Brillouin microscopy can assess mechanical properties of biological samples in a three-dimensional (3D), all-optical and hence non-contact fashion, but its weak signals often lead to long imaging times and require an illumination dosage harmful for living organisms. Here, we present a high-resolution line-scanning Brillouin microscope for multiplexed and hence fast 3D imaging of dynamic biological processes with low phototoxicity. The improved background suppression and resolution, in combination with fluorescence light-sheet imaging, enables the visualization of the mechanical properties of cells and tissues over space and time in living organism models such as fruit flies, ascidians and mouse embryos.

Brillouin microscopy (BM) can be used to assess the mechanical properties of biological samples in a 3D, all-optical, and hence non-contact fashion, but its weak signals require long imaging times and illumination dosages harmful to living organisms. Here, we present a line-scanning Brillouin microscope optimized for fast and high-resolution live-imaging of dynamic biological processes with low photo-toxicity. In combination with fluorescence light-sheet imaging, we demonstrate the capabilities of our microscope to visualize the mechanical properties of cells and tissues over space and time in living model organisms such as fruit flies, ascidians, and mouse embryos.

Brillouin microscopy is an emerging optical elastography technique capable of assessing mechanical properties of biological samples in a 3D, all-optical and hence non-contact fashion. The typically weak Brillouin scattering signal can be substantially enhanced via a stimulated photon-phonon process, which improves the signal-to-background ratio (SBR) as well as provides higher spectral resolution. However, current implementations of stimulated Brillouin spectroscopy (SBS) require high pump powers, which prohibit applications in many areas of biology, especially when studying photosensitive samples, or when live-imaging in 3D and/or over extended time periods. Here, we present a pulsed SBS scheme that takes full advantage of the non-linearity of the pump-probe interaction in SBS. In particular, we show that through quasi-pulsing and diligent optimization of signal detection parameters, the required pump laser power can be decreased ~20-fold without affecting the signal levels or spectral precision. Moreover, we devise a custom analysis approach that facilitates the analysis of complex, multi-peaked Brillouin spectra in order to harness the high spectral resolution of SBS for the specific identification of biomechanical components inside the point-spread function of the microscope. We then demonstrate the low-phototoxicity and high-specificity of our pulsed SBS approach by imaging sensitive single cells, zebrafish larvae, and mouse embryos as well as adultC. eleganswith sub-cellular detail. Furthermore, our method permits observing the mechanics of organoids andC. elegansembryos over time. We expect that the substantially lower photo-burden and improved SBR of pulsed SBS will facilitate studying biomechanics in 3D at high spatio-temporal resolution in living biological specimens in a non-invasive manner, opening up exciting new possibilities for the field of mechanobiology.

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