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Studying Single Red Blood Cells Under a Tunable External Force by Combining Passive Microrheology with Raman Spectroscopy

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Abstract

The dynamic micromechanical and structural properties of single human red blood cells are studied using a combination of dual trap optical tweezers and confocal Raman spectroscopy. Such a combination permits us to show a direct relationship between the rheological properties and chemical structure conformation. The frequency dependence of the complex stiffness of the cells was measured using both one and two probe response functions under identical experimental conditions. Both the microrheology and Raman measurements were performed at different stretching forces applied to the cell. A detailed analysis of the auto- and cross-correlated probe motions allows exploring the local and overall viscoelastic properties of the cells over a controlled range of the deformations. The observed growth of the cell viscoelasticity with stretching was associated with structural changes in the cell membrane monitored via the Raman spectroscopy.

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Acknowledgments

We acknowledge the financial support from MIIN FIS2008-00114 and FIS2011-24409, (Spain), Fundació Privada Cellex Barcelona, and discussions with S. Rao, M. Marro, F. Beunis, S. Campoy.

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Authors and Affiliations

  1. ICFO—The Institute of Photonic Sciences, Av. Carl Friedrich Gauss, num 3, 08860, Castelldefels, Barcelona, Spain

    Saurabh Raj, Michal Wojdyla & Dmitri Petrov

  2. ICREA—Institucio Catalana de Recerca i Estudis Avancats, 08010, Barcelona, Spain

    Dmitri Petrov

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  1. Saurabh Raj
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  2. Michal Wojdyla
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Correspondence to Dmitri Petrov.

Electronic supplementary material

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12013_2012_9439_MOESM1_ESM.eps

(Colour online). PSD of the single particle fluctuations (right trap) in the absence (black) andpresence of RBC and different forces applied to the cell (red—the cell at rest and blue—the cell stretched by16%). The spectrum obtained in the absence of RBC is fitted to the Lorentzian function (solid pink) and wasused for calibration purposes as described in the text. The inset shows the ratio of the PSD of the bead in thepresence of RBC to the PSD of the bead in the absence of RBC for 12 measured cells, demonstrating that thelow frequency effect is characteristic for all studied cells. The ratio exceeds unity for most of the cells at lowfrequency (EPS 223 KB)

12013_2012_9439_MOESM2_ESM.eps

Representative PSD of water (black) and Alsever’s (red) solution showing asmall difference due to the variation in the viscosity of these solutions. The experiments were performedunder the same conditions (excluding the bead polydispersity) using the beads from the same batch. Thesolid green line represents the Lorentzian fitting of the PSD in Alsever’s solution. As expected, the higherviscosity of Alsever’s solution leads to a lowering of the corner frequency (in this case from 59 Hz to 53Hz) and slightly higher PSD values below the corner frequency. The final value of the viscosity of Alsever’ssolution was found as an average value obtained from 40 PSD spectra for each solution (EPS 1258 KB)

12013_2012_9439_MOESM3_ESM.eps

Power spectral density of the relative fluctuations of the beads in the absence(black), r = 9.2 μm and presence of RBC and different forces applied to the cell (red circles—the cell at rest,r0 = 9.8 μm, green (right) triangles—the cell stretched by 12%, r1 = 10.6 μm, blue (up) triangles—the cellstretched by 16% , r2 = 10.8 μm). In the presence of RBC, PSDδ_x represents the PSD corresponding tothe fluctuations of the cell length; r is the centre-to-centre distance between the beads (EPS 132 KB)

12013_2012_9439_MOESM4_ESM.eps

Frequency dependence of the real (a) and imaginary (b) parts of an apparentsingle-particle response function (right trap) in the absence (black squares) and presence of RBC and differentforces applied to the cell (red circles - the cell at rest, green (right) triangles - the cell stretched by 12%, andblue (up) triangles - the cell stretched by 16%) (EPS 1212 KB)

12013_2012_9439_MOESM5_ESM.eps

Real part of the complex stiffness as a function of frequency measured viathe single particle (right trap) rheology (red circles—cell at rest, green (right) triangles—stretched by 12%,and blue (up) triangles—stretched by 16%). The actual complex stiffness was calculated using the responsefunctions corrected for the presence of two traps (EPS 1099 KB)

12013_2012_9439_MOESM6_ESM.eps

Imaginary part of the complex stiffness measured with the two particle microrheologyfor the cell at rest (red circles, r0 = 9.8 μm) and stretched by 12% (green (right) triangles,r1 = 10.6 μm) and 16% (blue (up) triangles, r2 = 10.8 μm); r is the centre-to-centre distance between thebeads (EPS 1102 KB)

Raman spectra of a single RBC at the relaxed (black lines) and stretched states (grey lines) (EPS 1085 KB)

12013_2012_9439_MOESM8_ESM.eps

Raman spectra intensity of 991 cm−1, 1442 cm−1 and 1530 cm−1 bands of single RBC plottedagainst the relative deformation (EPS 44 KB)

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Raj, S., Wojdyla, M. & Petrov, D. Studying Single Red Blood Cells Under a Tunable External Force by Combining Passive Microrheology with Raman Spectroscopy. Cell Biochem Biophys 65, 347–361 (2013). https://doi.org/10.1007/s12013-012-9439-x

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