PT  - JOURNAL ARTICLE
AU  - Dominic Waithe
AU  - Falk Schneider
AU  - Jakub Chojnacki
AU  - Dilip Shrestha
AU  - Jorge Bernardino de la Serna
AU  - Christian Eggeling
TI  - Advanced processing and analysis of conventional confocal microscopy generated scanning FCS data
AID  - 10.1101/163766
DP  - 2017 Jan 01
TA  - bioRxiv
PG  - 163766
4099  - http://biorxiv.org/content/early/2017/07/14/163766.short
4100  - http://biorxiv.org/content/early/2017/07/14/163766.full
AB  - 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.