A Practical Guide to Fluorescence Temporal and Spatial Correlation Spectroscopy

The aim of this article is to introduce the basic principles behind the widely used microscopy tool: fluorescence fluctuation correlation spectroscopy (FFCS). We present the fundamentals behind single spot acquisition (FCS) and its extension to spatiotemporal sampling, which is implemented through image correlation spectroscopy (ICS). The article is an educational guide that introduces theoretic concepts of FCS and some of the ICS techniques, followed by interactive exercises in MATLAB. There, the learner can simulate data time series and the application of various FFCS techniques, as well as learn how to measure diffusion coefficients, molecular flow, and concentration of particles. Additionally, each section is followed by a short exercise to reinforce learning concepts by simulating different scenarios, seek verification of outcomes, and make comparisons. Furthermore, we invite the learner throughout the article to consult the literature for different extensions of FFCS techniques that allow measurements of different physicochemical properties of materials. Upon completion of the modules, we anticipate the learner will gain a good understanding in the field of FFCS that will encourage further exploration and adoption of the FFCS tools in future research and educational practices.