  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 conducted on temperature-stiffened red blood cells show that
molecular rotors can probe their overall rigidity. A simple model allowed us to
separate the contribution of the cytosol from that of the membrane, providing a
quantification of cytosol rigidification with temperature, consistent with
independent measurements of the viscosity of hemoglobin solutions. Our
experiments demonstrate that the rotor can be used to quantify the
intracellular rheology of red blood cells at the cellular level, as well as the
heterogeneity of this stiffness in a blood sample. This technique opens up new
possibilities for biomedical applications, diagnosis and disease monitoring.
