Using molecular rotors to investigate hemoglobin concentration and heterogeneity in red blood cells

We explore the potential of using the DASPI molecular rotor [trans-4-[4-(Dimethylamino)styryl]-1-methylpyridinium iodide] to probe the rheology of red blood cells. Previous investigations have demonstrated the rotor’s capacity to penetrate cellular membranes and detect temperature-dependent modifications in membrane and cytosol mechanical properties. In this study, we demonstrate that the rotor has no specific affinity for intracellular proteins, leading to the establishment of a DASPI concentration equilibrium between the intracellular and extracellular environments. Using confocal microscopy and a simple geometric model of cells, we estimate that approximately 70% of the fluorescence intensity measured by widefield epifluorescence microscopy originates from the cytosol. We show that the rotor can differentiate healthy donors based on the variations in intracellular hemoglobin concentration and intracellular viscosity. It also detects intra-donor variability due to intrinsic properties of the cells. This study provides direct evidence for the use of the DASPI molecular rotor as a probe for rapid reading of cell rigidity and heterogeneity in blood samples, with potential applications in the study of erythrocyte pathologies.