Executive Summary of the Thesis

High-speed Wide-field Broadband Coherent Raman Microscopy

Laurea Magistrale in Engineering Physics - Ingegneria Fisica

Author: Andrea Rabolini

Advisor: Prof. Dario Polli
Co-advisor: Chiara Ceconello
Academic year: 2021-2022

1. Introduction gion (2700-3100 cm−1 ). However, SR suffers

from low scattering cross section (10−30 cm2 ),
Conventional bright-field microscopy is a widely
requiring long integration times. This limitation
used technique in life sciences providing high-
can be overcome with Coherent Raman Scat-
resolution images, but no information about
tering (CRS) techniques, exploiting third-order
chemical composition can be retrieved. Fluores-
non-linear optical effects. The coherent vibra-
cence microscopy exploits a fluorescent tag (a
tional response Ω is triggered employing two
fluorophore) which binds to a precise molecule,
spatially and temporally synchronized beams,
presenting high specificity and selectivity. How-
pump ωp and Stokes ωS = ωp −Ω. CRS presents
ever, the addition of a fluorescent marker can
several advantages, among which the higher sig-
perturb permanently the sample and compro-
nal with respect to SR, allowing high acqui-
mise its biological functions. To solve these
sition speeds, up to video-rate imaging. One
problems a label-free imaging technique is
of the most used CRS techniques is Coherent
needed, such as Spontaneous Raman (SR), an
anti-Stokes Raman Scattering (CARS). This is a
inelastic process that allows to study the unique
four-wave mixing process where the vibrational
vibrational spectrum of a chemical species. In
coherence set up by pump and Stokes is probed
SR, a molecule is excited to a virtual state
by a third beam (the probe, ωpr ), generating
by a quasi-monochromatic laser beam at a fre-
a fourth beam, the anti-Stokes, at frequency
quency ωp (pump), and it relaxes emitting a red-
ωaS = ωpr + Ω (in general, ωpr = ωp , there-
shifted photon at Stokes frequency ωS = ωp − Ω,
fore ωaS = ωp + Ω). The detected frequency ωaS
where Ω is the frequency of a molecular vi-
is at lower wavelengths with respect to pump
bration [1]. The vibrational spectrum can be
and Stokes, therefore these two pulses are cut
divided into three regions: the fingerprint re-
with a low-pass filter. A great disadvantage
gion (500-1800 cm−1 ), dense in peaks and it can
of CARS is the fact that the four beams can
provide detailed chemical information for bio-
also interact in a non-resonant way, giving rise
logical samples due to contributions from pro-
to the non-resonant background (NRB), which
teins and nucleic acids; the silent region (1800-
distorts the peaks and prevents a direct com-
2700 cm−1 ), where no natural peaks are present
parison with the SR signal. A way to retrieve
and the carbon-hydrogen (C-H) stretching re-
the CARS signal removing the NRB is to use

1
 Executive summary
phase-retrieval algorithms, like Kramers-Kronig
(KK) [2]. The CARS technique can reach very
high acquisition speeds, working in the single-
frequency approach where just one vibrational
mode is excited at once. In this way one chemi-
cal species at a time can be studied. Broadband
CARS (B-CARS) allows to acquire entire spec-
tra in a single shot. It has two main approaches:
hyperspectral, where a single vibrational mode
is excited at a time and the full spectrum can
be retrieved by detuning one narrowband beam;
and multiplex, where the Stokes is a broadband
pulse and several vibrational modes are excited
simultaneously.
A technique that allows high acquisition speeds
is wide-field CARS (WF-CARS), where pump
and Stokes have a FWHM of hundreds of µm,
enabling acquisition of wide portions of the
sample at once. Thus, video-rate imaging
(1 ms/frame) can be achieved more easily than
in scanning CARS and fast biological dynamics
can be investigated in real time [3].
2. Theory
If light intensities are higher than approximately
109 W/cm2 , the response of the material be-
comes non-linear and a perturbative approach
must be used. The polarization can be repre-
sented as P = P (l) + P (nl) , highlighting the lin-
ear (P (l) ) and non linear (P (nl) ) components.
The latter term can be written as the sum of
increasing non-linear order:
P (nl) = P (2) + P (3) + . . .
The second-order non-linear polarization, under
certain approximations, can be written as:
P (2) (t) = ϵ0 χ(2) E 2 (t)
where χ(3) is the third order susceptibility of the
material. The interaction of three monochro-
matic waves at frequencies ω1 , ω2 and ω3 in a
χ(2) material can be represented by a system of
coupled equations:

∂A1

 = −i d1 A3 A∗2 e−i∆kz

 ∂z

∂A2

= −i d2 A3 A∗1 e−i∆kz
∂z


 ∂A3 = −i d A A e+i∆kz



3 1 2
∂z
Andrea Rabolini
where Ai are the envelope function of the pulses,
ωi χ(2)
di = 4n(ω and ∆k = k3 − k2 − k1 . The ki
1 )c
terms are the wavenumbers of the waves. Let us
imagine an interaction of three pulses with fre-
quencies ω1 , ω2 and ω3 = ω1 + ω2 . The case in
which the wave at ω3 is much more intense than
the wave at ω2 is called Optical Parametric Am-
plifier (OPA) and it is an energy exchange be-
tween the ω3 wave (called pump) and the wave
at ω2 (called signal). A third wave at ω1 (the
idler) is generated due to conservation of energy.
To provide efficient signal generation, the phase-
matching condition ∆k = 0 must be fulfilled.
If the wave vectors of the three pulses are
not collinear, the process takes the name of
non-collinear OPA (NOPA). In this case, the
wavevector k1 is generated along a direction that
ensures conservation of momentum:
k3 = k1 + k2 (4)
Figure 1: Wave vector conservation in NOPA.
In NOPAs we can reach both phase matching
and group velocity matching by tuning angles θ
and Ω, thus obtaining a large gain over a large
bandwidth. However, not in all crystal it is
(1) possible to satisfy both conditions, due to de-
pendences on the refractive indexes and on the
wavelengths.
We will now focus on CARS, which is a third-
order non-linear process, thus we will analyze
(2)
the P (3) term. The third-order non-linear po-
larization, under certain approximations, can be
written as:
P (3) (t) = ϵ0 χ(3) E 3 (t) (5)
where χ(3) is the third-order susceptibility of the
material. Let us consider an interaction of an in-
coming electric field interacting with a χ(3) ma-
terial. The electric field has two components,
pump and Stokes:
(3)
1
E(z, t) = {Ap (z, t)ei(ωp t−kp z)
2 (6)
+AS (z, t)ei(ωS t−kS z) }
2
 Executive summary
where ωi are their central frequencies and ki
their wavenumbers. The CARS process arises
thanks to the coherence created by the interac-
tion between pump (ωp ) and Stokes (ωS ), when
the beating frequency Ω = ωp − ωS matches a
molecular vibration ΩR = Ω.
The resulting polarization is:
3ϵ0 χ(3) 2 ∗ i[ωaS t−(2kp −kS )z]
Pω(3)
aS
(z, t) = Ap AS e + c.c.
4 phase-matching condition, creating the CARS
(7)
which is oscillating at ωaS = 2ωp −ωS frequency.
The CARS intensity is:
 
2 ∆kL
ICARS (L) ∝ χ(3) Ip2 IS L2 sinc2
2 sample thickness (≈10 µm). This ensures effi-
where ∆k = 2kp − kS . The CARS signal scales
quadratically with the pump intensity Ip and lin-
early with the Stokes intensity IS . To obtain
high intensities, the phase-matching condition
(∆k = 0) should be fulfilled.
3. Wide-field CARS
Traditionally CARS microscopy is performed in
raster scanning fashion. The two beams are
tightly focused onto the sample employing high
numerical aperture (NA) objectives and signal is
generated in a small portion (i.e. a pixel) of the
sample. To generate an image, one must perform
raster scanning, either using galvanometric mir-
rors to move the excitation beams or piezoelec-
tric stage over which the sample is fixed. This
causes some problems, for example it is time-
consuming since one mirrors or the piezo have
to move over a region which can be 100×100
pixels or even more. In wide-field CARS (WF-
CARS), the pump and the Stokes on the sample
are bigger than in point-scanning CARS, typi-
cally hundreds of µm. Thus it is possible to ac-
quire whole regions even in a single shot, allow-
ing video-rate imaging. The major drawback of
WF-CARS is that wide-field illumination does
not present the spatial filtering capabilities of-
fered by confocal microscopes, thus giving lower
spatial resolution and lower axial sectioning ca-
pability. Two main approaches for WF-CARS
illumination have been implemented: the first
method consists in illuminating the sample with
a thin "sheet of light" (5-10 µm) by means of
a dark-field condenser with high NA (typically
NA=1.2-1.4) which creates a "cone" of light,
Andrea Rabolini
sending the pump on the sample almost from
the side; the phase-matching condition is ful-
filled on the whole sheet of light, suppressing
non-resonant CARS from planes above or be-
low the sample. In the second method, pump
and Stokes are in a non-phase-matching con-
figuration because in the sample, thanks to re-
flection and scattering of particles, some por-
tion of the incoming light is brought into the
signal. In our setup, we implement a non-phase-
matching configuration with collinearly focused
beams. The coherence length (i.e. the length in
which the maximum of the anti-Stokes signal is
generated) is ≈30 µm, which is higher than the
(8)
cient CARS signal generation even without the
fulfilment of the phase-matching condition.
4. Experimental setup
The setup is schematically represented in
Fig.2. The source is a Coherent Monaco, an
Ytterbium-amplified laser, emitting at 1035 nm
with a spectral bandwidth of 10 nm. For our ex-
periments, we used a total power of 40 W, with
a repetition rate of 2 MHz and pulse duration of
269 fs.
Figure 2: Scheme of the experimental setup.
Legend: M: mirror; L: lens; SPF: short pass fil-
ter; LPF: long pass filter; HWP: half wave plate;
PBS: polarizing beam splitter; OBJ: objective
lens.
To ensure enough Stokes power on the sample
and therefore enough CARS signal, the Stokes
beam has to be amplified: for this reason we
implemented a NOPA. After the laser output,
a half-wave plate (HWP) brings part of the
beam into vertical polarization. A first polar-
izing beam splitter (PBSW-1030) is mounted.
The reflected component gives rise to the pump
3
 Executive summary
for the OPA. Afterwards, another HWP rotates
the polarization in order to have again vertical
and horizontal polarization that are later sepa-
rated by a PBSW-1030. The reflected compo-
nent originates the pump for the CARS process,
while the transmitted one acts as the broadband
Stokes beam in the CARS process.
The spectrum of the pump of the CARS process
is shrunk by an etalon and reaches a delay line
(DL) that controls the delay between pump and
Stokes.
The transmitted component of the second
PBSW-1030 acts as the Stokes beam in the
CARS process. A DL tunes the delay between
the Stokes beam and the pump of the OPA to en-
sure temporal overlap. Two HWP and a PBSW-
1030 finely tune the power of the beam, which
is focused with a 75 mm lens on a 10-mm-thick
yttrium aluminium garnet (YAG) crystal that
provides white-light generation (WLG), broad-
ening the spectrum up to 1500 nm. WLG in
bulk media is a compact, robust, simple and
alignment-insensitive technique [2]. After the
YAG, a 75 mm lens is positioned slightly out of
focus so that the beam is not collimated but refo-
cused on a 2-mm-thick β-Barium Borate crystal
(BBO) crystal.
The beam reflected by the first PBSW1030 is fo-
cused by a f=300 mm lens on a 3-mm-thick BBO
crystal (cut at θ=23.4°, ϕ=90°), which provides
second harmonic generation (SHG). The angle
of the crystal was finely adjusted in order to ful-
fil type-I phase matching in the BBO, where the
seed is vertical and the second harmonic wave is
horizontal. The power on the BBO is 15 W and
each pulse has an energy of 7.5 µJ. The output
power of the SH is 3.76 W, meaning the effi-
ciency is about 25%. The green and the Stokes
beam are spatially and temporally overlapped
on the second BBO crystal, giving rise to NOPA
process with a non-collinear internal angle of 5°,
that was found to be optimal in our case [4].
The parametric amplification takes place in the
BBO (cut at θ=23.4°, ϕ=90°) crystal exploiting
type-I phase matching. The power of the beam
passes from 40 mW to almost 300 mW in the
1100-1300 nm region (Fig. 3). The most chal-
lenging part of my work was to carefully adjust
several different parameters in order to optimize
the NOPA process, by fine-tuning WLG and sec-
ond harmonic generation (SHG) for the green
4
Andrea Rabolini
Figure 3: Spectrum of the white-light continuum
(WLC) signal generated in the YAG crystal after
the FELH1050 (blue). Spectrum of WLC after
the OPA (red).
light.
After the OPA, a FELH1050 is placed and the
beam is collimated with a f=400 mm lens. Af-
terwards, there is a 4-f system, which consists
of a blazed grating, a 2-inch lens and a 5×5 cm
mirror. This system has paramount importance
in our setup since it allows us to separate and
select different wavelengths in a precise and re-
peatable way. Indeed, we would like to acquire
hypercubes where the frames are taken at a sin-
gle wavelength. By selecting a pixel in the frame,
we can reconstruct the spectra of the sample
analyzed. The selection is possible thanks to
a slit inserted on the mirror plane. The slit is
constituted by two sharp edges at a very nar-
row distance. To change the frequency of the
Stokes beam, we rotate the grating, thus chang-
ing the wavelength that is selected by the slit.
In this way we can calibrate the system, know-
ing at which angle correspond each wavelength.
The calibration of the 4-f system is fundamen-
tal since the hypercubes at single wavelength of
CARS signal are acquired with a camera, which
does not provide the wavelength of the incoming
beam. The 4-f system is also used to compress
the pulse duration (Fig. 4). The signal that ap-
pears at around 1000 nm is due to 3-color CARS,
an effect similar to CARS in which the Stokes
acts both as Stokes and probe beam. This ef-
fect is present only when the pulse is very com-
pressed in time (e.g. 20 fs). The optimization
of the 4-f system for these two applications was
one of the most challenging parts of my work.
The pump and Stokes beam are recombined
with a beam combiner and collinearly loosely
focused on the sample with a f=100 mm lens
 Executive summary
Figure 4: Plots representing CARS signal for dif-
ferent delays between pump and Stokes. When
the delay is changed, the CARS signal decreases
uniformly: this means that the chirp is well-
compensated. The sharp cut is due to the
FESH1000 filter.
in a non-phase-matching configuration [5]. Af-
ter the interaction with the sample, an objective
lens collects the light and collimates the beam.
The 100 mm lens produces spots on the sam-
ple of ≈100 µm for the Stokes and ≈200 µm
for the pump: these values are similar to those
present in literature [5]. The incoming power of
the pump beam is 1.5 W, the Stokes power is
≈40 mW without the slit and few µW with the
slit.
After the sample, a FESH1000 cuts the exciting
beams and passes only the CARS signal. A flip
mirror is used to decide whether to acquire the
spectrum of the signal or let the signal pass and
go into the camera (sCMOS, Prime BSI), with
which we acquired hyperspectral cubes. Using
the spectrometer with WF-CARS is useless be-
cause all the richness of information due to WF
illumination would be lost. We used the spec-
trometer to control the chirp of the signal or the
delay between pump and Stokes.
To acquire hypercubes, we select a region of in-
terest (ROI) on the camera and we acquire a
sequence of images at single wavelength. This
procedure is done also in scanning CARS mi-
croscopy [2], but the big advantage of our setup
is the reduced acquisition times thanks to WF
excitation.
5. Results
We performed measurements on some solvents
(DMSO and toluene) and on a sample containing
8-µm beads of PS and 10-µm beads of PMMA
in DMSO.
5
Andrea Rabolini
Figure 5: Broadband CARS image of 8-µm PS
and 10-µm PMMA beads in DMSO. The image
is 666×558 pixels.
In Fig. 5 we show a multiplex CARS image of
the beads. Knowing their real dimensions (8-
10 µm), we can estimate that the field of view
(FOV) is 100-130 µm. By adjusting the di-
mensions of pump and Stokes beams, it is pos-
sible to enlarge the FOV. We acquired hyper-
cubes at single wavenumber. The rotating grat-
ing was moved step-by-step in order to cover
the wavelength range interesting for our applica-
tions. Knowing the angle span and the number
of steps (350), each position of the rotation stage
is associated to a Stokes wavelength and conse-
quently to a CARS wavenumber. The frames
were acquired with an exposure time of the cam-
era of 2 ms. By selecting a single pixel on the
frame, we can look at the detected intensities
of that pixel along the frames (i.e. along differ-
ent wavenumbers) and reconstruct the spectrum
of that single pixel. The total time of acquisi-
tion of a single hypercubes is on the order of 3-4
minutes, but in the future by exploiting trigger
pulses we aim to reduce this time. The time to
acquire a frame is of the order of 650 ms. In
this time there are long dead times due to the
rotation stage that increase a lot the acquisition
times and the savings of the data in the disk.
Considering 650 ms as total time and a ROI of
200×200 pixels (which corresponds to an area
of 1.3×1.3 mm2 ), the acquisition time for each
pixel over the entire CARS spectrum is ≈6 ms,
which guarantees a video rate and is comparable
to raster scanning CARS. Without dead times,
the acquisition time can easily decrease to 50 ms
and is limited by the saving of data to disk. The
 Executive summary
total acquisition time can become almost 20 s,
leading to an acquisition time for each pixel over
the whole CARS spectrum of ≈437 µs, which is
better than the state of the art of WF-CARS.
The first sample analyzed is the DMSO. The re-
trieved spectrum is shown in Fig. 6.
Figure 6: CARS spectrum of DMSO recon-
structed started from the intensity of a chosen
pixel along 350 frames of a hypercube.
We performed the phase retrieval using a KK
algorithm. During the process, the retrieved
spectrum was slightly shifted in order to match
the spontaneous Raman peaks. For the phase-
retrieval algorithm, the pure NRB spectrum is
required: on the sample stage we positioned the
glass and the coverslip without the solvent.
The result is shown in Fig. 7 along with the
spontaneous Raman spectrum of DMSO. We can
see that there is a clear correspondence of the
peaks at 667 cm−1 , 1043 cm−1 and 1420 cm−1 .
6. Conclusions
This master thesis is focused on the design and
development of a wide-field broadband CARS
Figure 7: Spontaneous (blue) and retrieved (red)
Raman spectrum of DMSO. The retrieved spec-
trum is obtained by removing the NRB from the
spectrum in Fig. 6 with a Kramers-Kronig algo-
rithm.
Andrea Rabolini
home-made microscope. With respect to scan-
ning CARS, WF-CARS requires reduced times
of acquisition. This unlocks unprecedented ac-
quisition speed, useful to observe high-speed bi-
ological phenomena.
The setup has been tested to detect CARS spec-
tra of test samples both in multiplex and hyper-
spectral CARS configurations. We acquired also
some CARS hypercubes from which we recon-
structed the spectrum and applied a Kramers-
Kronig algorithm to retrieve the Raman signal.
We were able to acquire some images of a bio-
logical sample (NASH liver). We have obtained
video-rate imaging (6 ms/pixel) on test samples,
but the acquisition time can be reduced down to
≈0.5 ms/pixel. In the future we aim to acquire
hyperspectral images of biological samples at the
video-rate level, allowing us to study fast biolog-
ical processes.
An important improvement in the setup would
be the implementation of a Fourier-Transform
detection to more easily analyze the hypercubes.
In this way, the slit and the calibration of the
rotational stage are no more needed, improving
the precision and repeatability of measurements.
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