Detection of volume changes in calcein-stained cells using confocal microscopy

Calcein is an intracellular fluorescent probe that has been used as an indicator of cell volume in several previous studies. These studies have reported two different fluorescence responses depending on the optical setup used to collect the data: wide-field microscopy has resulted in a decrease in fluorescence upon cell shrinkage, whereas confocal microscopy has been shown to yield the opposite result. In this short communication, we have investigated the effect of optical setup on detection of cell volume changes in calcein-stained endothelial cells. A confocal microscope was used to collect the fluorescence data, and the pinhole diameter was varied in order to examine the effects of optical section thickness on fluorescence response. For large pinhole diameters - which correspond to relatively thick optical sections - fluorescence intensity decreased when cells were induced to shrink. In contrast, for small pinhole diameters the fluorescence intensity increased with cell shrinkage. The transition between these two types of fluorescence responses occurred when using a pinhole diameter of 285 μm, which corresponds with an optical section thickness slightly less than the height of the cells. Our results have implications for the design and interpretation of experiments involving the use of calcein as a cell volume indicator.