exc em
hemoglobinUV fluorescence
INTRACELLULAR RHEOLOGY OF RED BLOOD CELLS
Alice Briole, Thomas Podgorski, Bérengère Abou
Molecular rotors
➤ DASPI Molecular rotor
« Stiffening »
18°C 10µm
37°C 30°C 24°C
43°C
Rencontres du Non Linéaire 2021 [email protected]
RBCs are observed on an inverted fluorescence microscope in a 1mM DASPI solution
Temperature is controlled by an objective heater through the immersion oil in contact with the sample
Statistical sampling 5
donors Single donor
400 RBCs per temperature
80 RBCs per temperature
DASPI 0.1mM DASPI
1mM DASPI
1mM
T(°C)
•
The decrease in intensity with temperature reflects the decrease in cell stiffness•
The technique allows to evaluate the stiffness of individual cells (1dot = 1 RBC) and the stiffness heterogeneities within a sample (width of the intensity distribution)Our RBC model provides (not shown) :
•
The separation of membrane and cytosol contributions•
Cytosol fluorescence consistent with independent measurements of hemoglobinviscosity with temperature
Ethylene-glycol/
Glycerol mixtures
100 101 102 103
(mPa.s) 102
103 104 105
Intensity (a.u.)
16°C 20°C 25°C 30°C
1mM 0.1mM 0.01mM
0 0.5 1
DASPI (mM) 0
1000 2000 3000
C (a.u.)
- measured by microrheology
•
DASPI spectral properties are decoupled from those of hemoglobin•
Cells fluorescence increases with decreasing temperature i.e with stiffening•
DASPI penetrates homogeneously into RBCs (confocal microscopy images not shown)Intracellular rheology
DASPI: intracellular nanorheology probe
➤ Temperature-stiffened RBCs : membrane rigidity and cytosol viscosity decrease with temperature2,3
➤ Fluorescence intensity and RBC stiffness
➤ Viscosity probe in model fluids
Fluorescent molecule
R Cytosol
C = f (M,P,R)
x (µm)
The signal is composed of membrane and cytosol contributions. By working on
intensity profiles, we propose a geometric model of RBC for contribution separation
A. Briole, T. Podgorski, and B. Abou, Molecular Rotors as intracellular probes of Red Blood Cell stiffness, submitted to Soft Matter, 2021
