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Label-free, non-contact imaging with mechanical contrast and optical sectioning is a substantial challenge in microscopy. Spontaneous Brillouin scattering microscopy meets this challenge, but encounters a trade-off between acquisition speed and the specificity for biomechanical constituents with overlapping Brillouin bands. Stimulated Brillouin scattering microscopy overcomes this trade-off and enables the cross-sectional imaging of live Caenorhabditis elegans at the organ and subcellular levels, with both elasticity and viscosity contrasts at high specificity and with practical recording times. Stimulated Brillouin scattering microscopy overcomes the trade-off between acquisition speed and spectral resolution in spontaneous Brillouin scattering microscopy and allows visualization of elasticity and viscosity, as shown in C. elegans .

Single particle tracking in three dimensions is an indispensable tool for studying dynamic processes in various disciplines, including material sciences, physics, and biology, but often shows anisotropic three-dimensional spatial localization precision, which restricts the tracking precision, and/or a limited number of particles that can be tracked simultaneously over extended volumes. Here we developed an interferometric, three-dimensional fluorescence single particle tracking method based on conventional widefield excitation and temporal phase-shift interference of the emitted, high-aperture-angle, fluorescence wavefronts in a greatly simplified, free-running, triangle interferometer that enables tracking of multiple particles at the same time with <10-nm spatial localization precision in all three dimensions over extended volumes (~35 × 35 × 2 μm 3 ) at video rate (25 Hz). We applied our method to characterize the microenvironment of living cells and up to ~40 μm deep in soft materials. Conventional widefield excitation and temporal phase-shift fluorescence interference in a basic 4π-interferometer enable video rate, multiple particle tracking with isotropic, 3D precision over large volumes in cells and deep in soft materials.

Stimulated Brillouin scattering (SBS) microscopy is a nonlinear all-optical imaging method that provides mechanical contrast based on the interaction of laser radiation and acoustical vibrational modes. Featuring high mechanical specificity and sensitivity, three-dimensional sectioning, and practical imaging times, SBS microscopy with (quasi) continuous wave excitation is rapidly advancing as a promising imaging tool for label-free visualization of viscoelastic information of materials and living biological systems. In this article, we introduce the theory of SBS microscopy and review the current state-of-the-art as well as recent innovations, including different approaches to system designs and data analysis. In particular, various performance parameters of SBS microscopy and its applications in the life sciences are described and discussed. Future perspectives for SBS microscopy are also presented.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The field of Brillouin microscopy and imaging was established approximately 20 years ago, thanks to the development of non-scanning high-resolution optical spectrometers. Since then, the field has experienced rapid expansion, incorporating technologies from telecommunications, astrophotonics, multiplexed microscopy, quantum optics and machine learning. Consequently, these advancements have led to much-needed improvements in imaging speed, spectral resolution and sensitivity. The progress in Brillouin microscopy is driven by a strong demand for label-free and contact-free methods to characterize the mechanical properties of biomaterials at the cellular and subcellular scales. Understanding the local biomechanics of cells and tissues has become crucial in predicting cellular fate and tissue pathogenesis. This Primer aims to provide a comprehensive overview of the methods and applications of Brillouin microscopy. It includes key demonstrations of Brillouin microscopy and imaging that can serve as a reference for the existing research community and new adopters of this technology. The article concludes with an outlook, presenting the authors' vision for future developments in this vibrant field. The Primer also highlights specific examples where Brillouin microscopy can have a transformative impact on biology and biomedicine.

Optical imaging techniques with mechanical contrast, including passive microrheology, optical coherence elastography and Brillouin microscopy, are critical for material and biological discovery owing to their less perturbative nature compared with traditional mechanical imaging methods. An emerging optical microscopy approach for mechanical imaging is stimulated Brillouin scattering microscopy, which has been shown to be useful for biomechanical imaging with high sensitivity and specificity. However, the excitation energy used is high and the temporal resolution remains limited by the need to acquire full spectra. Here we develop Brillouin gain microscopy that detects the Brillouin gain at a specific mechanically contrasting frequency corresponding to a Brillouin acoustic-vibrational mode of interest in the sample. Brillouin gain microscopy affords a 200-fold improvement in temporal resolution compared with stimulated Brillouin scattering microscopy, down to 100 μs at excitation energy as low as 23 μJ. Using Brillouin gain microscopy, we demonstrate cross-sectional, all-optical mechanical imaging of materials as well as of the structure and dynamics in living systems with low excitation energy and at high temporal resolution. By measuring the Brillouin gain only at mechanical frequencies of interest, Brillouin gain microscopy enables Brillouin imaging with a temporal resolution of 100 µs with excitation energies of 23 µJ on biological samples.

Conventional low-magnification phase-contrast microscopy is an invaluable, yet a qualitative, imaging tool for the interrogation of transparent objects over a mesoscopic millimeter-scale field-of-view in physical and biological settings. Here, we demonstrate that introducing a compact, unbalanced phase-shifting Michelson interferometer into a standard reflected brightfield microscope equipped with low-power infinity-corrected objectives and white light illumination forms a phase mesoscope that retrieves remotely and quantitatively the reflection phase distribution of thin, transparent and weakly scattering samples with high temporal (1.38 nm) and spatial (0.87 nm) axial-displacement sensitivity and micrometer lateral resolution (2.3 μm) across a mesoscopic field-of-view (2.25 × 1.19 mm 2 ). Using the system, we evaluate the etch-depth uniformity of a large-area nanometer-thick glass grating and show quantitative mesoscopic maps of the optical thickness of human cancer cells without any area scanning. Furthermore, we provide proof-of-principle of the utility of the system for the quantitative monitoring of fluid dynamics within a wide region.

Brillouin microscopy is an emerging technique for all-optical biomechanical imaging without the need for physical contact with the sample or for an external mechanical stimulus. However, Brillouin microscopy often retrieves a single, averaged Brillouin frequency shift of all the materials in the sampling volume, introducing significant spectral artifacts in the Brillouin shift images produced. To enable the identification between single- and multi-peak Brillouin signatures in the sample voxels, we developed here a new physics-driven model selection framework based on information theory and an overfit Brillouin water peak threshold. The model selection framework was applied to Brillouin data of NIH/3T3 cells measured by stimulated Brillouin scattering microscopy, facilitating the improved quantification of the Brillouin shift of different regions in the cells, and substantially minimizing spectral artifacts in their Brillouin shift images.

Optical imaging with mechanical contrast is critical for material and biological discovery since it allows contactless light-radiation force-excitation within the sample, as opposed to traditional mechanical imaging. Whilst optical microscopy based on stimulated Brillouin scattering (SBS) enables mechanical imaging of materials and living biological systems with high spectrospatial resolution, its temporal resolution remains limited. Here, we develop Brillouin gain microscopy (BGM) with a 200-fold higher temporal resolution by detecting the Brillouin gain at a mechanically contrasting frequency in the sample with high sensitivity. Using BGM, we demonstrate mechanical imaging of materials, living organisms and cells at high spectro-spatiotemporal resolution.

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 high-frequency viscoelastic response to osmotic stress in the head of living worms, and in vivo mechanical contrast mesoscopy of developing nematodes