Quantitative Analysis of Viscoelastic Properties of Red Blood Cells Using Optical Tweezers and Defocusing Microscopy

Summary

Here, an integrated protocol based on optical tweezers and defocusing microscopy is described to measure the rheological properties of cells. This protocol has wide applicability in studying the viscoelastic properties of erythrocytes under variable physio-pathological conditions.

Abstract

The viscoelastic properties of erythrocytes have been investigated by a range of techniques. However, the reported experimental data vary. This is not only attributed to the normal variability of cells, but also to the differences in methods and models of cell response. Here, an integrated protocol using optical tweezers and defocusing microscopy is employed to obtain the rheological features of red blood cells in the frequency range of 1 Hz to 35 Hz. While optical tweezers are utilized to measure the erythrocyte-complex elastic constant, defocusing microscopy is able to obtain the cell height profile, volume, and its form factor a parameter that allows conversion of complex elastic constant into complex shear modulus. Moreover, applying a soft glassy rheology model, the scaling exponent for both moduli can be obtained. The developed methodology allows to explore the mechanical behavior of red blood cells, characterizing their viscoelastic parameters, obtained under well-defined experimental conditions, for several physiological and pathological conditions.

Introduction

Mature red blood cells (RBCs), also known as erythrocytes, are able to extend more than twice their size when passing through the narrowest capillaries of the human body¹. Such capacity is attributed to their unique ability to deform when subjected to external loads.

In recent years, different studies have characterized this feature in RBC surfaces^2,3. The area of physics that describes the elastic and viscous responses of materials due to external loads is called rheology. In general, when an external force is applied, the resulting deformation depends on th....

Protocol

Human blood samples were provided by adult men and women volunteers according to protocols approved by the Research Ethics Committee of the Federal University of Rio de Janeiro (Protocol 2.889.952) and registered in Brazil Platform under CAAE number 88140418.5.0000.5699. A written form of consent was issued to and collected from all volunteers. Those with any hemoglobinopathy and/or taking controlled medication were excluded. The entire process followed the guidelines approved by the institute's ethical committee.

Discussion

In this protocol, an integrated method based on optical tweezers and defocusing microscopy is presented to quantitatively map the viscoelastic properties of RBCs. Results for the storage and loss shear moduli, together with the scaling exponent that characterizes the soft glassy rheology of RBC are determined. Application of this protocol for different experimental conditions, such as in physiological situation⁸ or along each stage of P. falciparum intra-erythrocytic cycle

Materials

Name	Company	Catalog Number	Comments
35mm culture dishes	Corning	430165
Bovine serum albumin	Sigma-Aldrich	A9418
Coverslips	Knittel Glass	VD12460Y1A.01 and VD12432Y1A.01
Glass-bottom dishes	MatTek Life Sciences	P35G-0-10-C
Glucose	Sigma-Aldrich	G7021
ImageJ	NIH	https://imagej.nih.gov/ij/
Immersion oil	Nikon	MXA22165
Inverted microscope	Nikon	Eclipse TE300
KaleidaGraph	Synergy Software	https://www.synergy.com/
KCl	Sigma-Aldrich	P5405
KH2PO4	Sigma-Aldrich	P5655
Microscope camera	Hamamatsu	C11440-10C
Na2HPO4	Sigma-Aldrich	S5136
NaCl	Sigma-Aldrich	S5886
Neubauer chamber	Sigma-Aldrich	BR717805-1EA
Objective lens	Nikon	PLAN APO 100X 1.4 NA DIC H; PLAN APO 60x 1.4 NA DIC H and Plan APO 10x XXNA PH2
Optical table	Thorlabs	T1020CK
OT laser	IPG Photonics	YLR-5-1064-LP
Polystyrene microspheres	Polysciences	17134-15
rubber ring	Forever Seals	NBR O-Ring
Silicone grease	Dow Corning	Z273554
Stage positioning	PI	P-545.3R8S
Pipette	Gilson	P1000