Raster Image Correlation Spectroscopy Performance Evaluation

Abstract Raster image correlation spectroscopy (RICS) is a fluorescence image analysis method for extracting the mobility, concentration and stoichiometry of diffusing fluorescent molecules from confocal image stacks. The method works by calculating a spatial correlation function for each image and analyzing the average of those by model fitting. Rules of thumb exist for RICS image acquisitioning, yet a rigorous theoretical approach to predict the accuracy and precision of the recovered parameters has been lacking. We outline explicit expressions to reveal the dependence of RICS results on experimental parameters. In terms of imaging settings, we observed that a twofold decrease of the pixel size, e.g. from 100nm to 50nm, decreases the error on the translational diffusion constant (D) between 3- and 5-fold. For D = 1μm2s-1, a typical value for intracellular measurements, about 25-fold lower mean squared relative error was obtained when the optimal scan speed was used, although more drastic improvements were observed for other values of D. We proposed a slightly modified RICS calculation that allows to correct for the significant bias of the autocorrelation function at small (ii 50x50 pixels) sizes of the region of interest. In terms of sample properties, at molecular brightness e = 100kHz and higher, RICS data quality was sufficient using as little as 20 images, while the optimal number of frames for lower e scaled pro rata. RICS data quality was constant over the nM - μM concentration range. We developed a bootstrap-based confidence interval of D, that outperformed the classical least squares approach in terms of coverage probability of the true value of D. We validated the theory via in vitro experiments of enhanced green fluorescent protein at different buffer viscosities. Finally, we outlined robust practical guidelines and provide free software to simulate the parameter effects on recovery of the diffusion coefficient.