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

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.


Introduction
Brillouin light scattering (BLS) spectroscopy is an emerging technique in biomedical sciences, biophysics and biophotonics. It is making an impact in these fields as it probes micromechanical properties through an optical high-resolution and contact-free method. In BLS, micromechanical information is obtained via spectral analysis of inelastically scattered light from thermally induced acoustic waves in the GHz range. Longitudinal acoustic modes propagating at a speed of a few km/s give rise to Brillouin peaks in the range 10-20 GHz. The actual frequency shift depends on the stiffness and the linewidth on the attenuation of the acoustic waves in the material [1][2][3]. Viscoelastic materials such as biopolymers and biomaterials in general exhibit frequency-dependent mechanical responses, and their elastic moduli differ based on the spatial and temporal scale of the technique employed. In these materials, Brillouin measurements giving access to microscale mechanics yield longitudinal elastic moduli of the order of GPa [4,5], whilst traditional quasistatic mechanical testing give Young's moduli in the MPa range [6]. Whilst this indicates that the techniques probe different forms of elastic modulus, it is also apparent that measurements and molecular dynamics simulations on a nanometre scale also provide Young's moduli in the GPa range [7]. Traditionally Brillouin spectroscopy has been performed using tandem multipass Fabry-Pérot interferometers, which can achieve very high contrast and spectral resolution [8], the only limitation being the acquisition time of a single spectrum especially in mapping large samples. While coherent methods have been developed to improve speed and sensitivity [9], spontaneous Brillouin techniques are convenient in terms of simplicity of implementation and instrument costs. Here we use spontaneous Brillouin microscopy based on a double-stage cross-axis cascading virtually imaged phase array (VIPA) spectrometer [10,11] capable of acquiring hyperspectral maps of biomedical samples with good contrast and reduced laser power.
Our previous works have demonstrated the application of tandem Fabry-Pérot Brillouin spectroscopy to the studies of fibrous proteins of the extracellular matrix (ECM), providing access to the full elasticity tensor of collagen and elastin fibres [4] and the effects of hydration [6] and purification [5]. We have also applied high-resolution Brillouin microscopy to microbial biofilms [8], histological sections of epithelial tissue in Barrett's oesophagus [12,13] and to Alzheimer's brain in a mouse model of amyloidopathy [14,15]. This has demonstrated the versatility and the capability of the technique to spatially map stiffness in correspondence to specific molecular composition in tissues on a microscale.
In the present work, we apply VIPA Brillouin microscopy to collagen gelatin hydrogels at various concentrations and in the presence of a cross-linker, and present a new method to extract the relevant information contained in Brillouin images of biomaterials. Gelatin hydrogels are physical gels derived from bovine skin collagen that are devoid of the complex hierarchical structure (triple helices / fibrils / fibril bundles / fibres) typical of collagen-rich tissues [16], thus providing a homogeneous material for testing. Formalin is commonly used as a fixative for biological samples, to preserve cells and tissues. Here it is added prior to gelation, thus altering the gel structure. Although the method can readily be extended to other specimens, it is applied here to hydrogels that are tissue models with water concentration ranging between 96 and 82 wt. %.
The method that we present here for the first time is capable of automatically analysing Brillouin images, to identify peaks and to apply fit analysis to extract the relevant parameters for viscoelastic characterization. Further, the software automatically corrects for drifts in the pattern of scattered light and other visual distortions, and provides real-time, fully automated data analysis. The ability to complete processing in real time, i.e. faster than the image acquisition rate, will allow for high resolution scanning of large areas without the need to store vast amount of imaging data. Further, algorithm design has been developed with modern high speed parallel processing devices, such as Graphics Processing Units (GPUs), in mind so that the remarkable increase in speed that this type of hardware brings can be applied to the data analysis. The protocol was developed for dual-stage VIPA Brillouin images, however it can be easily adapted to analyse images from a single-stage spectrometer.
This paper is divided into three sections. In Section 2, we present the experimental system and the computer method developed for image acquisition and data processing. We also address issues related to laser drifting and how to correct for those without the need for an additional optical path. In Section 3, we present and discuss the results from image analysis of collagen gelatin hydrogels. Finally, Section 4 draws the conclusion.

Collagen gel preparation
Type B 225-Bloom gelatin derived from bovine skin (G9382, Sigma-Aldrich) was combined with appropriate quantities of Milli-Q water to prepare hydrogels of 4 to 18 wt. % gelatin giving a water concentration of 96 to 82 wt. %. Gelatin powder was dissolved in water at 55-65 °C under stirring for 60 minutes, resulting in a clear solution for all concentrations. Gels containing the cross-linker were prepared using the same protocol, with the addition of formalin (37 wt. % formaldehyde solution, Sigma-Aldrich) when the gel reached 50°C.
Hydrogels we water content poured into m room tempera mould was t Attofluor cell

Brillouin
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Conclusio
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e was develop n of spectral d gen gelatin hyd we can obtain nd bespoke soft he Brillouin fre ggests an increa ity of this meth mens close to ph elatins with 6 and nd 2s exposure tim d in Fig. 8