Confocal Brillouin microscopy for three-dimensional mechanical imaging

Abstract

Acoustically induced inelastic light scattering, first reported in 1922 by Brillouin1, allows non-contact, direct readout of the viscoelastic properties of a material and has widely been investigated for material characterization2, structural monitoring3 and environmental sensing4. Extending the Brillouin technique from point sampling spectroscopy to imaging modality5 would open up new possibilities for mechanical imaging, but has been challenging because rapid spectrum acquisition is required. Here, we demonstrate a confocal Brillouin microscope based on a fully parallel spectrometer-a virtually imaged phased array-that improves the detection efficiency by nearly 100-fold over previous approaches. Using the system, we show the first cross-sectional Brillouin imaging based on elastic properties as the contrast mechanism and monitor fast dynamic changes in elastic modulus during polymer crosslinking. Furthermore, we report the first in situ biomechanical measurement of the crystalline lens in a mouse eye. These results suggest multiple applications of Brillouin microscopy in biomedical and biomaterial science.

Figure 1. Principle and schematic of the experimental set-up

a, Illustration of Brillouin light scattering originating from acoustic phonons (waves). b, Schematic of spectrometer based on a VIPA. c, Schematic of the confocal Brillouin microscope system.

Figure 2. Brillouin spectra of various samples

a, Typical CCD output obtained from distilled water. b, The width of the transmission profile (dotted line) matched to the FSR to maximize the signal. c, Calibrated optical spectra: experimental data (red circles); overall curve fit based on lorentzian functions (solid green line); and a curve fit to the Stokes Brillouin spectrum (blue dashed line). The measured Brillouin shifts and linewidths are 7.46 GHz and 0.79 GHz for water, 5.57 GHz and 0.46 GHz for methanol, 9.23 GHz and 1.97 GHz for benzyl alcohol and benzyl benzoate (BABB), and 15.6 GHz and 0.26 GHz for acrylic glass (Plexiglas).

Figure 3. Cross-sectional Brillouin image of an intraocular lens

a, Picture of the sample used. To visualize the outline of the lens clearly, the picture was taken before filling the cuvette with index-matching viscous polymer. The imaged area is 3.2 mm (x) × 1.3 mm (z). b, Image based on measured frequency shifts, corresponding to a cross-sectional map of elastic modulus. c, Image created by using Brillouin scattering magnitudes as contrast. d, Representative cross-sectional line profiles taken along the dotted line in b and c. Arrows indicate which y axis scale applies.

Figure 4. Real-time monitoring during UV-induced crosslinking of polymer

Four distinct phases are observed, characterized by a constant Brillouin shift before curing (−100 s to 0 s), dynamic changes during UV illumination (0 s to 100 s), an increase during post-illumination crosslinking (100 s to 250 s), and a steady state after being fully cured (after 24 h). The elastic modulus was calculated from the measured Brillouin frequency shift.

Figure 5. In situ characterization of the crystalline lens in a mouse eye

a, Left: Schematic of the murine eye. Right: images of the crystalline lens extracted after measurement. The arrow indicates the beam entrance direction. b, Brillouin frequency shifts measured at various depths along the central optic axis (blue circles), showing a twofold increase from the outer layers (cortices) towards the lens centre (nucleus). Error bars represent the measurement uncertainty. C, cornea; A, aqueous humour; L, lens; V, vitreous humour; and R, retina.