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 Andrea Rabolini

phase-retrieval algorithms, like Kramers-Kronig where Ai are the envelope function of the pulses,
(KK) [2]. The CARS technique can reach very ωi χ(2)
di = 4n(ω and ∆k = k3 − k2 − k1 . The ki
1 )c
high acquisition speeds, working in the single- terms are the wavenumbers of the waves. Let us
frequency approach where just one vibrational imagine an interaction of three pulses with fre-
mode is excited at once. In this way one chemi- quencies ω1 , ω2 and ω3 = ω1 + ω2 . The case in
cal species at a time can be studied. Broadband which the wave at ω3 is much more intense than
CARS (B-CARS) allows to acquire entire spec- the wave at ω2 is called Optical Parametric Am-
tra in a single shot. It has two main approaches: plifier (OPA) and it is an energy exchange be-
hyperspectral, where a single vibrational mode tween the ω3 wave (called pump) and the wave
is excited at a time and the full spectrum can at ω2 (called signal). A third wave at ω1 (the
be retrieved by detuning one narrowband beam; idler) is generated due to conservation of energy.
and multiplex, where the Stokes is a broadband To provide efficient signal generation, the phase-
pulse and several vibrational modes are excited matching condition ∆k = 0 must be fulfilled.
simultaneously. If the wave vectors of the three pulses are
A technique that allows high acquisition speeds not collinear, the process takes the name of
is wide-field CARS (WF-CARS), where pump non-collinear OPA (NOPA). In this case, the
and Stokes have a FWHM of hundreds of µm, wavevector k1 is generated along a direction that
enabling acquisition of wide portions of the ensures conservation of momentum:
sample at once. Thus, video-rate imaging
k3 = k1 + k2 (4)
(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-
Figure 1: Wave vector conservation in NOPA.
sented as P = P (l) + P (nl) , highlighting the lin-
ear (P (l) ) and non linear (P (nl) ) components. In NOPAs we can reach both phase matching
The latter term can be written as the sum of and group velocity matching by tuning angles θ
increasing non-linear order: and Ω, thus obtaining a large gain over a large
bandwidth. However, not in all crystal it is
P (nl) = P (2) + P (3) + . . . (1) possible to satisfy both conditions, due to de-
pendences on the refractive indexes and on the
The second-order non-linear polarization, under
wavelengths.
certain approximations, can be written as:
We will now focus on CARS, which is a third-
order non-linear process, thus we will analyze
P (2) (t) = ϵ0 χ(2) E 2 (t) (2)
the P (3) term. The third-order non-linear po-
larization, under certain approximations, can be
where χ(3) is the third order susceptibility of the
written as:
material. The interaction of three monochro-
matic waves at frequencies ω1 , ω2 and ω3 in a P (3) (t) = ϵ0 χ(3) E 3 (t) (5)
χ(2) material can be represented by a system of
where χ(3) is the third-order susceptibility of the
coupled equations:
material. Let us consider an interaction of an in-

∂A1 coming electric field interacting with a χ(3) ma-

 = −i d1 A3 A∗2 e−i∆kz terial. The electric field has two components,

 ∂z
pump and Stokes:

∂A2

= −i d2 A3 A∗1 e−i∆kz (3)
∂z 1
E(z, t) = {Ap (z, t)ei(ωp t−kp z)


 ∂A3 = −i d A A e+i∆kz

2 (6)


3 1 2
∂z +AS (z, t)ei(ωS t−kS z) }

2
 Executive summary Andrea Rabolini

where ωi are their central frequencies and ki sending the pump on the sample almost from
their wavenumbers. The CARS process arises the side; the phase-matching condition is ful-
thanks to the coherence created by the interac- filled on the whole sheet of light, suppressing
tion between pump (ωp ) and Stokes (ωS ), when non-resonant CARS from planes above or be-
the beating frequency Ω = ωp − ωS matches a low the sample. In the second method, pump
molecular vibration ΩR = Ω. and Stokes are in a non-phase-matching con-
The resulting polarization is: figuration because in the sample, thanks to re-
flection and scattering of particles, some por-
3ϵ0 χ(3) 2 ∗ i[ωaS t−(2kp −kS )z] tion of the incoming light is brought into the
Pω(3)
aS
(z, t) = Ap AS e + c.c.
4 phase-matching condition, creating the CARS
(7) signal. In our setup, we implement a non-phase-
which is oscillating at ωaS = 2ωp −ωS frequency. matching configuration with collinearly focused
The CARS intensity is: 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
 
2 ∆kL
ICARS (L) ∝ χ(3) Ip2 IS L2 sinc2 (8)
2 sample thickness (≈10 µm). This ensures effi-
cient CARS signal generation even without the
where ∆k = 2kp − kS . The CARS signal scales
fulfilment of the phase-matching condition.
quadratically with the pump intensity Ip and lin-
early with the Stokes intensity IS . To obtain
high intensities, the phase-matching condition
4. Experimental setup
(∆k = 0) should be fulfilled. The setup is schematically represented in
Fig.2. The source is a Coherent Monaco, an
3. Wide-field CARS Ytterbium-amplified laser, emitting at 1035 nm
with a spectral bandwidth of 10 nm. For our ex-
Traditionally CARS microscopy is performed in
periments, we used a total power of 40 W, with
raster scanning fashion. The two beams are
a repetition rate of 2 MHz and pulse duration of
tightly focused onto the sample employing high
269 fs.
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 Figure 2: Scheme of the experimental setup.
are bigger than in point-scanning CARS, typi- Legend: M: mirror; L: lens; SPF: short pass fil-
cally hundreds of µm. Thus it is possible to ac- ter; LPF: long pass filter; HWP: half wave plate;
quire whole regions even in a single shot, allow- PBS: polarizing beam splitter; OBJ: objective
ing video-rate imaging. The major drawback of lens.
WF-CARS is that wide-field illumination does
not present the spatial filtering capabilities of- To ensure enough Stokes power on the sample
fered by confocal microscopes, thus giving lower and therefore enough CARS signal, the Stokes
spatial resolution and lower axial sectioning ca- beam has to be amplified: for this reason we
pability. Two main approaches for WF-CARS implemented a NOPA. After the laser output,
illumination have been implemented: the first a half-wave plate (HWP) brings part of the
method consists in illuminating the sample with beam into vertical polarization. A first polar-
a thin "sheet of light" (5-10 µm) by means of izing beam splitter (PBSW-1030) is mounted.
a dark-field condenser with high NA (typically The reflected component gives rise to the pump
NA=1.2-1.4) which creates a "cone" of light,

3
 Executive summary Andrea Rabolini

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. Figure 3: Spectrum of the white-light continuum
The transmitted component of the second (WLC) signal generated in the YAG crystal after
PBSW-1030 acts as the Stokes beam in the the FELH1050 (blue). Spectrum of WLC after
CARS process. A DL tunes the delay between the OPA (red).
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 light.
is focused with a 75 mm lens on a 10-mm-thick After the OPA, a FELH1050 is placed and the
yttrium aluminium garnet (YAG) crystal that beam is collimated with a f=400 mm lens. Af-
provides white-light generation (WLG), broad- terwards, there is a 4-f system, which consists
ening the spectrum up to 1500 nm. WLG in of a blazed grating, a 2-inch lens and a 5×5 cm
bulk media is a compact, robust, simple and mirror. This system has paramount importance
alignment-insensitive technique [2]. After the in our setup since it allows us to separate and
YAG, a 75 mm lens is positioned slightly out of select different wavelengths in a precise and re-
focus so that the beam is not collimated but refo- peatable way. Indeed, we would like to acquire
cused on a 2-mm-thick β-Barium Borate crystal hypercubes where the frames are taken at a sin-
(BBO) crystal. gle wavelength. By selecting a pixel in the frame,
The beam reflected by the first PBSW1030 is fo- we can reconstruct the spectra of the sample
cused by a f=300 mm lens on a 3-mm-thick BBO analyzed. The selection is possible thanks to
crystal (cut at θ=23.4°, ϕ=90°), which provides a slit inserted on the mirror plane. The slit is
second harmonic generation (SHG). The angle constituted by two sharp edges at a very nar-
of the crystal was finely adjusted in order to ful- row distance. To change the frequency of the
fil type-I phase matching in the BBO, where the Stokes beam, we rotate the grating, thus chang-
seed is vertical and the second harmonic wave is ing the wavelength that is selected by the slit.
horizontal. The power on the BBO is 15 W and In this way we can calibrate the system, know-
each pulse has an energy of 7.5 µJ. The output ing at which angle correspond each wavelength.
power of the SH is 3.76 W, meaning the effi- The calibration of the 4-f system is fundamen-
ciency is about 25%. The green and the Stokes tal since the hypercubes at single wavelength of
beam are spatially and temporally overlapped CARS signal are acquired with a camera, which
on the second BBO crystal, giving rise to NOPA does not provide the wavelength of the incoming
process with a non-collinear internal angle of 5°, beam. The 4-f system is also used to compress
that was found to be optimal in our case [4]. the pulse duration (Fig. 4). The signal that ap-
The parametric amplification takes place in the pears at around 1000 nm is due to 3-color CARS,
BBO (cut at θ=23.4°, ϕ=90°) crystal exploiting an effect similar to CARS in which the Stokes
type-I phase matching. The power of the beam acts both as Stokes and probe beam. This ef-
passes from 40 mW to almost 300 mW in the fect is present only when the pulse is very com-
1100-1300 nm region (Fig. 3). The most chal- pressed in time (e.g. 20 fs). The optimization
lenging part of my work was to carefully adjust of the 4-f system for these two applications was
several different parameters in order to optimize one of the most challenging parts of my work.
the NOPA process, by fine-tuning WLG and sec- The pump and Stokes beam are recombined
ond harmonic generation (SHG) for the green with a beam combiner and collinearly loosely
focused on the sample with a f=100 mm lens

4
 Executive summary Andrea Rabolini

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- Figure 5: Broadband CARS image of 8-µm PS
compensated. The sharp cut is due to the and 10-µm PMMA beads in DMSO. The image
FESH1000 filter. is 666×558 pixels.

in a non-phase-matching configuration [5]. Af- In Fig. 5 we show a multiplex CARS image of

ter the interaction with the sample, an objective the beads. Knowing their real dimensions (8-
lens collects the light and collimates the beam. 10 µm), we can estimate that the field of view
The 100 mm lens produces spots on the sam- (FOV) is 100-130 µm. By adjusting the di-
ple of ≈100 µm for the Stokes and ≈200 µm mensions of pump and Stokes beams, it is pos-
for the pump: these values are similar to those sible to enlarge the FOV. We acquired hyper-
present in literature [5]. The incoming power of cubes at single wavenumber. The rotating grat-
the pump beam is 1.5 W, the Stokes power is ing was moved step-by-step in order to cover
≈40 mW without the slit and few µW with the the wavelength range interesting for our applica-
slit. tions. Knowing the angle span and the number
After the sample, a FESH1000 cuts the exciting of steps (350), each position of the rotation stage
beams and passes only the CARS signal. A flip is associated to a Stokes wavelength and conse-
mirror is used to decide whether to acquire the quently to a CARS wavenumber. The frames
spectrum of the signal or let the signal pass and were acquired with an exposure time of the cam-
go into the camera (sCMOS, Prime BSI), with era of 2 ms. By selecting a single pixel on the
which we acquired hyperspectral cubes. Using frame, we can look at the detected intensities
the spectrometer with WF-CARS is useless be- of that pixel along the frames (i.e. along differ-
cause all the richness of information due to WF ent wavenumbers) and reconstruct the spectrum
illumination would be lost. We used the spec- of that single pixel. The total time of acquisi-
trometer to control the chirp of the signal or the tion of a single hypercubes is on the order of 3-4
delay between pump and Stokes. minutes, but in the future by exploiting trigger
To acquire hypercubes, we select a region of in- pulses we aim to reduce this time. The time to
terest (ROI) on the camera and we acquire a acquire a frame is of the order of 650 ms. In
sequence of images at single wavelength. This this time there are long dead times due to the
procedure is done also in scanning CARS mi- rotation stage that increase a lot the acquisition
croscopy [2], but the big advantage of our setup times and the savings of the data in the disk.
is the reduced acquisition times thanks to WF Considering 650 ms as total time and a ROI of
excitation. 200×200 pixels (which corresponds to an area
of 1.3×1.3 mm2 ), the acquisition time for each
5. Results pixel over the entire CARS spectrum is ≈6 ms,
We performed measurements on some solvents which guarantees a video rate and is comparable
(DMSO and toluene) and on a sample containing to raster scanning CARS. Without dead times,
8-µm beads of PS and 10-µm beads of PMMA the acquisition time can easily decrease to 50 ms
in DMSO. and is limited by the saving of data to disk. The

5
 Executive summary Andrea Rabolini

total acquisition time can become almost 20 s, home-made microscope. With respect to scan-
leading to an acquisition time for each pixel over ning CARS, WF-CARS requires reduced times
the whole CARS spectrum of ≈437 µs, which is of acquisition. This unlocks unprecedented ac-
better than the state of the art of WF-CARS. quisition speed, useful to observe high-speed bi-
The first sample analyzed is the DMSO. The re- ological phenomena.
trieved spectrum is shown in Fig. 6. 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
Figure 6: CARS spectrum of DMSO recon- hyperspectral images of biological samples at the
structed started from the intensity of a chosen video-rate level, allowing us to study fast biolog-
pixel along 350 frames of a hypercube. ical processes.
An important improvement in the setup would
We performed the phase retrieval using a KK be the implementation of a Fourier-Transform
algorithm. During the process, the retrieved detection to more easily analyze the hypercubes.
spectrum was slightly shifted in order to match In this way, the slit and the calibration of the
the spontaneous Raman peaks. For the phase- rotational stage are no more needed, improving
retrieval algorithm, the pure NRB spectrum is the precision and repeatability of measurements.
required: on the sample stage we positioned the
glass and the coverslip without the solvent. References
The result is shown in Fig. 7 along with the [1] Dario Polli et al. Broadband coherent ra-
spontaneous Raman spectrum of DMSO. We can man scattering microscopy. Laser & Pho-
see that there is a clear correspondence of the tonics Reviews, 12(9):1800020, 2018.
peaks at 667 cm−1 , 1043 cm−1 and 1420 cm−1 .
[2] Federico Vernuccio et al. Fingerprint multi-
6. Conclusions plex cars at high speed based on supercon-
This master thesis is focused on the design and tinuum generation in bulk media and deep
development of a wide-field broadband CARS learning spectral denoising. Opt. Express,
30(17):30135–30148, Aug 2022.

[3] Chiara Ceconello et al. Wide-field broad-

band cars microscopy. EPJ Web of Confer-
ences, 266, 10 2022.

[4] Andrea Villa et al. Broadly tunable mid-

infrared femtosecond pulses directly gener-
ated by an optical parametric amplifier. OSA
Continuum, 4(11):2837–2844, Nov 2021.

[5] Yujie Shen et al. Wide-field coherent anti-

Figure 7: Spontaneous (blue) and retrieved (red) stokes raman scattering microscopy based
Raman spectrum of DMSO. The retrieved spec- on picosecond supercontinuum source. APL
trum is obtained by removing the NRB from the Photonics, 3(11):116104, 2018.
spectrum in Fig. 6 with a Kramers-Kronig algo-
rithm.