# Off-Axis Illumination and Epicollection Confocal Brillouin Scattering Microspectroscopy

## TL;DR

A new optical setup improves the ability to measure the viscoelastic properties of biological fluids and tissues without labels or invasive methods.

## Contribution

A novel optical configuration combining off-axis illumination and epicollection reduces background noise in Brillouin scattering measurements.

## Key findings

- The new setup successfully analyzed BSA solutions buried under thick glass windows.
- Hydrated water molecules around BSA contribute to Brillouin scattering bandwidth.
- The method can detect protein conformational changes in fluids.

## Abstract

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.