Mapping Diffusion Coefficients of Fluorescent Dyes in Cardiomyocytes

Abstract

Intracellular compartmentation and macromolecular crowding affect numerous processes in the cell. Local concentrations of metabolites and available pathways control metabolic regulation. In cells with highly organized internal structure, such as mammalian cardiomyocytes, ATP and ADP experience considerable restrictions to diffusion. This has been verified by numerous indirect methods, but the intracellular structures responsible for these restrictions is unclear. Our aim was to determine the location and extent of these diffusion restrictions. We have employed a combination of experimental and mathematical approaches to analyze the movement of molecules in the cell. Diffusion coefficients were estimated by raster image correlation spectroscopy (RICS). With our custom built confocal microscope and control software, we have been able to measure regional diffusion coefficients in rat and mouse cardiomyocytes taking into account anisotropy of media. As verification of the method, we see a sharp change of the diffusion coefficient on the border of the cell. In order to extend the longevity of the cell and thereby increase the time available for data acquisition, we used microinjection instead of permeabilization for getting fluorescently labeled molecules into the cell. Our new experimental approach has allowed us to increase the resolution of the method to sub-micrometer range and decrease uncertainty of results approximately 3 times. Additionally, we have compiled a stochastic computational model to study the effect of regularly spaced barriers on diffusion and RICS measurements. Modelling results agree with our conclusions from experiments. We found that average diffusion coefficient in rat cardiomyocytes was ∼2 (longitudinal) and ∼3.5 (transverse) times smaller than the estimated diffusion coefficient in the surrounding solution. This relatively small effect on diffusion coefficients implies that movement of smaller molecules is restricted mostly by regional diffusion barriers and not by macromolecular crowding in the cell.