Researchers have applied line-scanning Brillouin microscopy (LSBM), a microscopy technique based on Brillouin scattering, to visualize the mechanical properties of living cells over space and time, and to provide fast 3D imaging with low phototoxicity. Researchers at the European Molecular Biology Laboratory (EMBL) developed the approach, and used it to noninvasively track the mechanical properties of developing embryos at high speed and resolution. The mechanical properties of a cell affect processes as diverse as embryonic development, tissue integrity, and the pathophysiology of diseases such as cancer. The methods used for measuring these properties are typically invasive and liable to damage living cells and tissues. More than 100 years ago, French physicist Léon Brillouin predicted that when light is shone on a material, it interacts with the naturally occurring thermal vibrations within it, exchanging some energy in the process. This, in turn, influences how the light is scattered. Measuring the spectrum of the scattered light will enable certain physical characteristics of that material. Microscopy based on this effect, Brillouin scattering, is useful for noninvasively viewing and assessing tissue mechanics. However, traditional Brillouin microscopy is slow and requires lengthy light exposures that can lead to cell damage. Instead of collecting information from one point at a time, LSBM uses a line-scanning approach to enable multiplexed signal acquisition. This allows the system to simultaneously sense hundreds of points and their spectra in parallel, which increases imaging speed by at least 100×. The researchers used LSBM to live-image the 3D mechanical properties of developing embryos in fruit flies, mice, and a marine organism. The system provided a field of view of about 186 × 165 × 172 µm, with spatial resolution down to 1.5 µm and temporal resolution down to 2 minutes. The researchers followed the dynamic mechanical changes in the developing embryos, in all three species, in 3D, and over a timescale of several hours. “We often tend to think of cells or tissues only in terms of their biological properties,” said Robert Prevedel, who led the research. “However, cells and tissues also have rich ‘mechanical’ lives. And these physical properties can help determine their biological function.” Line-scanning Brillouin microscopy (LSBM) can be used to noninvasively study developing embryos in 3D and across time, providing new biological insights. Courtesy of Joana Gomes Campos de Carvalho/EMBL. LSBM’s use of a dual-objective system in a 90° configuration and near-infrared illumination ensures minimal photodamage and phototoxicity. The overall design is optimized for high spatiotemporal resolution and high signal-to-noise ratio to enable long-term mechanical imaging of biological specimens with subcellular resolution. In addition, LSBM can be integrated with light-sheet microscopy (LSM) to enable simultaneous visualization of biomolecules using fluorescence. An in-built, concurrent selective plane illumination microscopy (SPIM) fluorescence imaging modality allows 3D, fluorescence-guided Brillouin image analysis, and it aids in data interpretation and in correlating and assigning mechanical properties to different molecular constituents or tissue regions. LSBM is also equipped with a GPU (graphics processing unit)-optimized, fast numerical fitting routine for real-time spectral data analysis and visualization. Using the technique, the researchers showed that a higher Brillouin shift is a common feature of tissue folding, independent of the geometry of the contractile domain. They visualized subcellular structures with different mechanical properties, as well as differential tissue mechanics deep inside an embryo. These observations, made in 3D without the need for tissue sectioning or invasive injections, represent a milestone for mechanobiology research, according to the researchers. LSBM, they said, provides more than a twentyfold improvement in volume imaging speed and lowers the illumination energy per pixel by at least tenfold, compared with previous implementations. The method can be used to reveal previously unknown biological mechanisms, said team member Juan Manuel Gomez. The low phototoxicity and improved acquisition speed of LSBM could lead to applications for imaging mechanical changes during development together with, for example, cytoskeletal or cell fate-specific fluorescence reporters, or by correlating 3D mechanical with molecular, genetic, or ultrastructural data sets. This, in turn, could provide insight into the interplay between genetics, biochemical signaling, and the role of biomechanics in animal development. The research was published in Nature Methods (www.doi.org/10.1038/s41592-023-01822-1).