Comparison of cell volume measurements by fluorescence and absorption exclusion microscopy

There are two light microscopic methods for cell volume measurement based on volume exclusion. One method, sometimes referred to as FLEX, utilises negative staining by an external fluorescent dye, and cell volume is found from attenuation of fluorescence under a wide-field microscope. The other method (TTD) is based on exclusion of an external absorbing dye, resulting in an increased intensity of transmission image. In this work, we compared these two methods. TTD measurements were consistent, reproducible and identical to those obtained by confocal scanning. In our hands, FLEX based on either sodium fluorescein of fluorescent dextran, usually resulted in underestimation of cell volume, which were insignificant in shallow chambers but became more severe with increased chamber depth. We have not been able to exactly pinpoint the source of the problem; it may have been undetected accumulation of dye in the cells or, more likely, some unappreciated aspects of image formation under epi-illumination. We also discuss applicability of both methods to in-flow volume measurements. LAY DESCRIPTION: Cell volume is a parameter important for many cell biological and physiological applications, and many different methods have been proposed for its measurement. Two light microscopic methods based on volume exclusion deserve special attention due to their speed and simplicity. In one of them (transmission-through-dye, or TTD), cells are placed in a shallow chamber, and a strongly absorbing external dye is added to the cell-containing medium. The sample is imaged in transmission at a wavelength of maximum dye absorption. Because cells with intact membranes exclude the dye, they appear brighter on a transmission image, and their contrast quantitatively reflects cell thickness. By summation of thickness values over the cell area, cell volume can be obtained. The other method sometimes referred to as FLEX utilises exclusion of a fluorescent dye. Cells appear darker than the background under wide-field fluorescence observation in accordance with their thicknesses, and cell volume is computed by thickness summation over the area, like in TTD. In this work, we compared the accuracy of TTD and FLEX for volume measurements. TTD and confocal scanning produced virtually identical results, which suggests that TTD data are accurate. On the other hand, cell volumes measured by FLEX were consistently smaller than by TTD. The discrepancy always increased with the depth of the chamber, although the exact relationship varied between experiments. By contrast, TTD results were insensitive to chamber depth. Thus, it appears that FLEX underestimates cell volume. The reason for that is not entirely clear. Accumulation of the fluorescent dye inside the cell could be a possibility, although we found no evidence for that. Most likely, the reason lies with some unappreciated aspects of wide-field fluorescence image formation, which has not been well-characterised for the type of negative staining used in FLEX. In our opinion, TTD is the method of choice, at least for stationary cells. On the other hand, due to linear dependence of intensity on volume, FLEX might offer advantages for high-throughput flow volume imaging, although realisation of such an experiment has yet to be worked out.