Cells in the mechanical spotlight

Brillouin microscopy, a technique that visualizes the transfer of energy from photons of light to phonons of mechanical energy (1), has emerged as a useful tool in the study of biological mechanics. Unlike other approaches to mechanical characterization, such as optical tweezers, atomic force microscopy (AFM), or rheology, Brillouin microscopy does not require physical contact between a probe and the specimen. Instead, the mechanical properties of the specimen are characterized by observing optical frequency shifts in the Brillouin scattered light from the specimen. These frequency shifts arise from energy transfer between the photons of the electromagnetic field and acoustic phonons of vibrational energy within the specimen. The magnitude of this Brillouin shift (ν_B) is related to the wavelength of incident light (λ), and the density ($\text{ρ}$), refractive index ($\text{n}$), and longitudinal modulus (Μ) of the material according to Eq. 1, while the linewidth of the Brillouin peak is related to the wavelength, density, refractive index, and viscosity (η) of the specimen according to Eq. 2 (2). The wavelength of the incident light is an experimentally controlled parameter; thus, under conditions wherein the specimen density and refractive index are known, or where the relative changes in these values can be controlled or assumed to be constant within the specimen, then the viscoelastic properties of the specimen can be estimated, or relative changes compared, by observing the magnitude of the Brillouin frequency shift and the width of this peak (2).

$$M=\frac{{\lambda }^2}{4}\frac{\text{ρ}}{n^2}{{\nu }_B}^2$$ (1)

$$\eta =\frac{{\lambda }^2}{2}\frac{\rho }{n^2}{\Delta }_B$$ (2)

Optical measurements of a sample’s mechanical properties are advantageous. Such indirect approaches reduce damage to the specimen and provide access to mechanical measurements where direct contact is not possible as with cells embedded in a 3D matrix. The principle of Brillouin microscopy was first demonstrated in the early 20th century and then subsequently used for measuring the Brillouin shift of the extracellular matrix, DNA films, and various tissues (3). With pixel dwell times on the order of 40–100 ms (compared with ∼1 μs for a typical laser scanning confocal), it is roughly 100,000× slower than confocal microscopy. Its spatial resolution is limited by the mean free path of acoustic photons in the specimen to approximately 1 μm (4). With these advantages and tradeoffs in mind, Brillouin microscopy has been successfully used to study the mechanical properties of live cells and tissues (3). In particular, Brillouin shifts have been correlated with in vivo stiffening of the corneal lens (5,6) and have been used to resolve the mechanics of subcellular structures and to correlate cell stiffness with extracellular matrix stiffening (7,8).

While the frequency shifts observed by Brillouin microscopy have been correlated to changes in cellular mechanical properties, one challenge is how to relate the observed frequency shifts and the longitudinal modulus (Μ) from Eq. 1 to the elastic or shear moduli, which are more commonly measured via other approaches. The longitudinal modulus is defined as the ratio of axial stress to axial strain under conditions when expansion in the lateral direction is restricted, e.g., by external constraints or when the inertia of the surrounding material itself prevents expansion at relevant timescales. It is related to the elastic modulus (Ε) and the Poisson ratio (ν) according to Eq. 3 (2). M approaches infinity when ν approaches 0.5. Thus, relating M to E can be very sensitive to the relative water content in the specimen (because water is nearly incompressible with ν ∼ 0.5).

$$M=\frac{E\left(1-\nu \right)}{\left(1+\nu \right)\left(1-2\nu \right)}$$ (3)

Furthermore, Brillouin microscopy probes material properties at GHz frequencies due to the wavelength of visible light used compared with Hz or kHz in AFM or optical tweezers and as the Poisson ratio is dependent on frequency, it is challenging to compare the elastic modulus to longitudinal modulus with this relationship. Thus, most existing approaches rely on empirically measured calibration curves to relate Brillouin frequency shifts to cellular mechanics, e.g., by calibrating the frequency shifts together with AFM, magnetic twisting cytometry, or optical tweezer measurements on accessible samples to confirm a positive correlation and then applying Brillouin microscopy to settings that are inaccessible to direct contact approaches.

In this issue of Biophysical Journal, Nikolić et al. have correlated Brillouin microscopy frequency shifts in cells to measurements of the storage and loss modulus from broadband optical tweezer experiments (9). They first demonstrate that cellular stiffness, as assayed by Brillouin frequency shifts, replicates prior findings wherein cellular stiffness increases when cells are cultured on stiffer substrates and decreases when the cytoskeleton is perturbed with cytochalasin D (10,11). Interestingly, neither Brillouin shifts nor optical tweezer measurements detected a difference in cellular stiffness between cells cultured on top of or within Matrigel hydrogels; however, Brillouin shifts were significantly different when the authors accounted for cellular morphology by classifying cells as either round, elongated, or protrusive, a result that could partly be explained by the fraction of the cell that is occupied by the rigid nucleus versus a softer cytoplasm. Finally, the authors applied Brillouin microscopy to multicellular spheroids and found that Brillouin shifts in multicellular contexts were less variable compared with single cells in suspension. An exciting facet of this work is that it provides an experimental framework for simultaneously studying Brillouin shifts and traditional viscoelastic properties via optical tweezers in complex multicellular environments. Further experiments using these methods will help elucidate the potential of Brillouin microscopy in challenging biological contexts, such as organoids and developing organisms. To summarize, the authors have demonstrated the ability to simultaneously measure mechanical properties of cells using optical tweezers and measure Brillouin shift and find the two measurements generally trend together. Furthermore, they have characterized Brillouin shift under variable cell morphologies and in suspended and spheroid cell populations. These results provide additional evidence that Brillouin microscopy can provide new information about the mechanics of living systems.

As Brillouin microscopy continues to develop, it will be important to connect how the probed material properties under gigahertz frequencies are related to a classical understanding of materials as determined with tools like AFM at much lower frequencies. In addition, given Brillouin microscopy’s noninvasive nature, future studies that connect these measurements to biological processes, such as cell migration, mechanotransduction, and morphological changes in a 3D context, will further increase its impact. Interestingly, Brillouin microscopy could serve the unique role of providing complementary measurements of cellular mechanical properties in experiments measuring cellular traction forces in 3D environments (12). In this way, Brillouin microscopy could provide a unique biophysical perspective for the interplay between cellular and ECM mechanics and cellular force generation in a fully 3D context.

Editor: Guy Genin.