Measurement of the thickness and volume of adherent cells using transmission-through-dye microscopy

Cell volume is one of the basic characteristics of a cell and is being extensively studied in relationship to a variety of processes, such as proliferation, apoptosis, fertility, or locomotion. At the same time, its measurement under a microscope has not been well developed. The method we propose uses negative transmission contrast rendered to cells by a strongly absorbing dye present in the extracellular medium. Cells are placed in a shallow compartment, and a nontoxic and cell-impermeant dye, such as acid blue 9, is added to the medium. Transmission images are collected at the wavelength of maximum dye absorption (630 nm). Where the cell body displaces the dye, the thickness of the absorbing layer is reduced; thus, an increase in cell thickness produces brighter images and vice versa. The absolute values for cell thickness and volume can be easily extracted from the image by computing the logarithm of intensity and dividing it by the absorption coefficient. The method is fast, impervious to instability of the light source, and has a high signal-to-noise ratio; it can be realized either on a laser scanning or a conventional microscope equipped with a bandpass filter. For long-term experiments, we use a Bioptechs perfusion chamber fitted with a 0.03-mm spacer and an additional port to enable rapid switching of solutions. To show possible applications of this method, we investigated the kinetics of the cell volume response to a hypotonic buffer and to the apoptotic agents staurosporine and ionomycin.