Molecular Optical Coherence TomographyContrast Enhancement and Imaging 47Amy L. Oldenburg, Brian E. Applegate, Jason M. Tucker-Schwartz,Melissa C. Skala, Jongsik Kim, and Stephen A. Boppart
47.1 Introduction
Histochemistry began as early as the nineteenth century, with the development of
synthetic dyes that provided spatially mapped chemical contrast in tissue [1]. Stains
such as hematoxylin and eosin, which contrast cellular nuclei and cytoplasm,
greatly aid in the interpretation of microscopy images. An analogous development
is currently taking place in biomedical imaging, whereby techniques adapted for
MRI, CT, and PET now provide in vivo molecular imaging over the entire human
body, aiding in both fundamental research discovery and in clinical diagnosis and
treatment monitoring. Because OCT offers a unique spatial scale that is intermedi-
ate between microscopy and whole-body biomedical imaging, molecular contrast
A.L. Oldenburg (*)
Department of Physics and Astronomy and the Biomedical Research Imaging Center, University
of North Carolina at Chapel Hill, Chapel Hill, NC, USA
e-mail: [email protected]
B.E. Applegate
Department of Biomedical Engineering, Texas A&M University, College Station, TX, USA
J.M. Tucker-Schwartz • M.C. Skala
Department of Biomedical Engineering, Vanderbilt University, Nashville, TN, USA
J. Kim
Department of Electrical and Computer Engineering, Bioengineering, Medicine, and the Beckman
Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign,
Champaign, IL, USA
S.A. Boppart
Biophotonics Imaging Laboratory, Beckman Institute for Advanced Science and Technology,
University of Illinois at Urbana-Champaign, Urbana, IL, USA
Departments of Bioengineering, Electrical and Computer Engineering, and Medicine, University
of Illinois at Urbana-Champaign, Urbana, IL, USA
# Springer International Publishing Switzerland 2015
W. Drexler, J.G. Fujimoto (eds.), Optical Coherence Tomography,DOI 10.1007/978-3-319-06419-2_48
1429
OCT (MCOCT) also has great potential for providing new insight into in vivo
molecular processes. The strength of MCOCT lies in its ability to isolate signals
from a molecule or contrast agent from the tissue scattering background over large
scan areas at depths greater than traditional microscopy techniques while
maintaining high resolution.
MCOCT involves the use of OCT image acquisition and/or processing tech-
niques to generate image contrast using endogenous molecular species or exoge-
nous molecular probes of interest [2]. It should be noted that several other chapters
in this book cover techniques that provide information about tissue composition
that will not be repeated here, including nonlinear interferometric vibrational
imaging (NIVI), second harmonic generation OCT, and optical coherence
elastography. This chapter does address spectroscopic OCT (SOCT) in the context
of chromophore and probe detection, while the reader should refer to related SOCT
chapters in this book for greater detail on SOCT processing methods.
This chapter is structured according to the physical mechanisms used for contrast,
starting from direct schemes whereby probes add to or subtract from the optical
backscattering spectrum that comprises the OCT signal (i.e., scattering- and
absorption-based contrast, respectively), then covering methods that indirectly mod-
ulate the OCT signal (i.e., pump-probe, magnetomotive, and photothermal OCT). It
should be noted that any scheme developed for MCOCT must be compatible with
interferometric detection, which precludes the use of several physical mechanisms
such as fluorescence emission. In this chapter, imaging technology development will
be emphasized and presented with selected examples of biomedical applications.
Interestingly, many endogenous and exogenous probes can be sensed by more than
one method. For example, photothermal contrast relies upon absorption, and there-
fore, the same agents provide contrast in both photothermal and spectroscopic OC-
T. The development of exogenous imaging probes (or contrast agents) that enable
MCOCT is also a rich and varied topic that poses particular challenges in materials
science and targeted delivery. Here we introduce a variety of molecular probes in the
context of specific MCOCT imaging strategies, while the reader is referred elsewhere
[3–6] for detailed information about probe development.
47.2 Scattering-Based Contrast
The native “contrast” observed in OCT is light that has been coherently
backscattered. All MCOCT methods are based upon modifying this backscattering
signal in a way that can provide additional, molecular information about the
sample. One of the most straightforward strategies is to increase the OCT signal
directly with a probe particle that exhibits high backscattering. Analogous methods
include the use of positive T1 contrast agents in MRI and echogenic microbubbles
in ultrasound. In fact, microbubbles also happen to have reasonably high
light scattering and were one of the first types of contrast agents studied with
OCT [7]. Oil-filled protein microspheres were subsequently found to offer flexibil-
ity in loading the shell or core with nanoparticles to further increase the optical
1430 A.L. Oldenburg et al.
scattering [8], and variants of these are in continued use with magnetomotive OCT,
as discussed below [9]. It is important to note, however, that any new contrast agent
must undergo thorough safety and efficacy testing before it can be used on humans,
such as that required by the Food and Drug Administration in the United States.
As such, the study of agents already approved for human use can more readily lead
to clinical translation. Interestingly, it was recently found that several commonly
used ophthalmic medications provide scattering-based OCT contrast [10]. As
shown in Fig. 47.1, OCT reveals the diffusion of several medications within the
anterior chamber after administration. They were also shown to enhance the
visibility of corneal incisions postoperatively, which may provide a method for
assessing wound integrity. Future adaptations of molecularly targeted agents may
further broaden the functionality of OCT in ophthalmology.
While scattering-based contrast agents are readily visible within the highly trans-
parent anterior segment of the eye, the ability to detect these types of agents endo-
scopically or on the skin is more challenging, as they must be distinguishable against
the already high optical scattering of the tissue.Mie theory provides exact solutions for
light scattering from spherical particles, shelled spheres, and spheroids [11].
As a general rule, there is a rapid increase in scattering with particle size (scattering
cross section, ss / d6) in the Rayleigh regime (d << l) and a rapid increase in
Lipid-based Artificial Tears
Control
Triamcinolone
Prednisolone
Fig. 47.1 OCT images of
fresh, ex vivo porcine eyes
after intra-cameral injection
of contrast agents. From topto bottom: Control,triamcinolone acetonide
40 mg/mL, prednisolone
acetate 1 %, and lipid-based
artificial tears. The agents
provide positive contrast
within the anterior chamber
that has a different pattern
depending on the type of
agent used. These differences
are expected because the
agents are comprised of
varying particulate or oil-in-
water suspensions, with
differing mean particle sizes,
reflectivities, and diffusivities
47 Molecular Optical Coherence Tomography Contrast Enhancement and Imaging 1431
scattering as the material refractive index is different (higher or lower) than that of the
(typically aqueous) medium. At the same time, one must weigh the choice of the
material and the particle size against the biocompatibility and the ability for the probes
to access their target, respectively, for the needed application.
One of the most useful types of OCT contrast agents, which will be discussed
many times throughout this chapter, is plasmonic gold nanoparticles in their various
forms (nanospheres, nanoshells, nanorods, etc.). This is because gold is highly
unreactive and consequently relatively biocompatible, while at the same time
providing a surface plasmon resonance (SPR) effect at the red and near-infrared
wavelengths used in OCT [12]. This SPR is evident as either a strong absorption or
scattering spectral peak, with a transition from predominantly absorbing (low
albedo) to predominantly scattering behavior (high albedo) as the particle size is
increased; for spheres, the transition occurs at d � 80 nm [12]. Nanoshells in
particular have been highly developed for scattering-based OCT contrast [13–15].
They are comprised of a silica core and gold shell and offer spectral tunability by
adjusting the core diameter and shell thickness [3]. Figure 47.2 displays a demon-
stration of enhanced OCT contrast in the tumors of mice systemically intravenously
Tumor Tissue + PBSc d
a b
Min
200 µm
Max
Normal Tissue + PBS
Tumor Tissue + Nanoshells
Normal Tissue + Nanoshells
Glass
Skin
Muscle
Glass
Skin
Tumor
Fig. 47.2 OCT images of tissues from mice with subcutaneous tumors systemically treated with
phosphate-buffered saline (PBS) as a control (a, c) and multifunctional nanoshells (b, d).Enhanced retention of nanoshells in the tumor in panel (d) provides better delineation of tumor
borders, as well as subsequent tumor-specific photothermal ablation (Reprinted with permission
from Ref. [13]. Copyright 2007 American Chemical Society)
1432 A.L. Oldenburg et al.
injected with nanoshells [13]. These results highlight the enhanced permeation and
retention (EPR) effect, whereby the permeable vasculature of tumors acts to trap
particles, providing selective targeting [16]. Importantly, EPR further enables
site-targeted treatment; in this example, the nanoshells were designed to be
nearly equally light scattering and absorbing, providing both imaging contrast
(via scattering) and photothermal therapy (via absorption).
It should also be noted that, in cases where the SPR is narrow compared to the
bandwidth of the light used in OCT, it may be possible to employ spectroscopic OCT
(SOCT) techniques to distinguish the SPR signature, providing enhanced specificity
against the tissue scattering background. While this idea has been explored [17],
a confounding factor that makes this method challenging is the highly modulated
backscattering spectrum typically obtained from Mie scatterers (d � l) within the
tissue. In current practice, SOCT techniques are much more commonly employed to
detect absorption-based contrast agents, which is the focus of the following section.
47.3 Absorption-Based Contrast
Light absorption is a very attractive molecular process to exploit for contrast, both
because of the potential signal strength and because essentially all molecular species
have the capacity to absorb light. The imaging light used in OCT is spectrally broad,
and hence, the backscattered spectrum may be utilized to identify the absorption
spectrum of endogenous or exogenous species present within the tissue. The group of
techniques designed to extract this information are collectively referred to as spec-
troscopic optical coherence tomography (SOCT) [18].
The different algorithms developed for SOCT diverge in how they deal with the
trade-off between spatial and spectral resolution. One approach is to use multiple
light sources to collect independent OCT images with different center wavelengths.
Relatively straightforward algorithms such as spectral triangulation [19] may then
be implemented to extract the depth-resolved backscattered spectrum. A judicious
choice of center wavelengths can facilitate the detection of highly peaked spectral
features with limited spectral resolution. This approach has largely been used to
detect dyes such as indocyanine green (ICG) [19].
An alternate approach is to directly utilize the broad spectral bandwidth of the
light source and use the short-time Fourier transform (STFT) to gain spectral
resolution at the expense of spatial resolution [20]. This approach has the advantage
that it is entirely a post-processing technique; hence, it can be tailored to maximize
contrast to a target contrast agent. Likewise, the time-frequency distribution (TFD)
need not be limited to the STFT but may optimized as well to maximize the spatial
and spectral resolution [21]. Wax and coworkers [22] have recently developed an
algorithm that incorporates two STFTs, one with a narrow spectral window and one
with a broad spectral window. The two TFDs are multiplied point by point to
generate a TFD with both high spatial and spectral resolution.
Exogenous chromophores for SOCT are largely repurposed, commercially
available fluorescent dyes. Utilizing these dyes carries with it the advantage of
47 Molecular Optical Coherence Tomography Contrast Enhancement and Imaging 1433
awealth of biological and chemical research aimed toward targeting particular disease
states of tissue, chemical species, or morphologies. Some examples include ICG [23],
photodynamic therapy-related dyes [24], and fluorescent microspheres [25].
Dyes typically also have strongly peaked spectra which enable detection via fairly
simple methodologies. For instance, a commercial NIR absorbing dye (H.W. Sands,
ADS7460) which exhibits a sharp absorption peak at 740 nm was used to produce
contrast in an 800 nm OCT system by effectively clipping the shorter wavelengths,
resulting in a redshift of scattered light [26].
The major endogenous chromophore is hemoglobin. Detection of hemoglobin
absorption with SOCT has been explored as a method to measure blood oxygen
saturation [27]. Wax and coworkers [22] recently measured the oxygen saturation
along with fluorescein dye injected into the bloodstream in a mouse window
chamber model using METRiCS OCT which uses the two TFD methods noted
above along with OCT imaging at nontraditional wavelengths. Their imaging
bandwidth spans the 455-695 nm range which overlaps strong peaks in the oxy-
and deoxyhemoglobin spectrum. Selected results from this work demonstrating the
endogenous and exogenous tissue contrast as well as SO2 measurements are shown
in Figs. 47.3 and 47.4.
Plasmonic gold nanoparticles have also been widely employed for absorption-
based contrast, where the SPR peaks are tuned within the OCT imaging band by
varying the size of specific geometrical features of the nanoparticles. For instance,
light-absorbing gold nanorods are tuned by varying their aspect ratio (length over
width) while maintaining a length typically <100 nm to favor absorption over
scattering. Many of the different particle geometries have been explored for
contrast in SOCT, including gold nanospheres [28], gold nanorods [28, 29], and
gold nanocages [30]. For example, gold nanorods were imaged after injection into
excised human breast carcinoma tissue [29]. As mentioned above, light-absorbing
Fig. 47.3 Conventional OCT (a) and METRiCS OCT (b) images, located above point (e) in the
en face (x–y) image in Fig. 47.4.White x and z scale bars, 100 mm (Reprinted with permission from
Ref. [22]. Copyright 2011 Macmillan)
1434 A.L. Oldenburg et al.
gold nanoparticles are, at the same time highly effective for photothermal
cancer therapy, where a high power laser is used to irradiate particle-laden
tumors [31]. The synergy between imaging and therapy, which allows us to monitor
permeation and diffusion of SPR particles into tissues before treatment, aids in
particle development for improved delivery and informs the design of more effec-
tive treatment protocols.
47.4 Pump-Probe OCT
Pump-probe optical coherence tomography (PPOCT) is fundamentally the fusion
of optical coherence tomography with pump-probe absorption spectroscopy.
Spatially resolving the pump-probe interaction can provide molecular contrast
for absorbing agents in a tissue sample similar to SOCT. The major advantages of
PPOCT are that contributions to the OCT light attenuation due to scattering and
absorption are easily separated and there is no compromise between spatial and
spectral resolution. The disadvantages are that the optical setup is more complicated
and typically requires pulsed light sources. Only under special circumstances could
PPOCT be accomplished with a swept OCT laser source or superluminescent diode.
Fig. 47.4 (a) En face METRiCS OCT image with arrows indicating points where the spectra areextracted. White x and y scale bars, 100 mm. (b–e) Spectral profiles from corresponding points in
(a). Measured spectral profiles (black) are superposed with the theoretical oxy- (dashed red) anddeoxy- (dashed blue) hemoglobin normalized extinction coefficients, and normalized absorption
of NaFS (dashed green). Also shown are the SO2 levels and the relative absorption of NaFS with
respect to total hemoglobin (e � NaFS/Hb). All spectra were selected from depths immediately
below each corresponding vessel (Reprinted with permission from Ref. [22]. Copyright 2011
Macmillan)
47 Molecular Optical Coherence Tomography Contrast Enhancement and Imaging 1435
A typical PPOCT system has the following features: The probe is the light in the
sample arm of the OCT interferometer, i.e., the same light used for OCT imaging
serves as the probe light. A separate pump beam co-propagates with the OCT light in
the sample arm. The pump is typically amplitude modulated at frequency f0. Transferof the modulation onto the backscattered probe (OCT) signal at f0 is then evidence ofabsorption of the probe light by some tissue absorber. In time-domain OCT
implementations, the PPOCT signal appears as sidebands on the Doppler carrier
frequency, fD � f0 [32]. In spectral-domain OCT, the PPOCT signal can be extracted
from an M-scan by Fourier transformation along the time axis (at each depth) and
filtering around f0. For the process to work, absorption of the pump by the contrast
agent must change the absorption/scattering properties at the probe wavelength.
The first experimental realization of PPOCT [33] demonstrated imaging of
methylene blue, a dye used for chromoendoscopy [34]. The specific physical
mechanism leading to PPOCT signal from methylene blue is well understood and
therefore serves as a germane example, where the energy level diagram is illus-
trated in Fig. 47.5a. The pump light drives a transition from the singlet ground
state (S0) to the first excited electronic state (S1). Molecular population in the
excited singlet state is transferred to the triplet state via a particularly efficient [35]
spontaneous process (S1 ! T1, t1-1). Methylene blue in its triplet state has
a resonant transition peaked at 830 nm (T1 ! T2). When the pump is on, an
830 nm probe can be absorbed by methylene blue, but when the pump is off, there
is no probe absorption. In reality the excited triplet state has a finite lifetime (analogous
to fluorescence lifetime) that is a function of the oxygen level in its local environment,
but varies from �200 ns to over 1 ms. Consequently, the pump and probe need not be
incident on the sample at the same time, but may be delayed in time by some fraction
of the excited state lifetime. Measurement of this characteristic lifetime may be used to
help differentiate among multiple chromophores. An example decay for methylene
blue is in Fig. 47.5b. The average decay time (lifetime) from T1 to S0 (t0�1) was
calculated to be 247 ns via tavg ¼ ∑ S � t/∑S, where S is the PPOCT signal at delay
time t [36]. In addition to the lifetime, the absorption spectrum at the pump or probe
may be measured by recording the PPOCT signal as a function of the pump or probe
wavelength, respectively.
Several molecular species in addition to methylene blue have been imaged using
PPOCT. Phytochrome A, a naturally occurring molecular switch which may be
reversibly optically pumped from one isomeric state to another, was imaged
in a tissue phantom [37]. Hemoglobin was measured in the gill filament arteries
of a zebrafish (Brachyrerio danio) using a time-domain 532 nm PPOCT system
with a 532 nm pump [32]. The same system was also used to image the fluorescent
protein DsRed in a transgenic zebrafish. Melanin was imaged in the first spectral-
domain PPOCT system in a phantom made from human hair embedded in chicken
breast tissue [38]. Melanin has also been imaged using a time-domain optical
coherence microscopy system [39]. Recent work [36] has demonstrated volumetric
imaging of microvasculature in Xenopus laevis using a two-color (532 nm pump,
830 nm probe) PPOCT system. Representative PPOCT cross sections overlain
on the OCT cross sections are shown in Fig. 47.6 along with volumetric
1436 A.L. Oldenburg et al.
reconstructions of the vasculature measured with PPOCT. They have also demon-
strated the use of the characteristic lifetime to differentiate PPOCT signals from
two different chromophores.
Figure 47.7 shows a pair of capillary tubes loaded with methylene blue/micro-
spheres and bovine whole blood in heparin. The top panel (a) is the standard OCT
image showing similar signal from both capillary tubes. A PPOCT image with 2 ns
pump-probe delay (Fig. 47.7b) appears very similar to the OCT image. However,
when the pump-probe delay is increased to 24.8 ms, the signal from the methylene
blue/microsphere-loaded capillary tube decays, leaving only signals from the blood-
filled capillary. Taking advantage of the difference in lifetime between two chro-
mophores is an effective strategy for imaging multiple chromophores with PPOCT.
Future research in PPOCTmay lead in several directions. There is a clear potential
for imaging vasculature. While Doppler-based OCT can also measure vasculature,
a major advantage of PPOCT is that the signal is independent of the angle of flow,
while the Doppler signal approaches zero when the illumination is orthogonal to
Fig. 47.5 (a) Molecular energy level diagram for the methylene blue PPOCT mechanism. Driven
transitions are indicated by straight arrows and spontaneous transitions as zigzag arrows.(b) Measured normalized decay of the PPOCT signal due to methylene blue as a function of
delay between the pump and probe pulse. The decay has a characteristic average lifetime of 247 ns
(Modified and reprinted with permission from Ref. [36]. Copyright 2013)
47 Molecular Optical Coherence Tomography Contrast Enhancement and Imaging 1437
the flow. The PPOCT signal is also molecularly specific, so it may be possible to
differentiate oxy- and deoxyhemoglobin and develop a PPOCT-based measure of
blood oxygen saturation. Furthermore, the imaging of exogenous contrast agents such
as methylene blue could potentially be used to tag and image specific molecular
species that are otherwise invisible to OCT. Such applications hinge on the demon-
stration of sufficient sensitivity eitherwithmethylene blue or some other discovered or
engineered contrast agent.
Fig. 47.6 (a–c) PPOCTB-scans overlaid on the
corresponding co-registered
OCT B-scans. Xenopus laevisvasculature is clearly
depicted. Arrows in (a) pointto capillaries that were not
visible in conventional OCT.
(d) Volumetric
reconstructions of the
microvasculature (Modified
and reprinted with permission
from Ref. [36]. Copyright
2013)
1438 A.L. Oldenburg et al.
47.5 Magnetomotive OCT
47.5.1 Theory and Instrumentation
Magnetomotive OCT (MMOCT) is a method for contrasting the distribution of
magnetic particles based on their induced motion within a temporally modulated,
magnetic field gradient [40]. Figure 47.8 illustrates the mechanism of MMOCT,
showing howmagnetic particles inside the imaging volume are mechanically pulled
toward an electromagnet placed in the imaging arm of an OCT system. Typically,
phase-sensitive OCT is then used to track the motion of light scattering tissue
structures that are mechanically coupled to the particles [41]. Modulation of
the electromagnet thus leads to phase modulation that can be band-pass filtered
at the modulation frequency to detect the magnetomotion. Because human
tissues are only very weakly magnetic (magnetic susceptibility |w| < 10�5),
Fig. 47.7 (a) OCT B-scan of
methylene blue (MB) andhemoglobin (Hb) in acrylic
capillary tubes. (b)Corresponding PPOCT
B-scans with a pump-probe
delay of 3 ns. (d)Corresponding PPOCT
B-scan with a pump-probe
delay of 24.8 ms. The absenceof the MB tube demonstrates
the potential for using the
lifetime as an effective
method for differentiating
multiple chromophores
47 Molecular Optical Coherence Tomography Contrast Enhancement and Imaging 1439
MMOCT provides high specificity against the tissue background, on the order
of 105 when using probes of w � 1 [42].
A class of biomedical imaging probes currently used in MRI, called superpar-
amagnetic iron oxides (SPIOs), are ideal for MMOCT because they are designed to
exhibit large w, avoid irreversible aggregation that is associated with ferromagnetic
agents, and are composed of iron oxide which has a proven safety profile.
FDA-approved MR liver contrast agents such as Feridex™, for example, have
been shown to provide excellent MMOCT contrast [43]. Another type of
MMOCT probe is protein microspheres encapsulating SPIO-containing ferrofluid,
which then offer flexibility in adding targeting ligands and therapeutic
payloads [44].
Implementing MMOCT on an existing phase-sensitive OCT system is relatively
straightforward. A small electromagnet can be placed on either the same side
(as shown in Fig. 47.8) or opposite side of the tissue to provide a magnetic field
gradient oriented along the imaging axis. Somewhat counterintuitively, the strength
of the magnetic field should only be on the order of 0.1T; higher fields will typically
saturate the magnetic particles and reduce the detection sensitivity [41, 43].
The absolute sensitivity of MMOCT can be determined by considering the balance
of forces between the diamagnetic tissue, which is pushed away from the magnet,
and from the paramagnetic particles, which are pulled toward the magnet.
Fig. 47.8 Mechanism of magnetomotive contrast in OCT. A solenoid placed in the imaging arm
of an OCT system provides a magnetic gradient force, F!, on magnetic particles inside tissue
according to the gradient of the magnetic field, ∇ B!, and the magnetization and volume of the
particles, M!and V, respectively. The resultant elastic displacement of mechanically coupled light
scattering structures, Dz, is sensed as a phase shift in the OCT interferogram, Df. w is the particle
magnetic susceptibility, m0 is the vacuum permeability, and n is the tissue refractive index at the
imaging beam wavelength, l
1440 A.L. Oldenburg et al.
For a typical SPIO particle, the minimum particle concentration needed to tip this
force balance in favor of motion toward the magnet is on the order of 10 mg Fe/g.
Another important consideration is the elastic property of the tissue medium.
Magnetic particles in liquid do not undergo a restoring force during magnetic
field modulation, moving only in one direction, and exhibit little contrast by
conventional band-pass-filtered MMOCT. In a solid medium, the compliance of
the tissue dictates the amount of displacement Dz, resulting in MMOCT contrast
that is weighted by both the local particle concentration and the local tissue
stiffness. Owing to the nanoscale displacement sensitivity afforded by phase-
sensitive OCT systems, the tissue stiffness is typically of little detriment to the
overall MMOCT sensitivity, and sensitivities as low as 27 mg Fe/g have been
reported in optomechanical tissue phantoms [41]. The high sensitivity and speci-
ficity afforded by MMOCT have recently led to several new molecular imaging
application areas, which will be reviewed below.
47.5.2 MMOCT of Atherosclerosis
Atherosclerosis is a disease in which an arterial vessel wall thickens as a result
of the accumulation of fatty materials, including macrophages. Atherosclerosis is
promoted by low-density lipoproteins (LDL) and cholesterol (crystals) and results
in calcification in advanced lesions [45]. Standard intravascular OCT imaging
has been extensively investigated for applications in cardiology such as imaging
intraluminal 3D structure and function at high resolution, evaluating arterial
stents, and visualizing atherosclerotic plaque [46–51]. The addition of contrast
agents for use with intravascular OCT can enable site-specific molecular cardio-
vascular imaging, just as has been shown for ultrasound imaging using gas-filled
microbubbles [52]. Targeted contrast agents may enable the early detection
and localization of atherosclerotic lesions which may not be clearly evident in
structural OCT imaging or in other imaging modalities. Therefore, the combina-
tion of intravascular OCT and targeted molecular contrast enhancement with
MMOCT can potentially improve the sensitivity of early atherosclerotic lesion
detection. Figure 47.9 shows representative MMOCT images from an ex vivo
hyperlipidemic rabbit aorta. The RGD (arginine-glycine-aspartic acid)-
functionalized protein microspheres [44] were fabricated to target the aVb3integrin overexpressed in atherosclerotic lesions [53]. These microspheres
were loaded with SPIOs and a fluorescent dye, enabling multimodal imaging
using MMOCT, MRI, ultrasound, and fluorescence imaging. The ex vivo aorta
sample was perfused in a custom-designed flow chamber at physiologically
relevant pulsatile flow rates and pressures. The functionalized microspheres
have been successfully targeted to fatty streaks present during early-stage
atherosclerosis. The future development of a MMOCT catheter or a new
solenoid configuration for current commercial intravascular OCT systems
may enable in vivo MMOCT imaging of atherosclerosis-targeted magnetic
probes.
47 Molecular Optical Coherence Tomography Contrast Enhancement and Imaging 1441
47.5.3 MMOCT of Breast Cancer
Breast cancer is one of the most commonly occurring cancers in women and has
a widespread effect on our society [54, 55]. Like other diseases, early detection is
the key in the treatment of breast cancer. Breast cancer screening methods include
manual clinical and self-breast exams, mammography, genetic screening,
ultrasound, and MRI. Breast cancer cells often overexpress aVb3 integrin and
HER-2/neu receptors which are considered to be biomarkers for targeted cancer
treatment [56]. Figure 47.10 shows an important step in breast cancer diagnosis
using MMOCT to contrast HER-2/neu-targeted SPIOs [57]. In this study, the
authors used a nitroso-methyl-urea (MNU) carcinogen-induced rat mammary
tumor model. In vivo MMOCT images of tumors from rats injected with targeted
SPIOs, nontargeted SPIOs, and saline exhibit an accumulation of SPIOs only in the
tumors of rats injected with targeted MNPs.
Fig. 47.9 Representative MMOCT and corresponding fluorescence confocal microscopy images
of hyperlipidemic rabbit aortas after administration of RGD microspheres. Parametric MMOCT
images are displayed showing the magnetomotive signal in green and the OCT signal in red. TheMMOCT signal in the targeted microsphere group was statistically significantly higher (p < 0.01)
than the nontargeted and control groups. Yellow lines in the aorta photos correspond to the imaging
locations. The dotted blue and red boxes are magnified to show the presence of individual
microspheres (white arrows). Scale bars are consistent across each row
1442 A.L. Oldenburg et al.
Fig. 47.10 In vivo (a) MMOCT and (b) OCT of rat mammary tumors. The magnetomotive signal
(green) is superposed on the OCT (red) in MMOCT images. Prussian blue (PB) sections of (c, d)tumors and (e, f) livers from rats after injection with (left) targeted SPIOs, (center) nontargeted
SPIOs, and (right) saline. PB sections in (d, f) at 40� from boxed regions in (c, e), at 10�.
(g) Immunohistochemical-stained sections of (left) tumor from a targeted SPIO injected rat,
(center) tail injection site from a targeted SPIO injected rat, and (right) tumor from
a saline injected rat (Reprinted with permission from Ref. [57]. Copyright 2010 National Academy
of Sciences)
47 Molecular Optical Coherence Tomography Contrast Enhancement and Imaging 1443
Furthermore, aVb3 integrin is also overexpressed in cancer cells [58]. As in the
previous section on atherosclerosis, the protein shell of microspheres can be
functionalized with the RGD tripeptide to target the aVb3 integrin. A recent study
reported MMOCT imaging of a rat mammary tumor containing RGD-targeted
magnetic protein microspheres [9]. MMOCT imaging was performed ex vivo on
the tumor approximately 4 h post injection of RGD-functionalized protein micro-
spheres. It was found that the aVb3 integrin-targeted microspheres preferentially
accumulated in the tumor.
These preliminary results demonstrate the feasibility of using targeted magnetic
nanoparticles and microspheres to detect breast cancer in OCT images. In addition,
the liquid-core protein microspheres can potentially also be used to deliver a drug
payload, such as the anticancer drug Taxol [59].
47.5.4 Platelets as Functional MMOCT Contrast Agents forThrombosis
Another way of achieving molecular specificity is to label cells with imaging agents
in vitro and to subsequently monitor the activity of the cells after in vivo admin-
istration. Platelets are cellular fragments present in blood that are responsible for
primary hemostasis (plug formation) as part of the blood clotting process [60].
Platelets respond to factors expressed when the blood vessel endothelium is dam-
aged, such as during trauma and atherosclerosis [61]. Therefore, platelets labeled
with SPIOs can be considered novel contrast agents for targeting localized endo-
thelial damage.
Interestingly, platelets have a unique method for nanoparticle uptake,
dubbed “covercytosis” [62], which is thought to be part of the body’s native
immune mechanism. As such, they avidly take up particles at levels reaching
hundreds [63], if not thousands [64], of femtograms of iron per platelet.
Platelets harvested from blood can also be partially fixed and lyophilized to
allow for long-term storage; these are known as rehydratable, lyophilized (RL)
platelets [65].
To date, in vitro and ex vivo studies have demonstrated the potential for
MMOCT to provide targeted imaging of reactive vascular sites using SPIO-labeled
RL platelets [43, 63]. In an ex vivo study, pig arteries were cannulated and
flowed with whole blood containing both native platelets and SPIO-RL platelets
at equal concentrations [43]. The endothelium of one set of arteries was lightly
injured, resulting in blood clotting at the injury sites. As shown in Fig. 47.11,
subsequent MMOCT of the luminal wall of these arteries revealed specific
magnetomotive contrast to injured artery only, due to the incorporation of
SPIO-RL platelets into the clots. These findings may be broadly translatable for
assessing internal bleeding and a broad spectrum of cardiovascular diseases such as
atherosclerosis.
1444 A.L. Oldenburg et al.
47.6 Photothermal OCT
Photothermal OCT (PTOCT) provides sensitivity and specificity of OCT to
absorbers in a sample through active detection of photothermal heating. Photon
absorption by an endogenous chromophore or exogenous contrast agent leads to
a temperature rise in the surrounding environment. These local temperature
changes cause thermoelastic expansion of the sample and shifts in the local index
of refraction [66, 67], which in turn cause changes in the local optical path length.
These small, typically nanometer-scale, local optical path length changes can be
resolved with phase-sensitive OCT. Unlike fluorescence-based imaging modalities,
most absorption-based contrast agents do not undergo photobleaching, allowing for
constant PTOCT signal over time, even at high irradiance.
47.6.1 Theory and Instrumentation
In PTOCT, a separate laser source for photothermal heating is incorporated into the
sample arm of the OCT system, either via direct, free beam coupling at the sample
arm optics or through shared sample arm fiber optics. The heating laser wavelength
is chosen to match the peak absorption of the desired imaging target. In most
applications of PTOCT, square or sine wave amplitude modulation of the heating
beam (e.g., using a mechanical optical chopper or acousto-optical modulator) is
performed during temporal sampling (M-mode scanning) of each spot. An example
PTOCT instrumentation diagram can be seen in Fig. 47.12, with the incorporation
of an amplitude-modulated 808 nm laser into the sample arm of a spectral-domain
Fig. 47.11 Representative MMOCT images of ex vivo porcine arteries after exposure to SPIO-
labeled RL platelets in a flow chamber, revealing specific contrast to injured vascular endothelium.
Arteries were subsequently longitudinally cut and are imaged with the luminal wall facing upward.
Inset: TEM image of an SPIO-labeled platelet containing hundreds of SPIOs in its surface-
connected open canalicular system
47 Molecular Optical Coherence Tomography Contrast Enhancement and Imaging 1445
Fig.47.12
(a)Experim
entalsetupofthePTOCTsystem
,wherePCdenotesthepolarizationcontroller.(b)Diagram
ofthedataprocessingmethodusedto
imagesentinel
lymphnodes
withPTOCT(A
daptedwithpermissionfrom
Ref.[68].Copyright2011American
Chem
ical
Society)
1446 A.L. Oldenburg et al.
OCT system [68]. Amplitude modulation of the heating beam allows for digital
lock-in techniques to be used during signal processing, which can detect and isolate
the active heating dynamics from the passive scattering signal. Modulation fre-
quencies as low as 25 Hz [69] and as high as 120 kHz [70] have been reported in
PTOCT and PTOCM applications.
In PTOCT, the signal is isolated from an oversampled M-mode scan by
obtaining the Fourier transform (in the time dimension) of the OCT phase data at
each point in depth. The PTOCT signal is then defined as the magnitude of this
Fourier-transformed phase data at the modulation frequency. More complex signal
processing considerations are often taken into account to remove artifacts, includ-
ing fifth- [69] or sixth- [71] order polynomial background subtraction of the phase
data to minimize 1/f noise, baseline subtraction of nearby frequency components in
the FFT data to account for the additive noise floor in the signal [69, 71–73], and
averaging of overlapping short-time Fourier transforms over the M-mode scan to
better estimate the noise floor [73]. Previous investigations into the PTOCT imag-
ing parameters have demonstrated that the PTOCT signal increases linearly
with both absorber concentration [67–69, 72–75] and photothermal laser power
[69, 73, 74], decreases logarithmically with increased amplitude modulation fre-
quency [73], and has a constant mean value but increased noise level in the presence
of weak reflections in the sample [73].
47.6.2 PTOCT Applications
PTOCT has been demonstrated in vitro, ex vivo, and in vivo both with
endogenous and exogenous forms of contrast. The most common use of PTOCT is
to visualize ultralow concentrations of highly absorptive contrast agents in tissue and
in vitro. In particular, PTOCT can exploit the rapidly advancing field of nanotech-
nology to image contrast agents with strong, wavelength-tunable absorption peaks.
Gold nanoparticles are the most common PTOCT contrast agents due to their
biocompatibility and well-established surface modification chemistry, which can
allow for specific imaging of molecular targets. In vitro molecular imaging of
60 nm diameter epidermal growth factor receptor (EGFR)-targeted gold nanospheres
has been performed in agarose phantoms using a 532 nm heating laser at 25 Hz
modulation, where EGFR+ cells exhibited a 300 % increase in PTOCT signal
compared to EGFR cells [69]. This study also demonstrated a sensitivity of 14 ppm
(w/w) to gold nanospheres within a scattering background. Although this is the only
demonstration of true molecularly targeted imaging using PTOCT to date, a number
of applications have used nontargeted NIR-resonant nanoparticles in systems ranging
from phantoms to in vivo. Early investigations into PTOCT were performed with
gold nanoshells having an SPR tuned to the near-infrared [67]. In subsequent studies
with ex vivo human breast tissues, direct injections of 120 nm silica core and 16 nm
gold shell nanoshells at a 5 � 109/mL concentration were visible 300–600 mm deep
into the tissue using PTOCT with an 808 nm laser modulated at frequencies from 5 to
20 kHz, with 22mWof power on the sample [71]. Gold nanorods have also generated
47 Molecular Optical Coherence Tomography Contrast Enhancement and Imaging 1447
interest as PTOCT contrast agents due to their tunability (based on the aspect ratio)
and particularly narrow SPR peak. Gold nanorods coated in poly(ethylene glycol)
(PEG) were found to have a significantly enhanced PTOCT signal at as low as 1 pM
concentration using 50 Hz modulation of an 808 nm laser interfaced with a 1,310 nm
OCT system [68]. The same system was used to image nonspecific uptake of gold
nanorods in sentinel lymph nodes (SLN). After injection with PEG-coated gold
nanorods, SLNs were dissected at varying time points from sacrificed mice and
imaged ex vivo after being embedded in 1 % agar gel. PTOCT was able to identify
the accumulation of nanorods within several SLN structures (Fig. 47.13, [68]).
Fig. 47.13 (a) Three-dimensional OCT projection image of a dissected sentinel lymph node
(SLN) at 48 h after gold nanorod injection. (b) 3D OCT view of SLN morphology with a cross-
sectional cut at a depth of 240 mm below the top surface. (c) 3D PTOCT view of SLN
corresponding to the cross-sectional cut displayed in (b) reveals structures within the SLN. (d)Schematic diagram and photograph of a dissected SLN. (Volume size ¼ 2.5 � 2.5 � 2.0 mm
(x–y–z)) (Adapted with permission from Ref. [68]. Copyright 2011 American Chemical Society)
1448 A.L. Oldenburg et al.
A separate study was able to image, in vivo, picomolar concentrations of PEG-coated
gold nanorods directly injected into a mouse ear [73].
Applications of PTOCT are not limited to gold nanoparticles, but include
any targets that have strong absorption at the wavelength of the heating laser.
PTOCT has also been demonstrated using carbon nanotubes [72], indocyanine
green encapsulated poly(lactic-co-glycolic) acid nanoparticles [76], gold-iron
oxide nanoroses [75], and iron oxide-silica-gold multifunctional nanoprobes [77].
PTOCT has also been used to characterize hemoglobin oxygen saturation using
dual-wavelength approaches to probe the absorption differences of oxy- and
deoxyhemoglobin in vitro and in vivo [78, 79].
47.6.3 Recent PTOCT Advances
Recent advances in the field of PTOCT have been working toward providing
quantitative molecular imaging. First, measurements of the local slope in the
axial dimension of a PTOCT image has found some success in correcting for
phase accumulation in PTOCT [74]. Second, a method to perform photothermal
optical lock-in optical coherence microscopy (poli-OCM) has been developed for
real-time photothermal imaging [70]. In poli-OCM, one creates phase modulations
in the reference arm that are matched to the amplitude modulation frequency of the
heating beam, resulting in a demodulated photothermal signal of the absorbers in
the sample. The signal due to scatterers is then isolated from the photothermal
Fig. 47.14 Demonstration of the contrast selective to gold nanoparticles offered by poli-OCM, as
compared to dfOCM. A square lattice of isolated 40 nm gold particles on a glass surface immersed in
intravenous perfusion fluid, imaged with dfOCM (a), and poli-OCM (b). (d, e) correspond to cross
sections along the lines indicated in (a, b). Graph (c) depicts the signal along the lines in (a, b), while(f) corresponds to the axial signal along the line highlighted in (d, e). Scale bars: 10 mm (Adapted
with permission from Ref. [70]. Copyright 2012 Optical Society of America)
47 Molecular Optical Coherence Tomography Contrast Enhancement and Imaging 1449
signal by setting the CCD integration time to a multiple of the modulation period.
This provides real-time photothermal imaging without the need for temporal
(M-mode) sampling or extensive digital processing. Pache et al. demonstrated
single particle detection of gold nanoparticles using poli-OCM with modulation
frequencies of 120 kHz while rejecting the scattering signal captured from their
dark-field optical coherence microscopy (dfOCM) system (Fig. 47.14, [70]).
Photothermal optical lock-in has yet to be demonstrated with a traditional OCT
system, but the underlying principles remain the same.
PTOCT is a promising imaging technique for isolating absorbers in a scattering
sample and thus provides specific and sensitive molecular imaging of both endog-
enous and exogenous contrasts. With recent advances in PTOCT optimization,
demonstrations in ex vivo and in vivo samples, and incorporation of optical lock-
in techniques, PTOCT promises to be not only sensitive and specific, but also a fast
method for imaging absorptive contrast agents in tissue.
47.7 Conclusion
It should be evident to the reader that MCOCT can be accomplished by
a wide variety of methods that each offer unique advantages, all of which
should be weighed when considering a specific biomedical application. All of the
methods presented here are in continued development, and we can expect to see
continued progress (greater sensitivity, specificity, imaging speeds, and higher reso-
lution) in the years to come. Also, new methods continue to emerge, such as imaging
thermal diffusion of micro- and nanoprobes [80, 81], which may allow one to sense
the local macromolecule concentration in bodily fluids such as mucus and blood.
Fundamentally, OCT provides a unique and flexible platform for exploring new
concepts in molecular imaging. Tying these imaging technology advances with
concomitant advances in nanotechnology and targeted delivery makes this a rapidly
changing field with many new opportunities for scientific discovery.
Acknowledgments Wewish to thank our many colleagues and collaborators conducting research
in this area and apologize that, due to length restrictions, we were unable to highlight more results.
We acknowledge Justis Ehlers at the Cole Eye Institute, Cleveland Clinic, for aiding in Fig. 47.1.
Some of the studies reported in this chapter were supported in part by a grant from the US National
Institutes of Health (R01 EB009073, S. A. B.).
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