Abstract
The data provided with this paper are image series of slowly diffusing GlyRa3 molecules, linked to either eGFP or mCherry fluorescent proteins, at the membrane of HEK cells, acquired on a Zeiss LSM880 confocal laser scanning microscope. Raster spectral image cross-correlation spectroscopy (RSICS) is applied to the data, a technique that exploits intensity fluctuations in confocal image series recorded using a spectral detector to study the diffusion and concentration of molecules, and interactions between them. First, spectral filters are created from reference image series containing GlyRa3 labeled with a single fluorophore. Once experimental data containing GlyRa3 labeled with both fluorophores is acquired, single images are either autocorrelated, or the cross-correlation is calculated between two images, each one containing the data for eGFP or mCherry labeled GyRa 3. Data is then fit with a one-component model assuming a two-dimensional Gaussian point spread function to obtain the diffusion coefficient, D, and average number of molecules in the focus, N. The software package PAM is used to analyze all the acquired data. The data can be used as a reference for artifact-free two-color ccRICS that contains slowly diffusing interacting molecules. Additionally, the analysis workflow described in this paper helps researchers avoid common errors during a RICS experiment.