Fig. 2
From: Confocal-based fluorescence fluctuation spectroscopy with a SPAD array detector
Overview of the analysis techniques (1) spot-variation FCS, (2) pair-correlation analysis, and (3) spatio-temporal image correlation spectroscopy (STICS) with image-derived mean squared displacement (iMSD) analysis. In spot-variation FCS, a the signal from different pixels is summed for each frame, b the correlation curves of the corresponding time traces are calculated and fitted, and c the resulting diffusion times τ are plotted as a function of the beam waist ω02. Free diffusion yields a directly proportional relationship between τ and ω02. Microdomains in the sample yield a positive intercept, τ(0) > 0, diffusion through a meshwork yields a negative intercept, τ(0) < 0. In two-focus FCS with pair-correlation analysis, d all 24 cross-correlations between the central pixel and the other pixels are calculated. e Cross-correlations between pixel pairs with the same interpixel distance are averaged, and the resulting 5 curves are simultaneously fitted in a global fit. f For STICS with iMSD analysis, each 5 × 5 frame is treated as an image. g, h The images are correlated in space and time, resulting in a series of 9 × 9 matrices. For free diffusion, the surface plots of these matrices show a Gaussian function of which the amplitude decreases and the width (σ2) increases linearly for increasing lag times. i The diffusion coefficient can be derived from the slope of the curve σ2(τ)
