Brillouin microscopy is a technique for mechanical characterization of biological material without contact at high three-dimensional resolution. Here, we introduce dual line-scanning Brillouin microscopy (dLSBM), which improves acquisition speed and reduces irradiation dose by more than one order of magnitude with selective illumination and single-shot analysis of hundreds of points along the incident beam axis. Using tumor spheroids, we demonstrate the ability to capture the sample response to rapid mechanical perturbations as well as the spatially resolved evolution of the mechanical properties in growing spheroids.
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The authors declare that all data supporting the findings of this study are available in the paper and its Supplementary Notes 1 and 2. Source data are provided with this paper.
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The authors thank G. Zanini for help with the photodamage experiment and J. Xu and S. He for preparing the PDMS sphere. This work was supported by grants from the National Science Foundation (DBI-1942003, CMMI-1929412 to G.S.), the National Institutes of Health (R21CA258008 to G.S., R01EY028666 to G.S., R01HD095520 to G.S., K25HD097288 to J.Z.) and the Intramural Research Program of the National Cancer Institute (to K.T.), the American Cancer Society Institutional Research Grant (1816016) to J.Z. and a Wayne State University Research Grant to J.Z.
J.Z., M.N and G.S. are inventors of patents related to the Brillouin technology. G.S. is a consultant for Intelon Optics. K.T. declares no competing interests.
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Extended Data Fig. 1 Detailed schematic of the illumination beam.
a, M, mirror; L1-L2, spherical lens with focal length of 20 mm and 80 mm, respectively; Obj1-Obj2, objective lenses (4×/0.1NA). b, Measured laser spectra with and without Bragg filters. c, Extinction spectrum of the Fabry-Perot etalon used in the setup. The value of zero in b and c represents the wavelength and frequency of the laser light, respectively.
Extended Data Fig. 2 Dual-line illumination.
Brillouin image sections (left panels) and the averaged profile (right panels) along illumination direction of left-line illumination (a), right-line illumination (b), and combined dual-line illumination (c). The red arrow indicates the illumination direction of the beam line. Scale bar, 10 µm. The color bar represents the Brillouin shift with the unit of GHz. The center and error band represents the mean value + /− SD.
Extended Data Fig. 3 Brillouin shift gradient caused by the mismatch of refractive index.
a, Brillouin image of a PDMS sphere immersed in 1% agarose gel. Arrow indicates the propagation of illumination beam. Black dashed line indicates the location of interest for b. Scale bar is 10 µm. b, Shift profile across the PDMS sphere shows the gradient along illumination direction. c, Theoretical calculation model. ‘A’ represent an arbitrary point on the illumination axis, θ is the actual scattering angle collected by the spectrometer at 90° geometry, and α is the azimuthal angle of the sphere. d, The shift gradients from the calculation and the experiment.
Extended Data Fig. 4 Characterization of Brillouin spectrometer.
a, Exemplary raw Brillouin spectrum of DI water acquired by the spectrometer of the dLSBM setup with single-line illumination. b, Brillouin shift is extracted by fitting the spectrum of a single pixel with a Lorentzian function. Peaks represent the Stokes and anti-Stokes components of the Brillouin frequency. c, Representative histogram distribution of the estimated Brillouin shift of the same point after repeated acquisition (n = 300). The solid line is fitting result by Gaussian profile. The standard deviation of the estimated Brillouin shift is 8 MHz. d, Spectral precision of all points on illumination axis. The average is 9.8 MHz. e, Signal-to-noise (SNR) ratio against light energy of dLSBM spectrometer for water sample. The fitted line has a slope of 0.49.f, The measured spectrum of the laser. Circles are measured data, and solid line is Lorentzian fit. g, Spectral precision against light energy of the dLSBM spectrometer in water and spheroid sample. h, Comparison of dLSBM and confocal Brillouin microscopy (CBM) regarding the spectral precision against total light dose for 3D mapping of 100 × 200 × 10 pixels. water is used as sample. The data point of CBM is adapted from ref. 27, and the dash line indicates the shot noise limited operation.
Extended Data Fig. 5
Brillouin image reconstruction of the 3D mapping of a spheroid.
Extended Data Fig. 6 Characterization of the spatial resolution of the dLSBM setup.
a, Brillouin measurement across the interface of PDMS and water. b, Measurement of Rayleigh scattering from a 0.5 µm bead.
Extended Data Fig. 7 Comparison of dLSBM and CBM.
a-e, five independent spheroids measured by dLSBM (NA = 0.3) and CBM (NA = 0.4). Scale bar is 5 µm. For each spheroid, the Brillouin shifts of all pixels from the image was plotted into a histogram. The histogram was then fitted by a combination of two Gaussian distributions.
Extended Data Fig. 8 Analysis of subcellular mechanical information of spheroids.
a-d, Co-registered fluorescence and Brillouin images of four representative spheroids. Each sub figure shows the bright field image, fluorescence image of the nuclei, and the Brillouin image acquired by the CBM, respectively. Red line in the image outlines the profile of the spheroid. The plots of ‘H-peak 2’ is the right peak extracted from the curve fitting of the Brillouin shift histogram. The plots of ‘fluor. nucleus’ represents Brillouin shift of the nucleus region. e, Results of all the spheroid samples (n = 14). n.s.: not statistically significant (p = 0.6039). Statistical significance is determined by performing two-sided two-sample t-test, and no adjustment was made. In all boxplots, the central mark indicates the median, and the bottom and top edges indicate the 25th and 75th percentiles, respectively. The whiskers extend to the most extreme data points not considered outliers.
Extended Data Fig. 9 Effect of light illumination on the viability and growth rate of the spheroids.
a, Fluorescent images of the spheroids in control and illuminated groups. Live cells fluoresce in green (Calcein-AM) and dead cells fluoresce in red (EthD-1). b, Spheroid area against day of growth. The upper panel is the representative time-lapse image of a spheroid that is illuminated on Day 2. The points in the lower panel represent the area of spheroids over time. Black curves are best fit for exponential growth. Control group (n = 10), Illuminated group (n = 7).
Extended Data Fig. 10 Temporal change of the projection area of the spheroids under osmotic shock.
Hyperosmotic shock (n = 5), no shock (n = 6), and hypoosmotic shock (n = 5). Error bound indicates ± s.e.m.
Supplementary Notes 1 and 2, Supplementary Figs. 1–3.
Supplementary Data 1
Source data for Supplementary Fig. 2.
Supplementary Data 2
Source data for Supplementary Fig. 3.
Supplementary Code 1
Matlab code for spectrum analysis.
Supplementary Code 2
Matlab code for image fusion.
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Source data for Fig. 1e,g,h.
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Source data for Fig. 2d,e,g–j.
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Zhang, J., Nikolic, M., Tanner, K. et al. Rapid biomechanical imaging at low irradiation level via dual line-scanning Brillouin microscopy. Nat Methods 20, 677–681 (2023). https://doi.org/10.1038/s41592-023-01816-z
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