It has been a long-standing effort to study the mechanical aspects of biological systems, such as the movement and structure of living organisms. Historically, however, these efforts were largely limited to physical perspectives. With the emergence of mechanobiology as a distinct field, the importance of biomechanics is increasingly appreciated by biologists, as it has become clear that the mechanical force and stiffness of cells and tissue can impact their biological processes. Unsurprisingly, this shift in perspective is closely connected to advances in enabling technologies for quantifying force and stiffness.

Atomic force microscopy (AFM) is considered the “gold standard” for measuring cell stiffness. Using a tiny tip with a diameter in the nanometer or micrometer range, AFM can perform indentation tests on single cells and locally quantify the apparent Young’s modulus. However, indentation testing requires samples to be placed on top of a rigid substrate, which makes the AFM not applicable to cells suspended in fluid or complex 3D biological tissues.

Over the past two decades, Brillouin microscopy has emerged as a promising technique to address this gap^[1,2]. Brillouin microscopy is based on Brillouin light scattering, an optical phenomenon discovered about a century ago. This inelastic scattering process involves the interaction of photons and phonons. Due to the Doppler effect, photons scattered by phonons carry information about acoustic velocity and, consequently, the elastic longitudinal modulus. Unlike AFM, Brillouin microscopy probes the mechanical properties of the material using only a laser beam, making it a non-contact and non-invasive method. While Brillouin instruments have been available for biological samples since the 1980s, they were only used for single-point quantification rather than imaging because traditional spectrometers were too slow.

The first breakthrough in speed occurred in 2008, when Scarcelli and Yun reported a confocal Brillouin microscope equipped with a new spectrometer that was 100 times faster than traditional ones^[3]. This innovation enabled them to acquire the first-ever Brillouin image of an intraocular lens. Since then, confocal Brillouin microscopy has been broadly used for probing the mechanical properties of many biological samples, including single cells, cellular spheroids, embryos, and tissue slices, with subcellular resolution. While acceptable in some circumstances, the acquisition speed of confocal Brillouin microscopy remains significantly lower than that of other imaging technologies such as fluorescence microscopy. One solution to the speed limitation lies in multiplexing. In this regard, line-scanning Brillouin microscopy pushed the acquisition speed to 1 ms per pixel^[4–6]. This effort has enabled live and time-lapse mechanical imaging of large samples like cellular spheroids and early-stage embryos within practical time frames. Intriguingly, a multiplexing strategy was also adopted in Brillouin light scattering anisotropy microscopy for collecting angle-resolved dispersion in a single shot^[7].

Another solution to the speed limitation lies in the stimulated scattering process. The above-mentioned Brillouin microscopy relies on spontaneous scattering, which is an incoherent process driven by random thermal fluctuation and has very low scattering efficiency (e.g., 10−10). In contrast, stimulated scattering is a coherent process, and the efficiency can reach 1 in theory. In 2020, Remer et al. successfully acquired Brillouin images of live biological samples using stimulated Brillouin microscopy (SBM), which was built upon the interaction of two counterpropagating continuous-wave (CW) laser beams^[8]. However, it was soon recognized that the CW SBM operated in a suboptimal regime of the stimulated gain curve, primarily constrained by the maximum laser power that could be applied without causing photodamage to samples. Shortly thereafter, two groups independently reported a pulsed scheme for SBM^[9,10]. By taking advantage of the high peak power and low duty cycle of laser pulses, pulsed SBM can achieve acquisition speed comparable to CW SBM while consuming much less average power. Nevertheless, since the pulses were generated by modulating a CW laser, further increase in peak power became challenging due to the available power of the laser. This constraint resulted in similar acquisition speed for SBM (i.e., 20 ms per pixel) and spontaneous Brillouin microscopy (i.e., 1–50 ms per pixel).

Recently published in Nature Photonics, the work by Qi et al. further advanced the speed of SBM by developing a much more powerful pulsed laser source^[11]. Different from the previous approach that generated pulses by modulating a 780 nm CW laser, the researchers first generated a nanosecond pulse from a 1560 nm fiber laser using a pulse generator, followed by two-stage pulse amplification and second-harmonic generation [Fig. 1(a)]. In this way, researchers can generate 780 nm laser pulses as short as 6 ns with a peak power of 267 W, which is 100-fold higher than that in prior systems. Combined with an auto-balanced detection system for noise suppression, the new pulsed laser enabled their SBM to achieve an acquisition speed of 200 µs per pixel using only 30 mW of average power on the sample.

This substantial improvement brings Brillouin microscopy one step closer to achieving the goal of imaging at speeds comparable to fluorescence confocal microscopy. Using the new instrument, researchers successfully demonstrated non-perturbative biomechanical imaging of various biological samples, including live single cells, organoids, zebrafish larvae, ovarian follicles, and C. elegans embryos. In addition to its higher speed, the pulsed SBM has better spectral resolution (and, thus, higher mechanical specificity) than spontaneous Brillouin microscopy, though it is slightly worse than that of CW SBM. As previously noted, however, SBM works in transmission mode that requires two counterpropagating laser beams to overlap precisely within the sample [Fig. 1(b)]. This optical configuration restricts imaging to thin (e.g., 100–200 µm) and transparent specimens. In contrast, spontaneous confocal Brillouin microscopy, which works in reflection mode, is not subject to such a limitation.

In summary, this work marks the latest advance in the field of Brillouin microscopy. Looking ahead, this timely technological innovation, together with the recent effort toward the standardization of Brillouin microscopy^[12], is expected to facilitate the widespread adoption of this emerging optical technology in biophysics and mechanobiology.