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Chinese Science Bulletin, Volume 64 , Issue 15 : 1610-1619(2019) https://doi.org/10.1360/N972018-01266

Measurement and analysis of cellular viscoelastic properties using atomic force microscopy

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  • ReceivedDec 18, 2018
  • AcceptedMar 4, 2019
  • PublishedApr 24, 2019

Abstract

Cell mechanics plays an important role in cellular physiological and pathological processes. During the formation and progression of tumors, the alterations in the mechanics of cancerous cells and tumor micro-environment promote the growth and migration of cancerous cells. Hence, investigating cell mechanics is of crucial significance in understanding the underlying mechanisms regulating life activities and diseases. The advent of atomic force microscopy (AFM) provides a powerful tool for detecting the mechanical properties of single cells. Compared with other single-cell mechanical analysis techniques, the advantage of AFM is that AFM is able to simultaneously obtain the topography and mechanics of cells, which is particularly useful for investigating the correlation between cell structures and cell mechanics. The biomedical applications of AFM in single-cell mechanics provide considerable novel insights into how cell and tissue mechanics affect tumor development and metastasis, contributing much to the communities of biomechanics and biophysics. However, current AFM single-cell mechanical experiments are commonly performed on measuring the elastic properties of cells and studies about the viscoelastic properties of cells are still scarce. In this work, AFM was utilized to measure and analyze the viscoelastic properties of cells. First, the detailed procedure of detecting the viscoelasticity of cells based on AFM indentation technique was established. AFM probe was controlled to perform approach-dwell-retract movement on cells in the vertical direction. During the approach-dwell-retract process, the deflection of AFM cantilever versus time was recorded, which yielded the force-time (F-T) curves. The original F-T curves were then normalized and fitted by two-order Maxwell model, which gave two cellular relaxation times (the first relaxation time τ1 and the second relaxation time τ2). The fitting results showed that the theoretical curve matched the experimental relaxation curve well, indicating that the two-order Maxwell model was suited for characterizing the relaxation behaviors of cells. Second, the established procedure was used to measure the relaxation time of six different types of cells, including mammalian adherent cells, mammalian suspended cells, normal cells, cancerous cells, cell lines cultured in vitro and primary cells prepared from bone marrow and peripheral blood of healthy volunteers. The fitting curves were consistent with the experimental relaxation curves for all of the six types of cells used here, indicating the effectiveness of the presented method for detecting the viscoelastic properties of cells. Besides, the results showed the various cellular relaxation times for the different types of cells. The statistical results showed that the first cellular relaxation times were in the range 0.01−0.03 s, which might correspond to the behaviors of cytoplasm. The second cellular relaxation time was in the range 0.2−0.4 s, which might correspond to the behaviors of the cytoskeleton. Finally, regression analysis was performed on the measured cellular relaxation times, showing the linear relationship between the first cellular relaxation time τ1 and the second cellular relaxation time τ2, and the regression coefficients were variable for different types of cells. The research improves our understanding of cellular viscoelasticity and also provides a novel idea to measure the viscoelastic properties of cells, which will have potential impacts on cell mechanics and biomedicine.


Funded by

国家自然科学基金(61873258,61503372)

中国科学院青年创新促进会项目(2017243)


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  • Figure 1

    (Color online) AFM experimental platform for measuring the viscoelastic properties of cells. (a) Actual photograph of AFM. (b), (c) AFM is controlled to probe individual living cells under the guidance of optical microscopy. (b) Raji cell (a type of mammalian suspended cell). (c) MCF-7 cell (a type of mammalian adherent cell)

  • Figure 2

    (Color online) Schematic diagram of detecting cellular viscoelasticity by controlling AFM probe to perform approach-dwell-retract movement on cells in the vertical direction. (a) The piezoelectric tube vertically drives AFM probe to gradually move to the cell. (b) The AFM probe contacts and indents the cell. (c) The AFM probe dwells on the cell surface to observe the cellular relaxation process. (d) The AFM probe retracts from cell surface after measurement

  • Figure 3

    (Color online) Flow chart of extracting cellular relaxation time from the recorded relaxation curve

  • Figure 4

    Procedure of measuring cellular relaxation time by AFM. (a) AFM height image of living MCF-7 cells. AFM indentation experiments are performed at the central areas (denoted by the green dots) of cells. (b) Original curves recorded during the approach-dwell-retract process of AFM probe performed on MCF-7 cells. The red curve corresponds to the deflection of AFM cantilever and the gray curve corresponds to the vertical position of AFM probe. (c) Original relaxation curve (denoted by the blue dashed box in (b)). (d) Normalized relaxation curve. (e) Fitting the normalized relaxation curve with two-order Maxwell model gives two cellular relaxation times (the first relaxation time τ1 and the second relaxation time τ2). (f) Original curves recorded on the substrate for control experiments. The blue curve is the deflection of AFM cantilever and the gray curve is the vertical position of AFM probe

  • Figure 5

    (Color online) Typical relaxation curves recorded on different types of cells and the Maxwell fitting curves. (a) HEK 293 cells. (b) MCF-7 cells. (c) MDA-MB-231 cells. (d) Raji cells. (e, f) Primary B cells prepared from the bone marrow (e) and peripheral blood (f) from lymphoma patients. The insets in (a)−(f) are the optical images of cells

  • Figure 6

    (Color online) Statistical comparison of the cellular relaxation times of different types of cells. (a) Cellular relaxation time τ1. (b) Cellular relaxation time τ2

  • Figure 7

    Regression analysis of the first relaxation time τ1 and the second relaxation time τ2 of cells showing the linear relationship between τ1 and τ2

  • Table 1   Cellular relaxation times of different types of cells (Mean±SD)

    细胞松弛时间

    HEK293

    MCF-7

    MDA-MB-231

    Raji

    骨髓原代B细胞

    外周血原代B细胞

    松弛时间τ1(s)

    0.0254±0.0043

    0.0244±0.0051

    0.0198±0.0046

    0.0284±0.0072

    0.0277±0.0071

    0.0287±0.0087

    松弛时间τ2(s)

    0.2656±0.0376

    0.3033±0.0654

    0.2706±0.0491

    0.2849±0.0879

    0.2634±0.0609

    0.2978±0.0419

  • Table 2   Linear relationship between the first relaxation time and the second relaxation time of cells

    回归分析

    HEK293

    MCF-7

    MDA-MB-231

    Raji

    骨髓原代B细胞

    外周血原代B细胞

    线性关联

    τ2=11.8665×τ1

    τ2=12.2897×τ1

    τ2=11.1736×τ1

    τ2=11.0472×τ1

    τ2=11.7918×τ1

    τ2=11.1592×τ1

    R2

    0.9494

    0.9244

    0.9098

    0.9459

    0.8979

    0.9504

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