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.
Highlights
fluorescence correlation spectroscopy (FCS) (Fluorescence Correlation Spectroscopy) is a wellestablished technique which allows the diffusion characteristics of fluorescently labelled molecules to be measured [1,2]
To validate FoCuS-scan and to understand the characteristics of diffusion across a range of physiologically and experimentally relevant rates we simulated Brownian motion and confocal scanning acquisition in 2-dimensions and compared the data to experiments performed on live cells under similar settings looking at the diffusion of a fluorescent DPPE-Atto647N lipid analogue (1,2-dipalmi toyl-sn-glycero-3-phosphoethanolamine tagged with the organic dye Atto647N) in the membrane
The distribution and form of the correlation functions are very similar between the actual live-cell data and those generated from the simulation with characteristic variances and shapes, but there are some minor deviations between the two sets of curves
Summary
FCS (Fluorescence Correlation Spectroscopy) is a wellestablished technique which allows the diffusion characteristics of fluorescently labelled molecules to be measured [1,2]. In FCS the molecular mobility is accessed by determining the time it takes the molecules under study to traverse through the observation spot of an optical microscope. Knowledge of both the average transit time and the size of the observation spot allows a straightforward determination of respective molecular diffusion coefficients as well as anomalies in diffusion. Conventional (point) FCS employs a single measurement spot at a time only. In this paradigm a laser is focused on a single spot in the sample and the emission light is recorded at this location throughout the duration of the experiment. With measurement times of seconds to tens of seconds a sequential approach like point FCS often results in averaging of possible diffusion dynamics and so obscuring the vital diffusion heterogeneities that are of interest
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