Noncontact label-free biomechanical imaging is a crucial tool for unraveling the mechanical properties of biological systems, which play critical roles in the fields of engineering, physics, biology and medicine; yet, it represents a significant challenge in microscopy. Spontaneous Brillouin microscopy meets this challenge, but often requires long acquisition times or lacks high specificity for detecting biomechanical constituents with highly overlapping Brillouin bands. We developed stimulated Brillouin scattering (SBS) microscopy that provides intrinsic noncontact biomechanical contrast and generates mechanical cross-sectional images inside large specimens, with high mechanical specificity and pixel dwell times that are >10-fold improved over those of spontaneous Brillouin microscopy. We used SBS microscopy in different biological applications, including the quantification of the high-frequency complex longitudinal modulus of the pharyngeal region of live wild-type Caenorhabditis elegans nematodes, imaging of the variations in the highfrequency viscoelastic response to osmotic stress in the head of living worms, and in vivo mechanical contrast mesoscopy of developing nematodes. Main TextLabel-free biomechanical imaging has long used a variety of techniques, including atomic-force microscopy, multiphoton microscopy, and optical coherence elastography [1][2][3][4][5][6] , that obtain mechanical images with high spatial resolution, but require contact or external mechanical stimulation of the sample. Spontaneous Brillouin microscopy 7-20 circumvents these requirements by measuring the so-called Brillouin shifts B and linewidths , which are the frequency shifts and linewidths of light backscattered inelastically from gigahertz-frequency longitudinal acoustic phonons characteristic to the different viscoelastic constituents of the material. However, spontaneous Brillouin microscopy often demands long acquisition times due to the low efficiency of spontaneous Brillouin scattering in biological matter, or suffers from limited mechanical specificity because of the relatively low spectrometer resolution of spontaneous Brillouin microscopes, making it difficult to specifically detect biomechanical constituents with highly overlapping Brillouin bands.
Stimulated Brillouin scattering (SBS) microscopy is emerging as a promising approach for mechanical imaging in biological settings. It is based on a spectroscopic backscattering SBS setup, but with scanning of the sample and using higher apertures of the excitation and collection optics for adequate spatial sampling. Here, we provide direct experimental measurements and theoretical predictions of the aperture-induced spectral effects of SBS microscopy in water—a key constituent of biological systems. It is shown that with increasing numerical aperture (NA), the Brillouin frequency shift and peak gain decrease, while the Brillouin linewidth broadens asymmetrically with the commencing of an extended tail in the low frequency region for NA > ∼0.5. Further, significant distortions of the Brillouin spectral line shape are predicted for NAs close to 1, affecting the ability to retrieve spectral parameters of the Brillouin medium precisely and accurately.
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