arXiv:physics/0312114v1 [physics.optics] 18 Dec 2003
Nonlinear optical contrast enhancement
for optical coherence tomography
Claudio Vinegoni, Jeremy S. Bredfeldt, and Daniel L. Marks
Beckman Institute for Advanced Science and Technology, University of Illinois at
Stephen A. Boppart
Department of Electrical and Computer Engineering
Beckman Institute for Advanced Science and Technology, Department of Bioengineering
College of Medicine
University of Illinois at Urbana-Champaign, 405 N. Mathews Ave., Urbana, IL 61801
Coherent Anti-Stokes Raman Scattering (CARS) and Second Harmonic
Generation (SHG) signals. Heterodyne detection is employed to increase
the sensitivity in both CARS and SHG signal detection, which can also be
extended to different coherent processes. The exploitation of the mentioned
optical nonlinearities for molecular contrast enhancement in Optical
Coherence Tomography (OCT) is presented. Numerical simulations for
both coherent nonlinear processes are performed in order to determine
the properties of the signal expected at the exit of the described nonlinear
We present a new interferometric technique for measuring
© 2008 Optical Society of America
OCIS codes: (111.4500) Optical coherence tomography; (300.6230) Spectroscopy, coherent
anti-Stokes Raman scattering; (190.4410) Nonlinear optics, parametric processes; (120.3180)
Interferometry; (040.2840) Heterodyne; (190.4160) Multiharmonic generation.
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Optical coherence tomography (OCT) is an emerging biomedical imaging technology that has
been applied to a wide range of biological,medical, and materials investigations.OCT was first
developed in the early 1990s for noninvasive imaging of biological tissue  and is capable of
imaging tissue microstructures at near histological resolutions . Axial resolution of 10 µm
is common for standard OCT, where for ultrahigh resolution OCT, an axial resolution in the 1
µm range has been recently achieved using broadband-continuumgeneration from a photonic
crystal fiber .
The advantages of OCT in biomedical imaging, compared to other imaging techniques, are
quite numerous . In particular, OCT can provide imaging resolutions that approach those of
conventional histopathology and can be performed in situ . Despite its advantages, a serious
drawback to OCT is that the linear scattering properties of pathological tissue probed by OCT
are often morphologicallyand/or optically similar to the scattering properties of normal tissue.
For example, although morphological differences between normal and neoplastic tissues may
be obvious at later stages of tumor development, it is frequently difficult to optically detect
early-stage tumors .
This implies a need for novel contrast enhancing mechanisms for OCT. Examples of meth-
ods that have been recently developed, include: spectroscopic OCT , pump and probe tech-
niques , and the use of engineeredmicrospheres or microbubbles. SpectroscopicOCT
(SOCT) measures the spectral absorption from tissues by measuring the spectral differences
between the source and the backscattered interference signal to provide information about the
properties of the scatterers in the sample. However, this technique is limited to the identifica-
tion of scatterers that have absorption within the bandwidth of the optical source. Pump and
probe contrast enhancement for OCT imaging relies on transient absorptions in the sample un-
der investigation that are induced by an external pump beam. Unfortunately it is necessary in
most cases to introduce different contrast agents depending on the excitation source and on the
transient spectra of the molecules under investigation . An alternative way to obtain contrast
enhancement in OCT includes the use of exogenous contrast agents such as engineered micro-
spheres. These microspheres can be targeted to cell receptors and change the optical scattering
or absorption characteristics in selected regions, providing molecular specific contrast .
As clearly evidencedabove,most of the current methods (if not all) that are currentlyused to
biology under investigation. It follows there is a need for new techniques that could help elim-
inate this limitation. In this paper, we propose and present new methods to achieve enhanced
OCT contrast, exploitingoptical nonlinearities. The nonlineareffects on which we focus in this
work in particular are Coherent Anti-Stokes Raman Scattering (CARS) and Second Harmonic
Generation (SHG), but the general idea could be easily extended to other nonlinear effects
such as Third Harmonic Generation (THG), Coherent Stokes Raman Spectroscopy (CSRS),
and stimulated emission in active materials (i.e. InGaAs, GaAs based materials, etc.).
2. CARS contrast enhancement
It is well known that the nonlinear polarization for a material can be expressed as a function of
the incident electric field vector¯E:
χ(1)·¯E +χ(2):¯E¯E +χ(3)...¯E¯E¯E +...
¯P = ε0
with¯P the induced polarization, χ(n)the n-th order nonlinear susceptibility, and ε0the vacuum
permittivity. This implies that for high intensities (i.e. in a nonlinear regime) the induced po-
larization is no longer directly proportional to the incoming electric field vector¯E. Usually the
first term χ(1)represents the main contribution to¯P and describes linear effects as absorption
or reflection. The second term is responsible for nonlinear effects like SHG and sum-frequency
generation which we will consider in Sect. 3. The third term χ(3)is responsible for phenom-
ena involving four photons, like CARS, four wave mixing (FWM), third harmonic generation
(THG), and nonlinear refraction.
In this section we focus on CARS, a spectroscopic technique that has recently received in-
creasing attention for its applications for vibrational imaging . In CARS spectroscopy, the
frequencies of two incident lasers, ωpand ωs(Pump and Stokes, respectively), are selected
such that the difference in frequency ωp-ωs= ωvis equal to the frequency of a Raman-active
vibrational mode present in the molecule under study . As evidenced from Eq.1, CARS is
a nonlinear, four-wave mixing process. It follows the CARS field is a result of the interaction
between four photons and is generated in the phase-matching direction at the anti-Stokes fre-
quency ωAS= 2ωp−ωs, implying that the CARS signal intensity is linearly dependent on the
Stokes field intensity and quadratically dependent on the pump field intensity. Note that CARS
is a coherent process, with the phase of the anti-Stokes field related to the phase of the excita-
tion field. Therefore, constructive interference of the anti-Stokes field causes the CARS signal
to be significantly larger than the spontaneous Raman signal, given the same average excitation
power . All these characteristics have allowed CARS to be successfully employed to pro-
vide vibrational contrast in scanning microscopy [9, 11, 12, 13]. CARS microscopy generally
involves scanning overlapped and tightly focused pump and Stokes lasers through a sample
while measuring the anti-Stokes signal amplitude point by point . The first CARS micro-
scope  used non-collinear pump and Stokes visible lasers to prove microscopic imaging of
the spatial distribution of deuterated molecular bonds in a sample of onion skin.
Fig. 1. Simplified energy diagram for CARS. ωPindicates the pump laser frequency, ωs
the Stokes laser frequency, ωASthe Anti-Stokes frequency (emitted CARS signal), and ωv
the Raman frequency corresponding to an active vibrational transition.
Picosecond lasers were used by Hashimoto et al. to improve the Raman spectral res-
olution and to further reduce the non-resonant background signal. Cheng et al. theoreti-
cally evaluated the use of CARS in microscopy and recently used polarization, epi-directional,
counter propagating, and forward CARS microscopy to study cell biology. Multiplex CARS
imaging has also been demonstrated to providecontrast based on one or more vibrational spec-
tral features .
In each of these CARS microscopy techniques, the anti-Stokes photons are counted in order
to estimate the density of the Raman scatterers in the focal volume of the microscope. Unfortu-
nately, the spectral phase information is lost in this process. In this paper we present a new in-
terferometric technique called Nonlinear Interferometric Vibrational Imaging (NIVI)  with
the capability for heterodyne detection and the possibility to obtain a full reconstruction of the
magnitude and phase of the sample Raman susceptibility [16, 17].
2.2. Experimental Setup
The laser system constructedto create the laser fields necessary to stimulate the CARS signal in
the samples is shown in Fig.2. A diode pumped frequency doubled Nd:YVO4laser (Coherent,
Verdi) was used to pump a mode-locked Ti:sapphire oscillator (KMLabs) operating at a center
wavelength of 807 nm, with a bandwidth of 30 nm, a repetition rate of 82 MHz, and an average
power of 300 mW. These pulses were sent to seed a regenerative chirped pulse amplifier (Co-
herent, RegA 9000)that producedapproximately70 fs, 5 µJ pulses with a repetitionrate of 250
kHz and an average power of 1.25 W. Around ten percent of this average power was used as the
pump beam; the remaining power was directed to an optical parametric amplifier (Coherent,
OPA 9400) which generated a 4 mW average power Stokes beam, tunable from 400-1200 nm
Oncethe pumpandtheStokes fieldswere generated,the twofields enteredthe interferometer
shown in detail in Fig.3. An excitation field consisting of two overlappedpulses centered at the
pump and Stokes wavelengths was divided by a beamsplitter into two separate interferometer
paths, which are referred to in Fig.3 as “sample arm” and “reference arm”.
A sample of a molecule was placed into each arm into which the split excitation fields were
focused. When the Stokes pulses were tuned such that the difference in frequency between the
pump and Stokes pulses was equal to a Raman active vibrational mode present in the molecule
in both the sample arm and the reference arm, an anti-Stokes signal was generated in each arm
(400 – 1200 nm)
Fig. 2. Setup used to generate the Stokes and the Pump excitation fields.
Fig. 3. Setup of the interferometric CARS measurement system [16, 17]. DM, dichroic mir-
ror; BS, beamsplitter; M, mirror; HPF, high-pass-filter; PH, pin-hole; PMT photomultiplier
tube; PC, personal computer.
of the interferometer. It will follow that the anti-Stokes fields, being coherent with the incident
fields, will interfere at the beamsplitter BS2 when temporally and spatially overlapped.
The pump and Stokes pulses, at 807 and 1072 nm respectively, were used to excite the
Raman-active vibrational mode of benzene at 3063 cm−1. The pulses were collinearly over-
lapped using a dichroic mirror and split with a 50:50 ultrafast beamsplitter (BS1) into the sam-
ple arm and the reference arm. The positions of the samples in the sample and reference arms
were chosen such that the distances of the samples from the beamsplitter BS1 were equal. The
two generated anti-Stokes pulses were then overlapped in time by adjusting the relative de-
lay (delay line) and in space by adjusting the position on a second beamsplitter (BS2). The
anti-Stokes signal was spectrally and spatially filtered. The delay line in the reference arm was
scanned by a computer-controlled single axis translation stage at a constant rate. The CARS
signal intensity was measured with a photomultiplier tube (PMT). The signal was filtered with
a low-pass anti-aliasing filter and sampled with a PC based data acquisition system (NI DAQ,
Initially the interferometer was calibrated with the pump signal only (λ = 807 nm). The ac-
quired interferogram (Fig.4) is shown for reference as a comparison with the following inter-
ferograms shown below. The interferogram was detected at the beamsplitter BS2 (Fig.3) and
was recorded as the pathlength of the reference arm was scanned. Having determined the in-
Results and discussion
-40 -200 2040 6080
?PUMP = 807 nm
Lc = 28 ?m
Fig. 4. Interferogram of thepump beam detected at the beamsplitter BS2of thesetup shown
in Fig.3. The envelope of the interferogram was fitted using the modulus of the degree of
the coherence function. In the inset is shown a detail of the interference pattern and its fit
by the real part of the degree of coherence function (open circles: experimental data; solid
line: fit). Lcis the coherence length of the pulse. λPUMPis the wavelength of the Pump
terferogram for the pump field, we inserted in the two interferometer arms two quartz cuvettes
filled with benzene. We first demonstratethat the signal collected from the cuvette was a CARS
signal. Figs. 5(a) and 5(b) show the observed relationship between the CARS and the pump
intensity (Stokes intensity fixed) and the CARS and the Stokes intensity (pump intensity fixed),
respectively. In agreement with the theory, the slope of the fitted lines verify the linear relation-
m = 0.99
Log (ICARS[arb. units])
m = 2.22
Log (ICARS[arb. units])
Fig. 5. Log-log plots of the intensity of the CARS signal as a function of (a) the intensity
of the Pump field and (b) the intensity of the Stokes field (solid lines, curve fitting). The
dotted line in (a) has a slope of 2. “m” is the angular coefficient of the solid lines .
ship between the anti-Stokes and the Stokes intensities, and the quadratic relationship between
the anti-Stokes and the pump intensities. This implies that our signal is a result of a four-wave
mixing process. In addition, we observed that this process is CARS resonance because the
anti-Stokes power is maximized when the Stokes wavelength is tuned to resonance with the
Raman-active benzene vibrational mode.
Established that the filtered signals from both the cuvettes were CARS signals, we detected
the resultingsignalat thebeamsplitterBS2 ofthesetupshowninFig.3.Themeasuredinterfero-
gram results from the interference between the two anti-Stokes signals and was recorded as the
pathlength of the reference arm was scanned. The function used to fit the experimental data is
the degree of coherencefunction, that under the assumption of a Gaussian spectral distribution,
is given by
where τ is the time, ω0is the center frequency and δω is the bandwidth of the CARS pulse.
This function is used to fit the interferogram in Fig.6. The real part and the modulus of the
coherence function, under the assumption of a Gaussian spectral distribution, are used to fit the
experimental data (interferogram and envelope respectively) and are plotted in Fig. 6.
The resulting coherence length LC, defined as the axial resolution of the interferometric
CARS measurement technique, is equal to
γ(τ) = exp
and was found to be equal to 32 µm.
-200 20 406080
?AS = 647 nm
LC = 32 ?m
Fig. 6. CARS interferogram of the pump beam detected at the beamsplitter BS2 of the
setup shown in Fig.3. The envelope of the interferogram was fitted using the modulus of
thedegree ofthecoherence function. Intheinset isshown adetailof theinterferencepattern
and its fit by the real part of the degree of coherence function (open circles: experimental
data; solid line: fit). Lcis the coherence length of the pulse. λASis the wavelength of the
The possibility to demodulate interferometrically the two anti-Stokes signals generated in
separate samples demonstrates the potential of CARS as a promising technique for providing
molecular contrast for OCT-like interferometric imaging systems. Interference indicates that
similar Raman-active vibrationalfrequencies were present in both the reference and the sample
arm. The “fingerprint” nature of Raman spectroscopy and the possibility to switch between
different molecular species in the reference arm, could permit selective detection and imaging
of different molecules in the sample.
Moreover, the possibility to interfere a weak CARS signal with another strong CARS signal
(produced in the reference arm), provides heterodyne sensitivity and an improved S/N ratio.
3.SHG contrast enhancement
Second Harmonic Generation (SHG), also known as frequencydoubling, has recently emerged
as a validimagingcontrastmechanismformicroscopicimagingof cell andtissue structuresand
functions . As mentioned in Sec. 2.1 SHG, in contrast to CARS, is a χ(2)process in which
two photons at the fundamental frequency are converted into a single photon at exactly twice
the frequency without having any absorption and/or re-emission from the sample . Even in
this case the intensity of the incident light is responsible for the induced nonlinear polarization,
with the result that the amplitude of the SHG signal is proportionalto the square of the incident
The first biological SHG imaging experiment  dates back to 1986 and involved the study
of orientation of collagen fibers in rat tail tendon . Since then, SHG microscopy has been
successfully applied in many different fields. In particular, SHG has proved to be highly effec-
tive in selectively probing interfaces, without being overwhelmed by the signal coming from
the bulk media . The reason for this is that second-order processes are electric dipole for-
bidden in centrosymmetric media. This implies bulk liquids and centrosymmetric crystalline
solids do not generate second-harmonic signals. Instead, at the interfaces, the molecular and
atomic species experience different interactions and the inversion symmetry, which is present
in the bulk, is broken . Another advantage of SHG microscopy is the high resolution typ-
ically achieved in nonlinear microscopy, and its applications for imaging structures in live tis-
sues consisting of endogenous proteins such as collagen. Note that the contrast mechanism is
obtained without requiring the presence of any exogenous labels .
For all these reasons, SHG microscopy is a good candidate for providing contrast enhance-
ment for OCT. In the next section we will demonstrate that the interferometerpresented in Sec.
2.1canbeanalogouslyusedforSHG heterodynedetectiondueto thefactthat SHG,like CARS,
is a process coherent with the excitation field.
The SHG interferometer is similar to the CARS interferometer and is shown in Fig.7. In this
Fig. 7. Setup of the interferometric SHG measurement system. Two different SHG crystals
(Type I) were inserted in the two arms of the interferometers. BS, beamsplitter; M, mirror;
IF, interference filter; PH, pin-hole; PMT, photomultiplier tube; PC, personal computer.
configuration, instead of having a Stokes and a Pump laser, only the Pump laser at 807 nm was
present. In the reference arm, a reference nonlinear crystal (BBO, Type I) with a thickness of
100 µm was present in which SHG signal was created. In the sample arm, a different nonlinear
crystal (BBO, Type I) with a thickness of 1 mm, was present. The signals generated in both the
crystals were overlapped as in the CARS configuration scheme.
Unique to the general methodology of our technique, when a SHG crystal is placed in the
reference arm, SHG signal created in the sample under investigation and present in the sample
arm can be heterodyne-detected,allowing for high sensitivity detection and OCT imaging.
3.3. Results and discussion
Fig.8 shows the measured interferogram resulting from the interference between the two SHG
signals. Thisresultindicatesthat twoSHG signals generatedin separatesamplesusingthesame
500 -50 -100-150
?SHG = 404 nm
LC = 52 ?m
ISHG (arb. units)
Fig. 8. SHG interferogram detected at the beamsplitter BS2 of the setup shown in Fig.7.
The interferogram was recorded as the pathlength of the reference arm was scanned. The
modulus of the degree of the coherence function was used to fit the envelope of the inter-
ferogram. The inset shows a detail of the interference pattern and its fit by the real part of
the degree of coherence function (open circles: experimental data; solid line: fit). Lcis the
coherence length of the pulse. SHG is the wavelength of the SHG signal.
pumplaser canbedemodulatedinterferometrically.Inthiscase as well, thepresenceofinterfer-
ence clearly demonstrates the potential of SHG and resonance-enhanced SHG as a promising
technique for providing molecular contrast for OCT-like interferometric imaging systems. The
presence of a nonlinear crystal in the reference arm will allow one to interferometrically de-
modulate the SHG signal created in the sample under investigation.
Simulations of the coherent nonlinear processes were performed to determine the properties
of the signals that could be expected from both the nonlinear interferometric setups (Fig.3, and
the slowly varying envelope approximation did not suffice for these simulations. Instead, we
utilized a method that approximated a continuous signal by sampling it spatially at regular
intervals. The evolution of the nonlinear signal was achieved by applying the nonlinearity at
each point in space and time, and propagating the signal forward in time in uniform steps.
Because of the low conversion efficiency,we assumed that the incident wave would not change
in time except for linear propagation effects, and so would experience no depletion in energy.
The model assumed that the incident and excited waves were one-dimensionalplane waves. As
a result, walk-off effects resulting from the divergence of the wave and ray (Poynting) vectors
that occur with a finite incident beam size in a birefringentmedium were not accounted for. For
the simulation of SHG, the nonlinearity was assumed to be instantaneous, but the dispersion of
the BBO crystal was accounted for, so that phase matching effects (at 28 degrees inclination of
the extraordinary axis) were properly simulated. The incoming wave was negatively chirped,
with an 807 nm center frequency and 30 nm bandwidth. Because of the rather thick crystal (1
mm) used in the experiment and present in the sample arm, 5 ps of simulation time was used
to interact the waves. Due to the thickness, phase matching can not be achieved over the entire
time= 500 fs
at time= 500 fs
0 50 -50
at time= 500 fs
Fig. 9. Simulation of SHG. Click on figure to view an mpeg movie (817 kB) of nonlinear
interferometry of the coherent SHG process.
incident bandwidth.The results of the simulation are shown in the first movie,a clip of which is
shown in Fig.9. The left graph shows the temporal spectrum of the incident beam in green, and
the second harmonic spectrum in black as the pulse evolves in time. The middle graph shows
the spatial envelope of the incident pulse in green and the created second harmonic pulse in
black. Finally, the right graph shows the autocorrelation of the second harmonic pulse at that
time, which corresponds to the interferogram that would be measured from two identical BBO
crystals of a thickness that corresponds to the propagation time of the simulation. The spatial
origin of the simulation moves with the reference frame of the incident pulse.
For the first 500 fs, the bandwidth is approximately√2 times that of the incident pulse. As
phase mismatch and group velocity mismatch start to occur, the nonlinear polarization adds
destructively with the second harmonic pulse. The resulting spectrum of the pulse becomes
quite nonuniform. Because the excited signal is simulated as a plane wave, all of the generated
frequency components overlap spatially and contribute to the autocorrelation of the measured
interference signal. Therefore the simulated autocorrelation has many sidelobes correspond-
ing to the nonuniform spectrum. In the experimental setup, a focused beam was used, and so
the walk-off separated the spectral components of the second harmonic pulse spatially. At the
pinhole PH present at the exit of the interferometer (Fig.7), only a limited bandwidth could
be focused through, and so the autocorrelation length was much longer than would have been
expected if phase matching occurred over the entire incident pulse bandwidth. We expect that
with a shorter crystal, phase-matching could be better achieved and the autocorrelation could
achieve its theoretical minimum for the bandwidth available from the incident pulse.
Simulating the CARS process require a different approach because unlike a nonresonant
nonlinear second-harmonic generation process, a resonant process has a “memory” associated
with the nonlinearpolarizationof the Raman process at each point in the medium.Walk-off did
not occur because the simulated medium, liquid benzene, is isotropic. The incoming radiation
was assumed to be two overlapped pulses of 30 nm bandwidth at 807 and 1072 nm, which
created a beat frequency at a vibrational frequency of the benzene. The vibration was assumed
to be a single vibration of Lorentzian profile with a much longer lifetime than the duration
of the incident pulse. At each time step the nonlinear Raman polarization at each point in
the medium was updated by driving it with the instantaneous intensity of the incident pulse
at that point. Fluctuations in the intensity of the incident field at the Raman frequency will
excite the nonlinear polarization at that point in the medium. This nonlinear polarization was
mixed with the incident beam to create the anti-Stokes signal. The second movie, shown in
at time=1000 fs
0.4 0.6 0.8-40
Pulse Position (microns)
at time=1000 fs
Raman Polarization Pos.
(microns) at time=1000 fs
(microns) at time=1000 fs
Fig. 10. Simulation of CARS. Click on figure to view an mpeg movie (1.713 Mb) of non-
linear interferometry of the coherent CARS process.
Fig.10, details the evolution of the simulation of the anti-Stokes pulse in liquid benzene over
1 ps. The upper left graph shows the temporal spectrum of the incident pulse in green, and
the created anti-Stokes spectrum in black. The upper right graph shows the spatial profile of
the magnitude of the incident pulse in green, and the magnitude of the created anti-Stokes
pulse in black. The lower left shows the spatial distribution of the magnitude of the Raman
polarization of the benzene medium. Finally, the lower right shows the autocorrelation of the
anti-Stokes pulse. Initially, as the pulse propagates the medium is not excited, and therefore
no anti-Stokes light can be created. As the pulse moves through a given point in the medium,
the medium is resonantly excited. The medium then mixes with the incoming pulse to generate
an anti-Stokes wave on the trailing edge of the incident pulse, where the maximum Raman
polarization coincides with the incident pulse. Because the anti-Stokes and incident pulses are
close in frequency, and dispersion is low in the near infrared, the anti-Stokes pulse can remain
in phase with the incident pulse for a long distance in the medium and phase matching is easier
to achieve. Because of the lack of walk-off, the simulation predicts well the autocorrelation
observed by experiment.
5. Conclusion Download full-text
In conclusion, we have described a novel technique for contrast enhancement in OCT based
on optical nonlinearities. The contrast mechanisms are based on resonant enhancement of the
third order and second order nonlinear susceptibility of the molecules under investigation. The
interference between two CARS signals generated in separate samples (or alternatively two
SHG signals), was observed,allowingforheterodynedetection.Numericalsimulationsforboth
coherent nonlinear processes were performed in order to determine the properties of the signal
expected at the exit of the described nonlinear interferometers, and the predicted results are in
agreement with the experimental data. The proposed interferometric scheme is very promising
for the development of a new molecular imaging technique (NIVI) based on nonlinear, low-
coherence interferometry [15, 17] and for SHG-OCT.
In this work, we focused on forward CARS and SHG, but epi-detected CARS and SHG are
coherent as well and are compatiblewith OCT coherence-rangingsystems. CARS and SHG in-
terferometry provide the advantages of interferometric detection and at the same time provide
OCT with molecular-specific contrast. These advantages could make CARS and SHG inter-
ferometry a powerful tool for biological imaging with OCT. Moreover, the same configuration
scheme could be exploited for Third Harmonic Generation (THG) microscopy , Coherent
Stokes Raman Scattering (CSRS) microscopy, and other coherent scattering processes.
This research was supported in part by a research grant entitled ”A Nonlinear OCT System
for Biomolecular Detection and Intervention” from NASA and the National Cancer Institute
(NAS2-02057, SAB). S.A. Boppart’s email address is firstname.lastname@example.org.