Mechanical Mapping of Spheroids Using Brillouin Spectroscopy

Chloe B. Rodgers; Giedrė Astrauskaitė; Rebecca E. Ginesi; Theodora Rogkoti; Greg Wardle; Troy R. Allen; Oana Dobre; Massimo Vassalli; Matthew Walker

doi:10.3791/67538

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Method Article

Mechanical Mapping of Spheroids Using Brillouin Spectroscopy

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DOI:

10.3791/67538

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December 12th, 2025

Chloe B. Rodgers^*¹ , Giedrė Astrauskaitė^*¹ , Rebecca E. Ginesi¹ , Theodora Rogkoti¹ , Greg Wardle² , Troy R. Allen² , Oana Dobre¹ , Massimo Vassalli¹ , Matthew Walker¹

¹Centre for the Cellular Microenvironment, Advanced Research Centre, 11 Chapel Lane, University of Glasgow, ²LightMachinery Inc.

^* These authors contributed equally

Summary

This article describes a protocol for mechanical characterisation of living spheroids within a 3D matrix using Brillouin micro-spectroscopy. This all-optical method enables spheroid visualisation with microscale resolution and quantitative mechanical properties. This approach has implications for mechanical phenotyping; for instance, determining pathological states of tumor spheroids within a 3D microenvironment.

Abstract

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.

Introduction

Culturing cells in 3D models is advantageous compared to the typical 2D cell culture methods, as it better recapitulates tissue structures and cell-cell interactions found in vivo. Spheroids are 3D cell aggregates that facilitate complex cell-cell interactions; these interactions are compromised in cells grown as a monolayer on tissue culture plastic. Spheroids can be grown within a scaffold that mimics the microenvironment where the cell originated from. For example, hydrogels are a popular biomaterial used to culture cells/spheroids in as they allow precise control of parameters such as stiffness, degradability, pore size, and the incorporation of growth fa....