Mechanical Mapping of Spheroids Using Brillouin Spectroscopy

Brillouin spectroscopy, an emerging technique gaining significant interest in biomedical science, allows researchers to gather information related to mechanics and structure by interrogating the viscoelastic and architectural properties of specimens in a non-destructive, contact-free manner. This approach evaluates the mechanical properties of 3D samples by measuring the interaction of visible light with thermally induced acoustic waves/phonons. The information that Brillouin spectroscopy provides has potential for in vivo assessment of biophysics and potential diagnosis of disease pathologies. A significant advantage of Brillouin spectroscopy is the capability to assess microscale mechanics inside a biological sample; other conventional techniques that can achieve this resolution, such as atomic force microscopy, can only probe samples in 2D since they require direct contact. This work describes the application of Brillouin micro-spectroscopy to investigate the biomechanics of living spheroids embedded within a 3D hydrogel matrix. Encapsulation of cellular spheroids within a 3D microenvironment establishes a spheroid system that closely recapitulates the interface between cells and the extracellular matrix in vivo. In our protocol, we describe spheroid sample preparation and measurements of Brillouin spectra with sequential fluorescence imaging. Additionally, we discuss procedures for spectral data analysis and technical details about the optical system.