We propose a new technique to measure the volume of adherent migrating cells. The method is based on a negative staining where a fluorescent, non-cell-permeant dye is added to the extracellular medium. The specimen is observed with a conventional fluorescence microscope in a chamber of uniform height. Given that the fluorescence signal depends on the thickness of the emitting layer, the objects excluding the fluorescent dye (i.e., cells) appear dark, and the decrease of the fluorescent signal with respect to the background is expected to give information about the height and the volume of the object. Using a glass microfabricated pattern with steps of defined heights, we show that the drop in fluorescence intensity is indeed proportional to the height of the step and obtain calibration curves relating fluorescence intensity to height. The technique, termed the fluorescence displacement method, is further validated by comparing our measurements with the ones obtained by atomic force microscopy (AFM). We apply our method to measure the real-time volume dynamics of migrating fish epidermal keratocytes subjected to osmotic stress. The fluorescence displacement technique allows fast and precise monitoring of cell height and volume, thus providing a valuable tool for characterizing the three-dimensional behaviour of migrating cells.

The insoluble (macrogel) and soluble fractions of 11 commercial natural rubber (NR) samples (Technically specified rubber) were separated. Nitrogen titrations and lipid extractions enabled a quantitative assessment of the proteins and extractable lipids in each fraction. Swelling was measured in tetrahydrofuran in order to evaluate the crosslink density (Mc –1) of each macrogel. While the soluble fraction had a high lipid concentration, the majority of non-isoprene compounds of the macrogel were found to be proteins, which accounted for 4.6 to 50.8% (w/w) of the macrogel. Indeed, the macrogels contained less than 0.5% (w/w) extractable lipids. However, our results showed that the soluble fraction contained large quantities of proteins (16–66% of the nitrogen content of the raw NR sample), probably structuring microaggregates. An exponential correlation (R2 > 0.96) was found between the crosslink density and the protein concentration of macrogel, suggesting that proteins are involved in the majority of crosslinks in macrogel. (Résumé d'auteur