Raster image correlation spectroscopy and number and brightness analysis.

The raster image correlation spectroscopy (RICS) and number and molecular brightness (N&B) methods are used to measure molecular diffusion in complex biological environments such as the cell interior, detect the formation of molecular aggregates, establish the stoichiometry of the aggregates, spatially map the number of mobile molecules, and quantify the relative fraction of molecules participating in molecular complexes. These methods are based on correlation of fluorescence intensity fluctuations from microscope images that can be measured in a conventional laser-scanning confocal microscope. In this chapter, we discuss the mathematical framework used for data analysis as well as the parameters need for data acquisition. We demonstrate the information obtainable by the N&B method using simulation in which different regions of an image have different numbers of interacting molecules. Then, using an example of two interacting proteins in the cell, we show in a real case how the RICS and N&B analyses work step by step to detect the existence of molecular complexes to quantify their properties and spatially map their interactions. We also discuss common control experiments needed to rule out instrumental artifacts and how to calibrate the microscope in terms of relative molecular brightness.