponsor
Marie Curie Career Integration Grant
Identifier
http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=WOS:000436917700008&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=1ba7043ffcc86c417c072aa74d649202
Grant Number
PCIG13-GA-2013-618914
Subjects
Science & Technology
Life Sciences & Biomedicine
Biochemical Research Methods
Biochemistry & Molecular Biology
Diffusion
Correlation
Scanning
Confocal
Spectroscopy
STED
FLUORESCENCE CORRELATION SPECTROSCOPY
IMAGE CORRELATION SPECTROSCOPY
STATISTICAL ACCURACY
STANDARD-DEVIATION
MEMBRANE DYNAMICS
CROSS-CORRELATION
DIFFUSION
BINDING
CELL
TRANSPORT
Publication Status
Published
Date Publish Online
2017-09-28