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Data Article
Brillouin microspectroscopy data of tissue-
mimicking gelatin hydrogels
Michelle Bailey
a
, Noemi Correa
a
, Simon Harding
b
,
Nick Stone
a
, Sophie Brasselet
c
, Francesca Palombo
a
,
*
a
School of Physics and Astronomy, University of Exeter, Exeter, EX4 4QL, UK
b
Machine Intelligence Ltd, South Zeal, EX20 2JS, UK
c
Institut Fresnel, CNRS, Aix Marseille University, Marseille, F-13013, France
article info
Article history:
Received 13 December 2019
Received in revised form 28 January 2020
Accepted 3 February 2020
Available online 8 February 2020
Keywords:
Brillouin scattering
Phonons
Biopolymers
Tissue phantoms
Collagen
Biomechanics
abstract
Brillouin spectroscopy, based on the inelastic scattering of light
from thermally driven acoustic waves or phonons [1], holds great
promise in the field of life sciences as it provides functionally
relevant micromechanical information in a contactless all-optical
manner [2]. Due to the complexity of biological systems such as
cells and tissues, which present spatio-temporal heterogeneities,
interpretation of Brillouin spectra can be difficult. The data pre-
sented here were collected from gelatin hydrogels, used as tissue-
mimicking model systems for Brillouin microspectroscopy mea-
surements conducted using a lab-built Brillouin microscope with a
dual-stage VIPA spectrometer. By varying the solute concentration
in the range 4e18% (w/w), the macroscopic mechanical properties
of the hydrogels can be tuned and the corresponding evolution in
the Brillouin-derived longitudinal elastic modulus measured. An
increase in Brillouin frequency shift with increasing solute con-
centration was observed, which was found to correlate with an
increase in acoustic wave velocity and longitudinal modulus. The
gels used here provide a viable model system for benchmarking
and standardisation, and the data will be useful for spectrometer
development and validation.
©2020 The Authors. Published by Elsevier Inc. This is an open
access article under the CC BY license (http://creativecommons.
org/licenses/by/4.0/).
*Corresponding author.
E-mail address: f.palombo@exeter.ac.uk (F. Palombo).
Contents lists available at ScienceDirect
Data in brief
journal homepage: www.elsevier.com/locate/dib
https://doi.org/10.1016/j.dib.2020.105267
2352-3409/©2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://
creativecommons.org/licenses/by/4.0/).
Data in brief 29 (2020) 105267
1. Data description
Microspectroscopic data of gelatin hydrogels at varying solute concentration up to 18% (w/w) were
acquired using a lab-built Brillouin microscope with a two-stage VIPA spectrometer previously
described [3] (see Table 1 for full specifications). Pseudo-colour images for different solute concen-
trations are presented in Fig. 1A. A Brillouin spectrum was extracted from each raw image and fit
analysis to a Lorentzian functionwas applied to both Stokes and anti-Stokes peaks (Fig. 1B) arising from
the interation of light with acoustic waves or phonons [1]. Average peak parameters were calculated
(see Methods and Fig. 3) and Fig. 1C shows a plot of the Brillouin frequency shift as a function of solute
concentration.
Using these data, the acoustic wave velocity Vwas determined (see below; Fig. 2A), which re-
produces values previously found in the cornea and lens of the eye [4], and from this the longitudinal
elastic modulus M
0
was derived. As already noted [2], the longitudinal modulus derived from Brillouin
scattering includes the contribution of the adiabatic bulk modulus that is of the order of GPa even in
water. A plot of M
0
vs. solute volume fraction xenables the Voigt model (rule of mixing) [5,6]tobe
applied to these data (Fig. 2B) according to the expression:
Specifications Table
Subject Biophysics
Specific subject area Brillouin scattering spectroscopy
Type of data Graph
Figures
Images
Table
How data were acquired Brillouin Microscopy: Olympus iX73 inverted microscope coupled to a cw 532 nm
Cobolt Samba laser, lab-built dual-stage Virtually Imaged Phase Array (VIPA)
spectrometer (two VIPA etalons; Light Machinery) and Andor ZYLA-4.2P-USB3 sCMOS
camera
Data format Raw
Analysed
Parameters for data collection Gelatin hydrogels were analysed at room temperature (20
C), approximately 24 h after
preparation.
Description of data collection Gelatin hydrogels at a concentration ranging between 4 and 18% w/w were placed onto
a glass cover slip and analysed using a lab-built Brillouin microscope with a 60(NA
1.20) water immersion objective.
Data source location University of Exeter
Exeter
UK
50.7184
N, 3.5339
W
Data accessibility Repository name: Open Research Exeter (ORE), University of Exeter, UK
Data identification number: 10.24378/exe.2144
Direct URL to data: https://doi.org/10.24378/exe.2144
Value of the Data
These data relate to gelatin hydrogels derived from denatured collagen that are biologically relevant homogeneous
materials, useful to extract and understand the information contained within Brillouin spectra.
They can benefit the whole BioBrillouin community, providing a benchmark for testing and validation of instruments.
They can also benefit life scientists, biologists and clinicians who are interested in novel biophotonic techniques.
In addition, these data can be used to draw comparisons between similar lab-built spectrometers, to gain further insights
and to promote the development of new concepts for faster high-contrast, high-resolution Brillouin spectroscopy.
The use of transparent homogeneous materials that are reasonably stable at ambient conditions adds additional value to
these data for system benchmarking and standardisation.
M. Bailey et al. / Data in brief 29 (2020) 1052672
M0¼MsxþMwð1xÞ(1)
where M
s
and M
w
are the longitudinal elastic moduli of the solute (gelatin) and water, respectively.
All the data presented in this article are available for download and reuse from the Open Research
Exeter (ORE) repository, University of Exeter UK.
2. Experimental design, materials, and methods
2.1. Hydrogel preparation
Gelatin from bovine skin, gel strength ~225 g Bloom, Type B (G9382, Sigma-Aldrich) was used to
prepare hydrogels with solute concentration in the range 4e18% (w/w). Gelatin powder was combined
with the appropriate quantity of distilled water to a total mass of 20 g and the mixture was held in a
Table 1
VIPA-Brillouin microscope system specifications.
Parameter Value
Laser wavelength 532 nm
Laser power (on the sample) 6 mW
Laser spectral linewidth (FWHM) <1 MHz
Scattering geometry 180
Objective lens 60x (NA 1.2) WI
Free spectral range 33 ±2 GHz
Spectral resolution 0.9 ±0.1 GHz
Finesse 38 ±6
SNR 17 dB (methanol)
Fig. 1. (A) Pseudo-colour images of the sCMOS outputs for gelatin hydrogels at varying solute concentration, from 0 to 18% w/w. (B)
Spectrum of an 8% gelatin hydrogel before calibration (black line) and Lorentzian fit for both anti-Stokes (AS) and Stokes (S) peaks
(red line; R
2
¼0.97). (C) Plot of the Brillouin frequency shift vs. solute concentration of the gelatin hydrogels. Error bars account for
drift in the calibration spectra during the course of the experiment and encompass intra-sample variability.
M. Bailey et al. / Data in brief 29 (2020) 105267 3
water bath (temperature 55e65
C) for 60 min under magnetic stirring. This was sufficient time for the
gelatin powder to fully dissolve at all concentrations prepared. All gels were left to set at room tem-
perature, covered in parafilm to reduce evaporation and measured approximately 24 h after gelation, as
preliminary testing established that this was sufficient time for the gels to stabilise. A small rectangular
piece of gel (<10 mm thickness) was cut from the bulk and placed on a round glass coverslip (Biochrom,
0.17 mm thickness) in an Attofluor cell chamber (Life Technologies) for microscopy measurements.
2.2. Brillouin measurements
Brillouin microscopy measurements were conducted using a lab-built setup [3] developed on the
basis of previous works [7,8], comprised of a 532 nm cw laser (Cobolt Samba), inverted microscope
(Olympus iX73) with 60(NA 1.20) water immersion objective (Olympus UPlanApo), and dual-stage
VIPA (LightMachinery, 30 GHz FSR) spectrometer with sCMOS camera (Andor ZYLA-4.2P-USB3). The
laser power measured at the sample was approximately 6 mW and the spectral resolution was eval-
uated as ~0.9 GHz. Full spectrometer specifications are listed in Table 1.
The sCMOS output of the dual-stage VIPA spectrometer presents square patterns arising from
different diffraction orders with Brillouin peaks along diagonals. Out of multiple diffraction orders, a
single square with linear edges and uniform intensity was selected. Brillouin measurements of the gels
were acquired with an exposure time of 3 s, as this was found to give the optimal trade-off between
acquisition time and signal-to-noise ratio. All measurements were conducted at room temperature
(20
C), taken in triplicate at varying locations within the sample, and average frequency shifts were
determined for each concentration. Brillouin peaks were identified and a Lorentzian fit was applied
using a method previously developed in our lab [3].
2.3. Calibration
Spectral calibration was performed using known values of frequency shift for methanol and water
(5.59 GHz and 7.46 GHz, respectively [9]). Calibration spectra from these standards were collected at
the beginning and end of all experiments. The parameters from initial and final calibration spectra
were averaged to account for drift during the experiment. To convert the peak position from a pixel to
frequency scale, a scaling factor Pwas determined according to the relation:
P¼2ð
n
w
n
mÞ
XmXw
;(2)
Fig. 2. Plot of (A) acoustic wave velocity Vvs. solute concentration and (B) longitudinal elastic modulus M0vs. solute volume
fraction. Red line: fit to the Voigt model applied to the data, M0¼6:73xþ2:14ð1xÞ;R
2
¼0.92. Shading: 95% confidence band of
the fit.
M. Bailey et al. / Data in brief 29 (2020) 1052674
where
n
m
and
n
w
are the known frequency shifts, and X
m
and X
w
the distance in pixels between peaks
from neighbouring dispersion orders (Fig. 3), with subscripts mand wdenoting methanol and water,
respectively.
The free spectral range (FSR) was taken to be a summation of the measured distance between
adjacent Brillouin peaks (X) and the frequency shift (
n
) of the standards: FSR ¼2
n
þPX (see Table 1).
The Brillouin frequency shift
n
B
for each gelatin sample was hence determined from the FSR and the
distance Xbetween adjacent Brillouin peaks according to the expression:
n
B¼1
2ðFSR PXÞ:(3)
2.4. Longitudinal elastic modulus
The acoustic wave velocity Vwas determined from the Brillouin frequency shift based on the
equation (valid for backscattering geometry):
V¼
ln
B
2n;(4)
where
l
is the incident wavelength and nthe refractive index of the sample. From these data, the
longitudinal elastic modulus M
0
was derived according to the relation:
M0¼
r
V2
;(5)
where
r
is the mass density of the sample. Refractive indices were measured using an Abbe refrac-
tometer, with distilled water as the calibration standard. Measurements revealed a linear relation
described by n¼0:00217xþ1:331 (R
2
¼0.996) where xis the solute concentration (w/w). Densities
were calculated by assuming ideal mixing:
r
¼ðmwþmsÞ
mw
r
wþms
r
s;(6)
Fig. 3. Calibration spectra and Lorentzian fit for methanol (red) and water (blue). Distances between Brillouin peaks, Xmand Xw,
were used to determine absolute peak positions. The free spectral range (FSR) between adjacent Rayleigh peaks is shown.
M. Bailey et al. / Data in brief 29 (2020) 105267 5
where mis the mass. Density was taken to be 1.00 g/cm
3
for water and 1.35 g/cm
3
for dry gelatin [10].
The density-to-square refractive index ratio, which is relevant in the relation between longitudinal
elastic modulus and the frequency shift (Eqs. (4) and (5)), was found to vary between 0.5628 and
0.5582 g/cm
3
hence accounting for only 0.8% change in the concentration range probed.
Acknowledgments
This work was supported by EPSRC and CRUK through the grants EP/M028739/1 and NS/A000063/1
(to F.P.) and a Leverhulme Trust Visiting Professorship (to S.B.). F.P. gratefully acknowledges support
through the COST Action BioBrillouin (CA16124) for helpful discussions on the topic of this article.
Conflict of Interest
The authors declare that they have no known competing financial interests or personal relation-
ships that could have appeared to influence the work reported in this paper.
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