Cell volume measurements by optical transmission microscopy.

Cell volume changes are critical in responses to osmotic stress and disease progression. Prior research has shown that volume shifts correlate with apoptosis and necrosis. However, measuring adherent cells remains challenging. Established methods include gravimetry and flow cytometry. These approaches may lack spatial resolution or require cell suspension. Optical techniques offer non-invasive alternatives. Yet, adapting them for adherent cells is limited. This gap motivated the development of a transmission microscopy-based method. This paper introduces a novel approach for adherent cell volume measurement.

The aim is to provide a practical method for measuring adherent cell volume using standard microscopy. Adherent cells are difficult to assess with traditional techniques. The study addresses the need for spatially resolved volume data. The method uses a dye that does not enter cells but absorbs strongly. This allows contrast proportional to cell thickness. The procedure avoids specialized equipment or complex protocols. The goal is to enable volume computation from transmitted light images. This approach could improve understanding of cell volume dynamics.

The method uses a strongly absorbing dye, Acid Blue 9, in a shallow chamber. Adherent cells are placed on a coverslip within the chamber. Transmitted light imaging occurs at 630 nm, the dye’s absorption peak. The dye’s presence creates contrast proportional to cell thickness. Image contrast is analyzed to derive thickness at each point. Volume is calculated from thickness maps across the cell area. The absorption coefficient is required for absolute units. A separate procedure measures this coefficient using the same setup.

The method successfully derives cell thickness from transmitted light images. Contrast at 630 nm reflects cell thickness with high accuracy. Volume is computed from thickness maps across the cell surface. The technique works with standard light microscopes and dyes. The absorption coefficient measurement procedure is validated. Absolute volume calculations require this coefficient. The method avoids cell detachment or complex labeling. Technical artifacts are minimized through controlled imaging conditions.

The study demonstrates a practical method for adherent cell volume measurement. The approach uses standard microscopy and a cell-impermeant dye. Contrast at 630 nm provides a direct measure of cell thickness. Volume computation follows from thickness maps. The method avoids specialized equipment or complex procedures. The absorption coefficient is essential for absolute units. The procedure for measuring this coefficient is described. This technique supports studies of cell volume dynamics in situ.