Image analysis applied to Brillouin images of tissue-mimicking collagen gelatins

Abstract

Brillouin spectroscopy is an emerging analytical tool in biomedical and biophysical sciences. It probes viscoelasticity through the propagation of thermally induced acoustic waves at gigahertz frequencies. Brillouin light scattering (BLS) measurements have traditionally been performed using multipass Fabry-Pérot interferometers, which have high contrast and resolution, however, as they are scanning spectrometers they often require long acquisition times in poorly scattering media. In the last decade, a new concept of Brillouin spectrometer has emerged, making use of highly angle-dispersive virtually imaged phase array (VIPA) etalons, which enable fast acquisition times for minimally turbid materials, when high contrast is not imperative. The ability to acquire Brillouin spectra rapidly, together with long term system stability, make this system a viable candidate for use in biomedical applications, especially to probe live cells and tissues. While various methods are being developed to improve system contrast and speed, little work has been published discussing the details of imaging data analysis and spectral processing. Here we present a method that we developed for the automated retrieval of Brillouin line shape parameters from imaging data sets acquired with a dual-stage VIPA Brillouin microscope. We applied this method for the first time to BLS measurements of collagen gelatin hydrogels at different hydration levels and cross-linker concentrations. This work demonstrates that it is possible to obtain the relevant information from Brillouin spectra using software for real-time high-accuracy analysis.

Predicting the Refractive Index of Tissue Models Using Light Scattering Spectroscopy

“…Type B gelatin (denatured collagen) was prepared to concentrations between 4 and 18% w/w as previously described. 1,26,27 The refractive index of all gelatin samples was measured by Abbe refractometry with a D line (589 nm) light source as part of a previous work. 1,26 The Raman spectra of gelatin at different concentrations were collected using two systems across different frequency ranges.…”

Brillouin imaging for studies of micromechanics in biology and biomedicine: from current state-of-the-art to future clinical translation

“…This has the potential to provide more conclusive insights into cellular responses and construct remodelling processes. In [83], Correa et al demonstrated an efficient Brillouin data collection and image analysis workflow on collagen gelatin hydrogels with different stiffness created by varying crosslinker concentration and formalin concentration. This is valuable for analysis of biologically-relevant tissue analogues with physiological hydration levels and subjected to routine fixation methods.…”

“…Data acquisition, storage and processing in BLS experiments is typically done by custom-built scripts and software, developed by each research group to meet particular requirements [83] with the exception of GHOST software which is supplied with TFP interferometers as a part of the commercial product. GHOST software, produced by Table Stable Ltd in conjunction with the University of Perugia, is a versatile multi-channel analyzer equipped with curve fitting and calibration features.…”

Brillouin Light Scattering Microspectroscopy for Biomedical Research and Applications: introduction to feature issue

“…Extracting the BLS spectra from a measurement performed using a VIPA cross-dispersion imaging spectrometer is not trivial and requires suitable calibrations with control samples and correct scaling function of the spectral dispersion axis. Here Correa et al [4] present a detailed workflow starting from the raw output of an sCMOS camera, through to extracting the measured spectra and fitting a corrected and filtered spectral projection to obtain the BLS frequency shift and peak width. This is demonstrated in the form of studies on tissuemimicking collagen gels.…”