A series of kinetic experiments show the existence of considerable ADP and ATP diffusion restrictions in rat cardiomyocytes (1,2). As an alternative method to estimate diffusion, we have extended raster image correlation spectroscopy — RICS (3) — to study diffusion in anisotropic media, such as heart muscle cells (4). On the basis of the measurements on two different dyes, our analysis suggests that the diffusion restrictions are localized in certain areas of the cell (5). However, their location, physiological role, and the intracellular structures responsible for these restrictions are still unclear. We hypothesize that diffusion restrictions in cardiomyocyte are in the form of membrane-like barriers, and present a theoretical foundation for the use of RICS to determine the presence and the locations of such barriers. With a Gaussian point spread function of the microscope, the pair correlation function (PCF) for an impermeable membrane is calculated analytically, thus giving a convenient theoretical basis for the interpretation of microscopy data. We demonstrate that, in the proximity of the membrane, the PCF is not symmetric, as opposed to the case of homogeneous media. As a result, this property of PCF can be used to detect barriers. Indeed, our preliminary experimental results support this theory. We recorded fluctuations in fluorescence within the permeabilized rat cardiomyocyte along a line that was partially in the cell and partially in the solution. Paired-correlation RICS analysis shows that in the cell region the symmetry of the correlation function is modulated, but not in the solution. These results can be interpreted as an evidence that periodic diffusion barriers exists in rat cardiomyocytes.