Name: Quantitative Analysis of Viscoelastic Properties of Red Blood Cells Using Optical Tweezers and Defocusing Microscopy (Video) | JoVE Journal
Uploaded: 2022-03-25T14:30:00.000+00:00
Duration: 8 min 3 s
Description: Learn to measure viscoelastic properties of red blood cells using optical tweezers and defocusing microscopy. This protocol characterizes erythrocyte mechanics and soft glassy behavior for physiological studies.

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The authors would like to acknowledge all the members of CENABIO advanced microscopy facility for all-important help. This work was supported by the Brazilian agencies Conselho Nacional de Desenvolvimento Cient&#237;fico e Tecnol&#243;gico (CNPq), Coordena&#231;&#227;o de Aperfei&#231;oamento de Pessoal de N&#237;vel Superior (CAPES) - Financial Code 001, Funda&#231;&#227;o de Amparo &#224; Pesquisa do Estado do Rio de Janeiro (FAPERJ), and Instituto Nacional de Ci&#234;ncia e Tecnologia de Fluidos Complexos (INCT-FCx) together with Funda&#231;&#227;o de Amparo &#224; Pesquisa do Estado de S&#227;o Paulo (FAPESP). B.P. was supported by a JCNE grant from FAPERJ.

<ol><li>Fowler, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+human+erythrocyte+plasma+membrane%3A+a+Rosetta+Stone+for+decoding+membrane-cytoskeleton+structure%5BTitle%5D)+AND+%22Current+Topics+in+Membranes%22%5BJournal%5D)">The human erythrocyte plasma membrane: a Rosetta Stone for decoding membrane-cytoskeleton structure.</a> Current Topics in Membranes. 72, 39-88 (2013).</li>
<li>Tomaiuolo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biomechanical+properties+of+red+blood+cells+in+health+and+disease+towards+microfluidics%5BTitle%5D)+AND+%22Biomicrofluidics%22%5BJournal%5D)">Biomechanical properties of red blood cells in health and disease towards microfluidics.</a> Biomicrofluidics. 8 (5), 051501(2014).</li>
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<li>Amin, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microrheology+of+red+blood+cell+membranes+using+dynamic+scattering+microscopy%5BTitle%5D)+AND+%22Optics+Express%22%5BJournal%5D)">Microrheology of red blood cell membranes using dynamic scattering microscopy.</a> Optics Express. 15 (25), 17001-17009 (2007).</li>
<li>Puig-de-Morales-Marinkovic, M., Turner, K. T., Butler, J. P., Fredberg, J. J., Suresh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Viscoelasticity+of+the+human+red+blood+cell%5BTitle%5D)+AND+%22American+Journal+of+Physiology+Cell+Physiology%22%5BJournal%5D)">Viscoelasticity of the human red blood cell.</a> American Journal of Physiology Cell Physiology. 293 (2), 597-605 (2007).</li>
<li>Gomez, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+cell+geometry+in+the+evaluation+of+erythrocyte+viscoelastic+properties%5BTitle%5D)+AND+%22Physical+Review+E%22%5BJournal%5D)">Effect of cell geometry in the evaluation of erythrocyte viscoelastic properties.</a> Physical Review E. 101 (6-1), 062403(2020).</li>
<li>Gomez, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plasmodium+falciparum+maturation+across+the+intra-erythrocytic+cycle+shifts+the+soft+glassy+viscoelastic+properties+of+red+blood+cells+from+a+liquid-like+towards+a+solid-like+behavior%5BTitle%5D)+AND+%22Experimental+Cell+Research%22%5BJournal%5D)">Plasmodium falciparum maturation across the intra-erythrocytic cycle shifts the soft glassy viscoelastic properties of red blood cells from a liquid-like towards a solid-like behavior.</a> Experimental Cell Research. 397 (2), 112370(2020).</li>
<li>Pompeu, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protocol+to+measure+the+membrane+tension+and+bending+modulus+of+cells+using+optical+tweezers+and+scanning+electron+microscopy%5BTitle%5D)+AND+%22STAR+Protocols%22%5BJournal%5D)">Protocol to measure the membrane tension and bending modulus of cells using optical tweezers and scanning electron microscopy.</a> STAR Protocols. 2 (1), 100283(2021).</li>
<li>Agero, U., Mesquita, L. G., Neves, B. R., Gazzinelli, R. T., Mesquita, O. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Defocusing+microscopy%5BTitle%5D)+AND+%22Microscopy+Research+and+Technique%22%5BJournal%5D)">Defocusing microscopy.</a> Microscopy Research and Technique. 65 (3), 159-165 (2004).</li>
<li>Agero, U., Monken, C. H., Ropert, C., Gazzinelli, R. T., Mesquita, O. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+surface+fluctuations+studied+with+defocusing+microscopy%5BTitle%5D)+AND+%22Physical+Review+E%22%5BJournal%5D)">Cell surface fluctuations studied with defocusing microscopy.</a> Physical Review E. 67 (5), 051904(2003).</li>
<li>Roma, P. M. S., Siman, L., Amaral, F. T., Agero, U., Mesquita, O. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Total+three-dimensional+imaging+of+phase+objects+using+defocusing+microscopy%3A+Application+to+red+blood+cells%5BTitle%5D)+AND+%22Applied+Physics+Letters%22%5BJournal%5D)">Total three-dimensional imaging of phase objects using defocusing microscopy: Application to red blood cells.</a> Applied Physics Letters. 104 (25), 251107(2014).</li>
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<li>Nans, A., Mohandas, N., Stokes, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Native+ultrastructure+of+the+red+cell+cytoskeleton+by+cryo-electron+tomography%5BTitle%5D)+AND+%22Biophysical+Journal%22%5BJournal%5D)">Native ultrastructure of the red cell cytoskeleton by cryo-electron tomography.</a> Biophysical Journal. 101 (10), 2341-2350 (2011).</li>
<li>Ayala, Y. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rheological+properties+of+cells+measured+by+optical+tweezers%5BTitle%5D)+AND+%22BMC+Biophysics%22%5BJournal%5D)">Rheological properties of cells measured by optical tweezers.</a> BMC Biophysics. 9, 5(2016).</li>
<li>Ayala, Y. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+cytoskeletal+drugs+on+actin+cortex+elasticity%5BTitle%5D)+AND+%22Experimental+Cell+Research%22%5BJournal%5D)">Effects of cytoskeletal drugs on actin cortex elasticity.</a> Experimental Cell Research. 351 (2), 173-181 (2017).</li>
</ol>

The authors have no financial interests in the products described in this manuscript and have nothing else to disclose.

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<sup class="xref...

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 surfaces2,3. 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 the material&#39;s properties and can be divided into elastic deformations, that store energy, or viscous deformations, that dissipate energy4. All cells, including RBCs, exhibit a viscoelastic behavior; in other words, energy is both stored and dissipated. The viscoelastic response of a cell can thus be characterized by its complex shear modulus G*(&#969;) = G&#39;(&#969;) + iG&#34;(&#969;), where G&#39;&#160;(&#969;) is the storage modulus, related to the elastic behavior, and G&#34; (&#969;)&#160;is the loss modulus, related to its viscosity4. Moreover, phenomenological models have been used to describe cell responses, one of the most used is called the soft glassy rheology model5, characterized by a power-law dependence of the complex shear modulus with the load frequency.
Single-cell-based methods have been employed to characterize the viscoelastic properties of RBCs, by applying force and measuring displacement as a function of the imposed load2,3. However, for the complex shear modulus, few results can be found in the literature. Using dynamic light scattering, values for RBC storage and loss moduli were reported varying from 0.01-1 Pa, in the frequency range of 1-100 Hz6. By using optical magnetic twisting cytometry, an apparent complex elastic modulus was obtained7, and for comparison purposes, a multiplicative factor was claimed to possibly clarify the discrepancies.
More recently, a new methodology based on optical tweezers (OT) together with defocusing microscopy (DM), as an integrated tool to quantitatively map the storage and loss of shear moduli of human erythrocytes over time-dependent loads, was established8,9. In addition, a soft glassy rheology model was used to fit the results and obtain a power-law coefficient that characterizes the RBCs8,9.
Overall, the developed methodology8,9, the protocol for which is described in detail below, clarifies previous discrepancies by using the measured values for the form factor, Ff, that relates forces and deformations to stresses and strains in the RBC surface and can be utilized as a novel diagnostic method capable of quantitatively determining the viscoelastic parameters and soft glassy features of RBCs obtained from individuals with different blood pathologies. Such characterization, using the protocol described below, may open up new possibilities to understand the behavior of RBCs from a mechanobiological perspective.

<table><tbody><tr><td>35mm culture dishes</td><td>Corning</td><td>430165</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A9418</td><td></td></tr><tr><td>Coverslips</td><td>Knittel Glass</td><td>VD12460Y1A.01 and VD12432Y1A.01</td><td></td></tr><tr><td>Glass-bottom dishes</td><td>MatTek Life Sciences</td><td>P35G-0-10-C</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G7021</td><td></td></tr><tr><td>ImageJ</td><td>NIH</td><td>https://imagej.nih.gov/ij/</td><td></td></tr><tr><td>Immersion oil</td><td>Nikon</td><td>MXA22165</td><td></td></tr><tr><td>Inverted microscope</td><td>Nikon</td><td>Eclipse TE300</td><td></td></tr><tr><td>KaleidaGraph</td><td>Synergy Software</td><td>https://www.synergy.com/</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P5405</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>P5655</td><td></td></tr><tr><td>Microscope camera</td><td>Hamamatsu</td><td>C11440-10C</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S5136</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S5886</td><td></td></tr><tr><td>Neubauer chamber</td><td>Sigma-Aldrich</td><td>BR717805-1EA</td><td></td></tr><tr><td>Objective lens</td><td>Nikon</td><td>PLAN APO 100X 1.4 NA DIC H; PLAN APO 60x 1.4 NA DIC H and Plan APO 10x XXNA PH2</td><td></td></tr><tr><td>Optical table</td><td>Thorlabs</td><td>T1020CK</td><td></td></tr><tr><td>OT laser</td><td>IPG Photonics</td><td>YLR-5-1064-LP</td><td></td></tr><tr><td>Polystyrene microspheres</td><td>Polysciences</td><td>17134-15</td><td></td></tr><tr><td>rubber ring</td><td>Forever Seals</td><td>NBR O-Ring</td><td></td></tr><tr><td>Silicone grease</td><td>Dow Corning</td><td>Z273554</td><td></td></tr><tr><td>Stage positioning</td><td>PI</td><td>P-545.3R8S</td><td></td></tr><tr><td>Pipette</td><td>Gilson</td><td>P1000</td><td></td></tr></tbody></table>

quantitative analysis of viscoelastic properties of red blood cells using optical tweezers and defocusing microscopy

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

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.

Figure 1 represents the schematics of the OT system used for the rheology measurements. Figure 2 shows the schematics of the microrheology experiment with both spheres and a representative RBC is also shown. Figure 3 shows a typical curve for the amplitudes of both spheres as a function of time when the sinusoidal movements are produced by the piezoelectric stage. While the reference sphere (Figure 3 - ...

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.

The viscoelastic properties of erythrocytes have been investigated by a range of techniques. However, the reported experimental data vary. This is not ...

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Biology

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Learn to measure viscoelastic properties of red blood cells using optical tweezers and defocusing microscopy. This protocol characterizes erythrocyte mechanics and soft glassy behavior for physiological studies.

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

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