Three dimensional biomechanical imaging by stimulated Brillouin scattering microscopy
Development of a stimulated Brillouin scattering microscope enables viscoelasticity imaging of live Caenorhabditis elegans in three dimensions with high mechanical specificity and practical recording times.
We started to develop stimulated Brillouin scattering (SBS) microscopy about five years ago. Our first SBS spectrometer was published in Optics Letters , following resubmission. It was based on schemes of SBS spectroscopy devised in the nineties, still with the important difference that our SBS spectrometer was aimed at high-sensitivity mechanical measurements not only in liquids but also in scattering media in the biological window (780 nm). We were excited about the initial results, but recognized that in order to convince the broad scientific community of its importance it would be necessary to convert the SBS spectrometer into a microscope and ascertain its applicability to an important biomechanical imaging application.
To this end, we first had to improve the acquisition time of the spectrometer substantially without compromising the precision of the measurements. We came up with the idea of using an atomic filter in the detector instead of the typical schemes of noise rejection used in SBS spectroscopy. This enabled to reduce unwanted noise including back reflections into the detector while increasing the SBS power recorded. The idea worked well, as described in our publications in APL Photonics  and in the Journal of Visualized Experiments . Yet, the acquisition time of a SBS spectrum (10 ms in water) was a bit longer than desired (few ms). To further decrease the acquisition time while preserving a high measurement precision level, we optimized the detected SBS power (by working in a true backscattering geometry) and the detected signal-to-noise ratio (by operating in the shot-noise limit) . These improvements allowed us to finally initiate SBS imaging in biological settings with practical acquisition rates.
We chose to image Caenorhabditis elegans worms because of their importance in biomedical research and since they have not been still interrogated by Brillouin microscopy. Many months were needed to develop and optimize the sample mounting and preparation protocol as well as the imaging procedure and the phototoxicity assay. The latter were further extended to SBS imaging at the subcellular level and to phototoxicity assessment at the molecular level during the review process, which greatly improved this work.
Originally, we focused on the analysis of the frequency shift in the SBS spectra of the worms, which is related to the high-frequency elasticity of the sample. Quite quickly we realized that the high sensitivity and the high spectral resolution SBS data allow us to distinguish between spectral components of different mechanical constitutes of the sample as well as to retrieve reliably and robustly also the linewidth and the peak gain of these spectra, which are related to the high-frequency viscosity of the sample. Importantly, after extracting all these spectral parameters, we were able to evaluate also the mass density of the sample given its refractive index. These advances provided a means to overcome the tradeoff between imaging speed and spectral resolution (or mechanical specificity) in conventional Brillouin microscopy and enabled to obtain, in practical measurements times, a unique three dimensional image dataset of the mechanical properties of live Caenorhabditis elegans, which up to now was impossible to achieve with conventional approaches.
We hope that this work will encourage scientists to further develop SBS microscopy and to use it for new discoveries in the life and medical sciences.
 Remer, I. & Bilenca, A. Background-free Brillouin spectroscopy in scattering media at 780 nm via stimulated Brillouin scattering. Opt. Lett. 41, 926–929 (2016).
 Remer, I. & Bilenca, A. High-speed stimulated Brillouin scattering spectroscopy at 780 nm. APL Photonics 1, 061301 (2016).
 Remer, I., Cohen, L. & Bilenca, A. High-speed continuous-wave stimulated
Brillouin scattering spectrometer for material analysis. J. Vis. Exp. 127,
 Remer, I., Shaashoua, R., Shemesh, N., Ben-Zvi, A. & Bilenca, A. High-sensitivity and high-specificity biomechanical imaging by stimulated Brillouin scattering microscopy. Nat Methods (2020).