Label-Free Quantification of Protein Density in Living Cells.

Intracellular water content, W, and protein concentration, P, are essential characteristics of living cells. Healthy cells maintain them within a narrow range, but often become dehydrated under severe stress; moreover, persistent loss of water (an increase in P) can lead to apoptotic death. It is very likely that protein concentration affects cellular metabolism and signaling through macromolecular crowding (MC) effects, to which P is directly related, but much remains unknown in this area. Obviously, in order to study the biological roles and regulation of MC in living cells, one needs a method to measure it. Simple and accurate measurements of P in adherent cells can be based on its relationship to refractive index. The latter can be derived from two or more (depending on the algorithm) mutually defocused brightfield images processed by the transport-of-intensity equation (TIE) that must be complemented by a determination of volume. Here, we describe the experimental considerations for both TIE imaging and for a particular method of cell volume measurement, transmission-through-dye (TTD). We also introduce an ImageJ plugin for solving TIE. TIE and TTD are fully compatible with each other as well as with fluorescence. A similar approach can be applied to subcellular organelles; however, in this case, the volume must be determined differently.© 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Sample preparation for TIE with or without TTD Basic Protocol 2: Acquisition of TIE and TTD images Basic Protocol 3: Calibration of TIE Basic Protocol 4: Measurement of the absorption coefficient of the medium used for TTD Basic Protocol 5: Image processing using Fiji Support Protocol 1: Installation and use of TIE plugin Support Protocol 2: Automation of the double TTD/TIE processing using a Fiji macro.