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Noncontact three-dimensional mapping of intracellular hydromechanical properties by Brillouin microscopy

Abstract

Current measurements of the biomechanical properties of cells require physical contact with cells or lack subcellular resolution. Here we developed a label-free microscopy technique based on Brillouin light scattering that is capable of measuring an intracellular longitudinal modulus with optical resolution. The 3D Brillouin maps we obtained of cells in 2D and 3D microenvironments revealed mechanical changes due to cytoskeletal modulation and cell-volume regulation.

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Figure 1: Principle and validation of Brillouin microscopy.
Figure 2: Brillouin microscopy measurements of mechanical changes in vitro and in a cell.
Figure 3: Effect of the extracellular matrix on cell stiffness.

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Acknowledgements

We thank A.C. Martin, F.M. Mason, E. Moeendarbary, M.C. Gather and K. Franze for helpful discussions, as well as H. Oda, K. Sawicki and K. Berghaus for help with hydrogel measurements. This work was supported in part by the National Institutes of Health (grants K25-EB015885 and R21-EY023043 to G.S.; grants R01-EY025454 and P41-EB015903 to S.H.Y.; and grant R33 CA174550 to R.D.K.), the National Science Foundation (grant CBET-0853773 to S.H.Y.) and a Human Frontier Science Program Young Investigator Grant (RGY0074/2013 to G.S.).

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Authors

Contributions

G.S. and S.H.Y. conceived the project. G.S., W.J.P., R.D.K. and S.H.Y. devised the research plan. G.S. developed the instrument and performed the experiments. W.J.P. and K.P. developed cell protocols and performed cell-related control measurements. H.T.N., W.J.P. and A.J.G. designed and performed indentation experiments. G.S., W.J.P. and S.H.Y. wrote the manuscript with input from all other authors.

Corresponding authors

Correspondence to Giuliano Scarcelli or Seok Hyun Yun.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Longitudinal modulus.

Illustration of cytoplasm, axial stress and axial compression of the medium. The ratio of stress to strain defines the longitudinal modulus. The Brillouin frequency shift is related to the local longitudinal modulus of the medium.

Supplementary Figure 2 Apodized VIPA spectrometer.

(a) Schematic of the double-stage apodized VIPA spectrometer. (b) As a result of multiple reflections within the etalon, the beam output from the VIPA has an exponential profile. (c) Measured VIPA output beam profile (solid lines) shows an exponential envelope. (d) The gradient intensity filter converts the exponential profile to an apodized beam shape that has reduced high spatial-frequency components. (e) Measured VIPA output beam profile (solid lines) compared to the exponential envelope without the filter (dotted line). (f) The resulting spectrum shows increased spectral extinction by ~15 dB at 7.5 GHz away from the elastic scattering peaks and >20 dB in a frequency band between 8 and 20 GHz.

Supplementary Figure 3 Calibration and analysis of Brillouin spectrum.

Spectrometer calibration. (a) CCD frame obtained from a sample. (b) CCD frame from two reference materials. (c,d) Lorentzian curve fit (blue) to the measured data (red). ΩX is determined from ΩACR-S and ΩMETHA.

Supplementary Figure 4 Accuracy of Brillouin-shift estimation.

(a) Logarithmic plot of the instrument SNR versus the total number of photons collected (proportional to the product of illumination power and acquisition time). Circles: measurement data; dotted line: linear fit. (b) Representative distribution of Brillouin shifts estimated from N = 300 sequential spectra of a methanol sample recorded at 4 mW and 50 ms.

Supplementary Figure 5 Computation of longitudinal modulus.

Brillouin-measured longitudinal modulus versus AFM-based Young’s moduli of NIH 3T3 fibroblast cells at different osmotic pressure conditions. The magenta dots take into account the variation of ρ/n2 between the samples; the turquoise dots assume constant ρ/n2. Lines are linear fits.

Supplementary Figure 6 AFM-based micro-indentation to estimate Young’s modulus.

The Young’s modulus of fibroblasts was measured via AFM-based indentation in different sucrose concentrations (mean ± s.e.m.; number of cells per concentration N ≥ 8; the Young’s modulus of each cell is the average of the Young’s modulus at M 4 indentation sites; *P < 0.05 using t-test).

Supplementary Figure 7 AFM force-displacement curves.

The force-displacement curves for cells at different concentrations of sucrose in the cell medium.

Supplementary Figure 8 Hertz model fit of AFM indentation.

A typical force-displacement curve (black) and the corresponding best fit curve (red) from the thin-layer Hertz model are shown for a cell in 300 mM sucrose.

Supplementary Figure 9 Brillouin-mechanical validation in polyacrylamide gels.

A log-log linear relation of the longitudinal modulus and shear modulus of polyacrylamide (PA) gels. The measurement data are for cross-linked PA gels at three different polymer concentrations (5%, 7.5%, 10% wt) and several concentrations of bis-acrylamide cross-linker (0.1–0.6% wt).

Supplementary Figure 10 In situ measurement of PEG hydrogel cross-linking.

(a) Real-time monitoring of photopolymerization induced by UV light illumination (0–10 s), which reveals a delayed onset of polymerization (4 s) and increased degree of cross-linking during and after illumination. Control, without UV illumination. Inset, polymerization induction time at various UV power levels; circles, experimental data; dashed line, theoretical model. (b) Brillouin shift of PEG at various concentrations before (squares) and after (diamonds) photopolymerization. Error bars, s.e.m. (N = 10).

Supplementary Figure 11 Brillouin shift of cells at different plating times.

Average Brillouin shift of NIH 3T3 fibroblasts cultured on top of polyacrylamide gels and measured 8 h after plating time (N = 6) and 12 h after plating time (N = 3). For comparison, cells measured 18 h (N = 8) and 24 h (N = 12) after plating are shown.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–11 and Supplementary Notes 1–8 (PDF 1439 kb)

Supplementary Software

Matlab code to analyze data. (ZIP 2 kb)

Supplementary Data

Raw data to run Matlab code. (ZIP 1284 kb)

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Scarcelli, G., Polacheck, W., Nia, H. et al. Noncontact three-dimensional mapping of intracellular hydromechanical properties by Brillouin microscopy. Nat Methods 12, 1132–1134 (2015). https://doi.org/10.1038/nmeth.3616

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