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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.
Image analysis applied to Brillouin images of
tissue-mimicking collagen gelatins
NOEMI CORREA,1 SIMON HARDING,2 MICHELLE BAILEY,1 SOPHIE
BRASSELET,3 AND FRANCESCA PALOMBO1,*
1School of Physics and Astronomy, University of Exeter, Stocker Road, EX4 4QL Exeter, UK
2Machine Intelligence Ltd, EX20 2JS South Zeal, UK
3Aix Marseille Univ, CNRS, Centrale Marseille, Institut Fresnel, F-13013 Marseille, France
*f.palombo@exeter.ac.uk
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.
Published by The Optical Society under the terms of the Creative Commons Attribution 4.0 License.
Further distribution of this work must maintain attribution to the author(s) and the published article’s title,
journal citation, and DOI.
1. 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 [1–3]. 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].
Vol. 10, No. 3 | 1 Mar 2019 | BIOMEDICAL OPTICS EXPRESS 1329
#353393
https://doi.org/10.1364/BOE.10.001329
Journal © 2019
Received 4 Dec 2018; revised 13 Jan 2019; accepted 20 Jan 2019; published 19 Feb 2019
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.
2. Materials and methods
2.1 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.
Vol. 10, No. 3 | 1 Mar 2019 | BIOMEDICAL OPTICS EXPRESS 1330
Hydrogels w
e
water conten
t
p
oured into
m
room temper
a
mould was
t
Attofluor cell
2.2 Brillouin
The Brillou
i
spectrometer
inverted mic
r
in Fig. 1.
Fig.
1
shutt
(10:9
optic
The light
at 532nm, wi
t
through a sh
u
a 10x beam
e
(Thorlabs) a
t
sputtered enh
b
eam towar
d
UPlanApo)
w
x, y ~270nm
objective and
(Thorlabs) a
n
directed tow
a
Thorlabs, f
=
etalons (Ligh
t
dispersion o
r
spectrometer.
camera (An
d
e
re prepared
w
t
adjusted to
m
m
oulds at 40°
C
a
ture. App
r
oxi
m
t
ransferred to
chamber (Lif
e
microscop
y
i
n mic
r
ospec
t
of the type p
r
r
oscope with e
p
1
. Schematic diag
r
e
r; FW: neutral d
e
0
) non-polarising
b
a
l masks.
source emplo
y
t
h 300mW ou
t
u
tter and a mot
o
e
xpander (Th
o
t
the entrance
anced silver r
e
d
s the object
w
as used for t
h
and z ~1μm.
B
directed back
n
d focused into
a
rds the VIP
A
=
200mm), tw
o
t
Machinery, 3
0
r
der by the
V
The spectral
r
d
or Zyla 4.2P
-
w
ith concen
t
ra
t
m
aintain a co
C
and covered
w
m
ately 24 ho
u
a round glas
s
e
Technologie
s
t
roscopy syst
e
r
eviously pres
e
p
i-configurati
o
r
am of the VIPA
e
nsity filter whe
e
b
eam splitter; C1
y
ed was a sin
g
tp
ut power an
d
o
rized neutral
d
o
rlabs), and t
h
of the micr
o
e
flective mirro
ive. A 60x,
h
e Brillouin m
e
B
ackscattered
l
to the beam s
p
a 75μm pinh
o
A
spectrometer
.
o
spherical le
n
0
GHz FSR).
T
V
IPAs and
t
r
esolution was
-
USB3), with
t
ions of form
a
nstant 10 wt.
w
ith Parafilm
u
rs after gelati
o
s
coverslip (
B
s
) for microsc
o
e
m comprise
d
e
nted [17], co
u
o
n. A schemat
i
Brillouin micros
c
e
l; BX: 10x bea
m
2: cylindrical len
s
g
le-frequency
D
d
<1 MHz spe
c
d
ensity filter
w
h
en through a
o
scope. Withi
n
r (R ~99.6%
a
1.20 NA w
a
e
asurements.
T
l
igh
t
from the
p
litter. The tra
n
o
le (Thorlabs).
.
This consist
e
n
ses (S1/S2, T
T
wo xy slits (
O
t
o reject any
~0.6 GHz. T
h
13μm x 13μ
m
a
lin in the ran
g
% gelatin co
to prevent dr
y
o
n, a small pie
B
iochrom, 0.1
o
py.
d a lab-
b
ui
l
u
pled to an O
ic diagram of
c
ope system. Acr
o
m
expander; M1–
6
s
es; S1–5: spheric
a
D
PSS laser (C
o
c
tral linewidth.
w
heel (Thorlab
(10:90) non-
p
n
the micros
c
a
t 530nm) was
ate
r
-immersio
n
T
he theoretica
l
sample was c
o
n
smitted light
The filtered l
i
e
d of two cyl
i
T
horlabs, f =
2
O
ptoSigma) w
e
unwanted s
t
h
e detector wa
s
m
pixel size.
g
e 1 to 16 wt
.
o
ncentration.
G
y
ing, then left
t
e
ce of gelatin
f
7mm thickne
s
l
t double-sta
g
lympus IX73
the apparatus
o
nyms denote SH
6
: mirrors; NPBS
al lenses; MK1–2
o
bolt Samba)
.
The laser be
a
b
s) before goin
g
p
olarising bea
m
c
ope frame, a
used to direc
t
n
objective
(
l
spatial resol
u
o
llected using
was collected
i
ght was colli
m
i
ndrical lense
s
2
00mm) and t
w
e
re employed t
o
t
ray light thr
o
s an ai
r
-coole
d
Exposure tim
.
%, with
G
els were
t
o cool at
f
rom each
s
s) in an
g
e VIPA
two-deck
is shown
:
:
:
operating
a
m passed
g
through
m
splitter
a
Chroma
t
the laser
(
Olympus
u
tion was:
the same
by a lens
m
ated and
s
(C1/C2,
w
o VIPA
o
select a
o
ugh the
d
sCMOS
m
es at the
Vol. 10, No. 3 | 1 Mar 2019 | BIOMEDICAL OPTICS EXPRESS 1331
camera vary
b
sample was
i
100mm x 75
m
were used fo
r
source (LED
)
System c
a
(30GHz) and
2.3 Image a
n
Brillouin im
a
visualization
time. Any las
corrected for.
collection an
d
Figure 2
s
image throug
h
Fig.
e br
gravi
Visualiza
t
visualization
however it e
n
correspondin
g
b
etween 1 an
d
i
n the range
3
m
m travel ran
g
r
scanning the
)
for bright fiel
d
a
libration was
validated usin
g
n
alysis
a
ges were ac
of both white
er drift, and h
e
This implem
e
d
analysis.
s
hows a block
h
to finding th
e
2
. Block diagram
s
o
ken down into
f
t
y.
t
ion of Brillo
u
of low-contra
s
n
ables a visu
a
g
processed i
m
d
4 s, dependi
n
3
–5 mW. Tw
o
g
e; PInano z p
i
sample and f
o
d
transmissio
n
performed u
s
g
methanol an
d
quired and p
light and ra
w
e
nce changes t
o
e
ntation provi
d
diagram of th
e
e
Brillouin fre
q
s
howing the stage
f
our major parts (
c
u
in spectra. T
h
s
t images. Thi
s
a
l inspection
o
m
ages are show
n
n
g on the sa
m
o
PI microsc
o
i
ezo well plate
o
cusing. The
m
n
images.
s
ing the nomi
n
d
wate
r
as sta
n
r
ocessed usin
w
Brillouin i
m
o
the Rayleig
h
d
es a consiste
n
e
processing st
q
uency values
s of the data anal
y
c
oloured boxes).
h
e first step
i
s
operation ha
s
o
f the outpu
t
s
n
in Fig. 3.
m
ple being tes
t
o
pe stages (P
I
scanner syste
m
m
icroscope als
o
n
al free spect
r
n
dards.
n
g bespoke s
o
m
ages at each
s
h
peak positio
n
n
t, use
r
-indep
e
t
eps that are a
p
and other line
y
sis protocol. Th
e
WCoG denotes
w
i
n image anal
y
s
no effect on
t
s
ignal. A typi
c
t
ed. Laser po
w
I
Line xy stag
e
m
, 220μm tra
v
o
includes a
w
r
al range of t
h
o
ftware whic
h
s
ample positi
o
n
s, will be auto
e
ndent approa
c
p
plied going fr
shape parame
t
e
data analysis ca
n
w
eighted centre o
f
ysis is to im
p
t
he actual spe
c
c
al sCMOS o
u
w
er at the
e
system,
v
el range)
w
hite light
h
e VIPAs
h
enables
o
n in real
o
matically
c
h to data
om a raw
t
ers.
n
f
p
rove the
c
tral data,
u
tput and
Vol. 10, No. 3 | 1 Mar 2019 | BIOMEDICAL OPTICS EXPRESS 1332
Fig.
water
colou
Raw Brill
and vertical l
can make it
visualization
p
seudo colo
u
according to:
where
0
I is t
h
standard devi
a
0 and 1.
I'
is
a
colour value
normalizatio
n
also worth p
o
image, howe
v
automated al
g
I
dentifica
t
will first loca
t
3
. (a) A typical s
C
acquired with a 6
r
image. (c) Contr
a
ouin images t
e
ines in the i
m
difficult to vi
s
of Brillouin s
p
u
r image (Fig.
h
e original int
e
a
tion of the in
t
a
perceptual i
n
based on a
n
n
is purely to
a
o
inting out th
a
v
er here they
g
orithms to fin
d
t
ion of Raylei
g
t
e the pixels t
h
C
MOS output of
t
0x wate
r
-immersi
o
a
s
t
-enhanced ima
g
e
nd to have sa
t
m
ages, someti
m
s
ually identif
y
p
ectra, the sC
M
3(b)) and the
n
()
0
0,
I'= I 1,
I
o
f
=
e
nsity value,
μ
t
ensities
0
I, a
n
tensity value,
b
n
appropriate
a
ssist the user
a
t in principle
were used t
o
d
the data reg
a
g
h peaks.
To a
u
h
at represent th
t
he spectrometer,
o
n objective and
2
g
e.
t
urated Raylei
g
m
es noisy bac
k
y
Brillouin si
g
M
OS output o
f
n
normalised
b
0
0
I
I
,
2
μ
ασ
μ
ασ
μασ
ασ
<−
>+
−+
μ
is the mean
o
n
d
α
is an arbit
r
b
etween 0 and
colour scale
.
and does not
Rayleigh pea
k
o
locate the d
a
a
rdless of drift
s
u
tomatically e
x
e Rayleigh pe
a
showing the Bril
2
s exposure time.
(
g
h peaks, as s
e
k
ground and l
o
g
nals. To addr
e
f
the spectro
m
b
y the standar
otherwise
o
f the intensit
y
r
ary scaling c
o
1, which can
t
.
Note that
t
take part in t
h
k
s can be opti
c
a
ta in the im
a
s
in the image.
x
tract spectros
c
a
ks within the
i
l
louin spectrum o
f
(
b) Correspondin
g
e
en from the
h
o
w contrast da
t
ess the need
f
m
eter is conver
t
r
d deviation (
F
y
of the imag
e
o
nstant rangin
g
t
hen be conve
r
t
his enhanced
h
e data proces
s
c
ally removed
a
ge, and allo
w
c
opic data, th
e
im
age (Fig. 4)
f
g
h
orizontal
ta, which
f
or better
t
ed into a
F
ig. 3(c)),
(1)
e
,
σ
is the
g
between
r
ted into a
contrast
s
ing. It is
from the
w
for the
e
software
.
Vol. 10, No. 3 | 1 Mar 2019 | BIOMEDICAL OPTICS EXPRESS 1333
Fig.
meth
Corre
denot
data.
the x
-
maxi
The auto
m
filter to the i
m
illustrated in
image. From
found (Fig. 4
(
can be furth
e
computed fo
r
curves arisin
g
may not be a
shape. The i
n
Rayleigh pea
k
around each
o
data.
After fin
d
then applied
image by sli
g
location, the
d
hence to pro
v
in the image
d
squared patte
r
Extractio
n
image contai
n
spectrum is f
o
the image, a
n
spectrum (Fi
g
allows for sp
e
4
. (a) A typical s
C
a
nol acquired w
i
sponding denois
e
e
d by blue crosse
(e) Spectral data
r
-
axis. (f) Brilloui
n
m
um intensity rec
o
m
atic identific
a
m
ages (Fig. 4
(
F
ig. 4(b). We
this image, th
e
(
c)). Using the
e
r related to e
r
both the hori
g
from the ord
e
good approxi
m
n
tersection of
k
s. Further re
f
o
f the intersec
t
d
ing the Rayle
i
to the image
a
g
htly shifting t
h
d
ata can be tra
n
v
ide a consiste
n
d
ue to laser d
r
r
n.
n
of Brillouin
n
ing only the
o
und by takin
g
n
d this value
g
. 4(f)). The l
o
e
ctra to be dire
C
MOS output of
t
i
th a 60x wate
r
e
d thresholded i
m
s at the corners o
r
otated (by 45° a
n
n
spectrum in wh
i
o
rded by the pixel
s
a
tion of Rayle
(
a)) and then
a
chose to use
a
e
centre pixels
centre of gra
v
ach correspo
n
zontal and ve
r
e
rs of diffracti
o
m
ation if the
s
these lines g
i
f
inement of t
h
t
ions and com
p
i
gh line coord
i
a
s ‘warp affin
h
e pixels relat
i
n
sformed to fi
t
n
t display of i
m
r
ift or other in
s
spectra.
N
ex
t
spectroscopic
g
the maximu
m
is retained a
o
cation of the
R
ctly compared
t
he spectrometer,
-immersion obje
c
m
age. (c) Final
c
f the image. (d)
C
n
ticlockwise) so t
h
i
ch the in
t
ensity
f
s
in each column
o
igh peaks star
t
a
threshold in
a
threshold of
0
of all lines f
o
v
ity of this bin
a
n
ding corner.
F
r
tical axes. H
e
o
n are approx
i
s
elected patter
n
i
ves a good a
p
h
e corner posi
t
p
uting the wei
g
i
nates, a hom
o
e’ transforma
t
i
ve to their or
i
t
a correctly or
m
aging data. T
h
s
tabilities of t
h
t
, the image i
s
data, as show
m
intensity rec
o
s the intensit
y
R
ayleigh peak
s
, as shown in
F
showing the Bril
c
tive and 2s e
x
c
olour image. R
a
C
olour image wit
h
h
at the spectral a
x
f
or each channel
c
o
f the image.
r
ts by first ap
p
order to pro
d
0.95 of the m
a
o
rming the ed
g
a
ry image as a
F
or each corn
e
e
re an assump
t
i
mated as strai
n
deviates sig
n
p
proximation
t
ions is perfo
r
g
hted centre o
f
o
graphy matri
x
t
ion. This tran
i
ginal location
r
iented square
a
his also corre
c
h
e syste
m
that
s
cropped an
d
w
n in Fig. 4(d)
o
rded by the p
y
for each c
h
s
in these ima
g
F
ig. 5.
l
louin spectrum o
f
x
posure time. (b
)
a
yleigh peaks ar
e
h
selected spectra
l
x
is coincides wit
h
c
orresponds to th
e
p
lying a Gaus
s
d
uce a binary
i
a
ximum inten
s
g
es of the squ
a
guide, the ce
n
e
r, a line of
b
t
ion is made i
n
ght lines. Not
e
n
ificantly fro
m
for the positi
o
r
med by takin
g
f gravity of th
e
x
can be com
p
sformation co
r
n
. By setting t
h
a
t a known lo
c
c
ts for any dis
p
cause distorti
o
d
rotated to p
r
and 4(e). Fr
o
ixels in each
c
h
annel of the
g
es is consiste
n
f
)
e
l
h
e
s
ian noise
i
mage, as
s
ity in the
a
re can be
n
tre pixels
b
est fit is
n
that the
e
that this
m
a square
o
n of the
g
an area
e
original
p
uted and
r
rects the
h
e desired
c
ation and
p
lacement
o
ns to the
r
oduce an
o
m this, a
c
olumn of
Brillouin
n
t, which
Vol. 10, No. 3 | 1 Mar 2019 | BIOMEDICAL OPTICS EXPRESS 1334
Fig.
objec
Setting t
h
spectrometer,
frequency sc
a
where ν
Β
is t
h
peaks, and
i
i
s
Separatin
g
given pixel i
n
Adjacent poi
n
p
reselecting
a
In this s
y
adequate to
a
work, we wil
l
Brillouin
p
p
roceed with
of the Brillo
u
and compare
d
b
oth Stokes a
n
5
. Brillouin spec
t
t
ive and 2s expos
u
h
e distance
b
it is possibl
e
a
le (GHz) thro
u
h
e Brillouin f
r
s
the pixel ind
e
g
data from t
h
n
dex with the
a
n
ts above this
a
n appropriate
t
y
stem, a linea
r
a
chieve results
l
investigate a
n
p
eak analysis
.
line shape an
a
u
in peaks. Her
e
d
the results w
i
n
d anti-Stokes
t
rum of methano
l
u
re time. Dashed l
i
b
etween Brill
o
e
to convert
t
u
gh the expres
s
FS
R
B
ν
=
r
equency shift
,
e
x.
h
e background
a
verage of the
threshold are
c
t
hreshold, the
p
r
relationship
comparable t
o
n
onlinear cali
b
.
Once the re
l
a
lysis. Various
e
we used Lor
e
i
th those from
peaks of met
h
l
and water acq
u
i
nes denote lines
o
o
uin
p
eaks
e
t
he dispersion
s
ion:
(
0
10
R
min ,iR
R
RR
⋅−
,
R
0
and
R
1
ar
noise is achi
e
surrounding i
n
c
onsidered to
b
p
rocess is full
y
between freq
u
o
those previo
u
b
ration to gene
r
l
evant peaks
h
function type
s
e
ntzian functi
o
a Gaussian fi
t
h
anol and wate
r
u
ired with a 60x
o
f average intensit
y
e
qual to the
axis from a
)
1
R
i
r
e the pixel p
o
e
ved by comp
n
tensities. Thi
s
b
e part of the
y
automated.
u
ency and po
s
usly publishe
d
r
alize the appr
o
h
ave been ide
n
s
can be imple
m
o
ns and nonlin
e
t
. Figure 6 illu
s
r.
wate
r
-immersio
n
y
.
nominal FS
R
pixel index s
c
o
sitions of the
p
aring the inte
n
s
is illustrated
same peak. A
p
s
ition of the
l
d
(see below).
oach further.
n
tified, it is
po
m
ented for cu
r
e
ar least squar
e
s
trates the fit
r
n
R
of the
c
ale to a
(2)
Rayleigh
n
sity at a
in Fig. 5.
p
art from
l
ines was
In future
o
ssible to
r
ve fitting
e
s fitting,
r
esults for
Vol. 10, No. 3 | 1 Mar 2019 | BIOMEDICAL OPTICS EXPRESS 1335
Fig.
meth
0.982
have
In additio
error (RMS)
a
The fit result
s
owing to con
v
3. Results
a
We validated
from the liter
a
Tab
l
[1
8
Sample
Methanol
Water
We notic
indicating th
a
Figure 7 s
6
. Fit results for (
a
nol and water. L
o
(methanol), 0.99
4
b
een converted to
n to the peak
a
nd the regres
s
s
show that a
L
v
olution with t
h
a
nd discussi
o
the accuracy
o
a
ture, listed in
e 1. Brillouin fre
q
8
].
b
Data derive
d
Wavelength
(
532
e a close co
r
a
t the method c
hows typical
B
left) Stokes and
(
o
rentzian and Gau
4
and 0.989 (wat
e
positive shifts.
parameters, t
h
s
ion coefficie
n
L
orentzian fit
w
h
e instrument
a
o
n
o
f spectral axi
s
Table 1.
q
uency shift and
d
using a high-res
nm) Fre
q
5.6
a
7.5
b
r
respondence
b
an now be ap
p
B
rillouin imag
e
(
right) anti-Stoke
s
ssian functions w
e
e
r), respectively.
N
h
e fitting algo
r
t (
R
2
) which a
r
w
orks better th
a
a
l function.
s
calibration u
s
linewidth for me
t
olution tandem
F
q
uency shift (GH
z
5.5
7.3
b
etween our
p
lied for the ch
a
e
s of collagen
g
s
parts of the Bril
e
re used resulting
N
ote that negativ
e
rithm provide
s
r
e used to ass
e
a
n a Gaussian
f
s
ing methanol
t
hanol and wate
r
F
abry-Pérot inte
r
z
)
-
0.
6
data and tho
s
aracterization
o
g
elatins at two
l
louin spectrum o
f
in R
2
= 0.996 an
d
e frequency shift
s
s
the root me
a
e
ss the quality
f
it, but only m
a
[18] and wate
r
.
a
Data from ref
r
ferometer [4].
Linewidth (GH
z
-
6
5
b
s
e from the
l
of samples of
i
protein conce
n
f
d
s
a
n square
of the fit.
a
rginally,
r [4] data
z
)
0.7
0.9
l
iterature,
i
nterest.
n
trations.
Vol. 10, No. 3 | 1 Mar 2019 | BIOMEDICAL OPTICS EXPRESS 1336
Fig.
60x
Fitting re
s
Fig.
10 wt
Here, we
collagen co
n
concentratio
n
p
reviously re
p
ranging betw
e
These res
u
stiffness of
t
p
lausibly due
4. Conclusi
o
In summary,
visualization
liquids and a
p
linker. We h
a
simplified op
t
The resul
t
formalin con
t
work demon
s
b
iologically r
e
7
. Brillouin spectr
u
w
ate
r
-immersion o
b
s
ults for these
d
8
. Plot of frequen
c
. % collagen conc
e
note an incr
e
n
centration (F
i
n
(Fig. 8(b)).
p
orted for sim
i
e
en
ca.
2 and
3
u
lts suggest th
t
he gel, and
t
to a different
g
o
n
new softwar
e
and extractio
n
p
plied to colla
g
a
ve shown that
t
ical system a
n
t
s show that t
h
t
ent, which su
g
trates the abil
i
e
levant speci
m
u
m of collagen g
e
b
jective, and 3s a
n
d
ata are plotte
d
c
y shift vs. (a) col
l
e
ntration for the g
e
e
ase in frequ
e
i
g. 8(a)) and
The frequenc
i
lar gels [19].
3
GPa were est
i
at increasing
c
t
hat the effec
g
el structure.
e
was develop
n
of spectral
d
g
en gelatin hy
d
we can obtain
n
d bespoke sof
t
h
e Brillouin fr
e
g
gests an incre
a
i
ty of this met
h
m
ens close to p
h
e
latins with 6 and
n
d 2s exposure ti
m
d
in Fig. 8.
l
agen concentrati
o
e
latin hydrogels.
e
ncy shift of
t
upon additi
o
y shifts obta
i
From the data
i
mated.
c
ollagen or fo
r
t is less pro
n
e
d for VIPA
d
ata in real ti
d
rogels, both i
n
accurate spec
t
t
ware algorith
m
e
quency shift
a
se in stiffnes
s
h
od to determi
n
h
ysiological h
y
12 wt. % collag
e
m
e, respectively.
o
n and (b) formal
i
t
he Brillouin
p
o
n of formal
i
i
ned here are
in Fig. 8, lon
g
r
malin concen
t
n
ounced in f
o
Brillouin ima
g
i
me. This wa
s
n
the presence
t
ral data from
t
m
s.
increases wit
h
s
as the water
c
n
e the Brillou
i
y
dration levels
e
n acquired with
a
i
n concentration a
t
peak upon in
c
i
n at constan
consistent
w
g
itudinal elast
i
t
ration leads t
o
o
rmalin-contai
n
g
e analysis to
s
validated on
and absence
o
t
issue phanto
m
h
increasing c
o
c
ontent is red
u
i
n spectral sig
n
s
.
a
t
c
rease of
n
t protein
w
ith those
i
c moduli
o
elevated
n
ing gels
improve
standard
o
f a cross-
m
s using a
o
llagen or
u
ced. This
n
atures of
Vol. 10, No. 3 | 1 Mar 2019 | BIOMEDICAL OPTICS EXPRESS 1337
Funding
UK Engineering and Physical Sciences Research Council; Cancer Research UK
(NS/A000063/1).
Acknowledgement
S.B. was supported by a Leverhulme Trust Visiting Professorship. The authors thank Dr
Kareem Elsayad at Vienna Biocenter Core Facilities for his help with instrument development
and Prof Daniele Fioretto at the University of Perugia, Italy, for helpful discussions.
Data from this work are available upon request to the corresponding author:
f.palombo@exeter.ac.uk.
Disclosures
The authors declare that there are no conflicts of interest related to this article.
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Vol. 10, No. 3 | 1 Mar 2019 | BIOMEDICAL OPTICS EXPRESS 1338
... 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 concentrations are presented in Fig. 1A. ...
... 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 60Â (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 evaluated as~0.9 ...
... 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]. ...
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