Molecular rotors as intracellular probes of red blood cell stiffness

The deformability of red blood cells is an essential parameter that controls the rheology of blood as well as its circulation in the body. Characterizing the rigidity of the cells and their heterogeneity in a blood sample is thus a key point in the understanding of occlusive phenomena, particularly in the case of erythrocytic diseases in which healthy cells coexist with pathological cells. However, measuring intracellular rheology in small biological compartments requires the development of specific techniques. We propose a technique based on molecular rotors - viscosity-sensitive fluorescent probes - to evaluate the above key point. DASPI molecular rotor has been identified with spectral fluorescence properties decoupled from those of hemoglobin, the main component of the cytosol. After validation of the rotor as a viscosity probe in model fluids, we showed by confocal microscopy that, in addition to binding to the membrane, the rotor penetrates spontaneously and uniformly into red blood cells. Experiments on red blood cells whose rigidity is varied with temperature, show that molecular rotors can detect variations in their overall rigidity. A simple model allowed us to separate the contribution of the cytosol from that of the membrane, allowing a qualitative determination of the variation of cytosol viscosity with temperature, consistent with independent measurements of the viscosity of hemoglobin solutions. Our experiments show that the rotor can be used to study the intracellular rheology of red blood cells at the cellular level, as well as the heterogeneity of this stiffness in a blood sample. This opens up new possibilities for biomedical applications, diagnosis and disease monitoring.