Off-Axis Illumination and Epicollection Confocal Brillouin Scattering Microspectroscopy

Viscoelastic properties of tissues and biologically relevant fluids therein are important to maintaining their structures and functions. Confocal Brillouin scattering microspectroscopy (μCBS) is a promising tool to address such local viscoelastic properties in nonlabeling and noninvasive manners. However, since the intensity of Brillouin scattering is extremely weak, the strong background by reflected light and Rayleigh scattering often hinders the quantitative analyses of viscoelastic properties in buried tissues and biological fluid. Here, we report an optical configuration for the reduction of such strong backgrounds based on the combination of off-axis illumination and epicollection for μCBS. As a test sample, aqueous solutions of bovine serum albumin (BSA) with relevant electrolytes were measured through thick glass windows (1.25 mm) of quartz cuvettes. Such thick windows act as strong reflectors and scatterers for the focused illumination light due to interfacial reflections and multiple reflections therein. The series of Brillouin scattering spectra of BSA solutions up to 20 wt % buried under the thick window were successfully analyzed owing to the effective reduction of those strong backgrounds. Further, it was clarified that hydrated water molecules surrounding BSA non-negligibly contribute to Brillouin scattering bandwidth, indicating that it would be also applicable to detect conformational changes of proteins in fluids. The off-axis illumination and epicollection optical configuration would further enhance the μCBS's ability for its future application in biochemistry and biomedicine to assess the viscoelastic properties of tissues with complex structures and biologically relevant fluids, such as blood and lymph flowing therein.