In vitro investigation of the mechanics of fixed red blood cells based on optical trap micromanipulation and image analysis

Abstract

Erythrocyte deformability correlates with various diseases. Single-cell measurements via optical tweezers (OTs) enable quantitative exploration but may encounter inaccuracies due to erythrocyte life cycle mixing. We present a three-step methodology to address these challenges. Firstly, density gradient centrifugation minimizes erythrocyte variations. Secondly, OTs measure membrane shear force across layers. Thirdly, MATLAB analyzes dynamic cell areas. Results combined with membrane shear force data reveal erythrocyte deformational capacity. To further characterize the deformability of diseased erythrocytes, the experiments used glutaraldehyde-fixed erythrocytes to simulate diseased cells. OTs detect increased shear modulus, while image recognition indicates decreased deformation. The integration of OTs and image recognition presents a comprehensive approach to deformation analysis, introducing novel ideas and methodologies for investigating erythrocytic lesions.

Fig. 1.

Results of erythrocyte gradient centrifugation, with left, center and right images showing the results at 1, 7, and 14 days, respectively.

Fig. 2.

Schematic diagram of the AOD scanning optical tweezers used for the experiment (left). Schematic diagram of the erythrocyte stretching process within the sample chamber (right).

Fig. 3.

Optical tweezers light field center potential energy curve.

Fig. 4.

CMOS shot of a single erythrocyte bound to a silica pellet and stretched by light tweezers. Top and bottom images were taken before and after light tweezers stretching, respectively.

Fig. 5.

Mechanical measurements of the erythrocyte stretching process. (A) Linear fit plot of the stretch force versus cell elongation. (B) Statistical plot of cellular shear modulus. The control group (CG) referred to erythrocytes not treated by gradient centrifugation, while UL and LL corresponded to the shear modulus of the upper and lower cell layers after gradient centrifugation; ★ represents statistically significant differences in means at p < 0.05.

Fig. 6.

(A) Shear modulus of erythrocytes in glutaraldehyde solutions of different concentration gradients (0.005%, 0.01%, 0.015%). (B) Values for the shear moduli of cell membranes in the control (non-glutaraldehyde-treated erythrocytes) and experimental (0.01% glutaraldehyde-treated) erythrocytes at different ex vivo time periods. The folded line is the ratio of the mean shear modulus of the control to that of the experimental groups.

Fig. 7.

Dynamic identification and area statistics of erythrocyte deformation processes. (A) binarized image of the erythrocyte stretching process. (B) Scatter plot of erythrocyte area versus stretching time for the control group. (C) Scatter plot showing the area of erythrocytes affected by glutaraldehyde solution versus stretching time. (D) Statistical results of the maximum degree of change in erythrocyte area in the control and experimental groups.