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A multiphoton microscope platform for imaging the mouse eye
Omid Masihzadeh,1 Tim C. Lei,2 David A. Ammar,3 Malik Y. Kahook,3 Emily A. Gibson1
1Department of Bioengineering, University of Colorado Anschutz Medical Campus, Aurora, CO; 2Department of Electrical
Engineering, University of Colorado Denver, Denver, CO; 3Department of Ophthalmology, University of Colorado Hospital Eye
Center, Aurora, CO
Purpose: To demonstrate the ability of multiphoton microscopy to obtain full three-dimensional high-resolution images
of the intact mouse eye anterior chamber without need for enucleation.
Methods: A custom multiphoton microscope was constructed and optimized for deep tissue imaging. Simultaneous two-
photon autofluorescence (2PAF) and second harmonic generation (SHG) imaging were performed. A mouse holder and
stereotaxic platform were designed to access different parts of the eye for imaging. A reservoir for keeping the eye moist
was used during imaging sessions.
Results: Non-invasive multiphoton images deep inside the anterior chamber of the mouse eye were obtained without the
need for enucleation. The iris, corneal epithelium and endothelium, trabecular meshwork region and conjunctiva were
visualized by the 2PAF and SHG signals. Identification of the anatomy was achieved by the intrinsic properties of the
native tissue without any exogenous labeling. Images as deep as 600 microns into the eye were clearly demonstrated. Full
three-dimensional image reconstructions of the entire anterior chamber were performed and analyzed using custom
software.
Conclusions: Multiphoton imaging is a highly promising tool for ophthalmic research. We have demonstrated the ability
to image the entire anterior chamber of the mouse eye in its native state. These results provide a foundation for future in
vivo studies of the eye.
The anterior chamber of the mouse eye is a complex
structure that closely resembles that of the human eye [1].
Many human ophthalmic diseases can be modeled and studied
in the mouse eye including that of elevated intraocular
pressure (IOP) and glaucoma [2]. The location of the
conventional aqueous humor outflow system, important in the
regulation of intraocular pressure (IOP) of the eye, between
the cornea and the iris presents challenges for structural and
functional testing which are aimed at better understanding the
pathophysiology of glaucoma. Previous studies have shown
that structural abnormalities in this region can lead to elevated
IOP and development of glaucoma [3]. Therefore, early
detection and clinical intervention in the prevention of this
disease would greatly benefit from high resolution structural
and functional imaging capabilities.
Current clinical techniques for imaging the eye include
optical coherence tomography (OCT), confocal reflectance
microscopy, ultrasound biomicroscopy and fluorescence
imaging. In comparison with multiphoton microscopy, OCT
has less spatial resolution, approximately 2–10 μm lateral and
is not capable of achieving subcellular resolution. Confocal
reflectance microscopy does allow subcellular level
resolution, but is not capable of providing data on the
Correspondence to: Emily A. Gibson, University of Colorado
Anschutz Medical Campus, Department of Bioengineering, Mail
Stop 8607, 12700 E 19th Avenue, Research 2 - Room P15-6C03,
Aurora, CO, 80045-2560; Phone: (303) 724-3678; FAX: (303)
724-5800; email: Emily.Gibson@UCDenver.edu
functional status of imaged tissues. Ultrasound
biomicroscopy is limited in resolution to ~25 μm and is also
not capable of imaging subcellular structures or cellular
function. For these reasons, multiphoton microscopy is a
promising tool for ophthalmic imaging and could provide
benefit in both research as well as clinical environments [4,
5]. Specific imaging of the aqueous outflow system and
trabecular meshwork (TM) has been performed with spectral
domain OCT [6,7]. However, OCT is still not capable of
obtaining functional information about the cells and collagen
network in the outflow region and is limited in resolution.
Three-dimensional micro-computed tomography (3D
MicroCT) imaging with resolution on the order of a few
microns has been performed on fixed human eye sections
showing the outflow structures similar to histological imaging
with light microscopy [8]. In contrast, multiphoton
microscopy provides sub-cellular resolution and functional
imaging capabilities using either intrinsic optical properties
of molecules or optical markers such as fluorescent labels. We
have previously demonstrated that multiphoton imaging can
resolve the collagen structure of the TM without exogenous
labeling as well as image epithelial cells in the TM by their
intrinsic autofluorescence generated predominately by the
cofactor nicotinamide adenine dinucleotide phosphate
(NAD(P)H) [9]. Progression of primary open angle glaucoma
(POAG) is indicated by a deterioration in the TM structure
and in loss of epithelial cells in the TM, as found by electron
microscopy studies [10,11]. A reduction in the dimensions of
Schlemm’s canal in POAG has also been observed by
Molecular Vision 2012; 18:1840-1848 <http://www.molvis.org/molvis/v18/a190>
Received 11 April 2012 | Accepted 1 July 2012 | Published 4 July 2012
© 2012 Molecular Vision
1840
histological studies [12]. Therefore, imaging this region of the
eye may allow for an improved understanding of the
pathophysiology of glaucoma, better diagnostic capabilities,
and potential for assisting in new therapeutic drug discovery
studies.
Multiphoton microscopy (MPM) is being increasingly
used for ophthalmic research [9,13-25]. MPM provides sub-
cellular resolution and considerable penetration depth as
compared to confocal microscopy and is a powerful multi-
modal tool for visualization of ocular structures by their
inherent optical properties, without the need for labeling
contrast agents [26,27]. Two-photon autofluorescence
(2PAF) and second harmonic generation (SHG) are examples
of different multiphoton imaging modalities. While both
involve the simultaneous interaction of two infrared photons
with molecules in the sample, they are fundamentally different
phenomena and reveal different information about the
molecular-level structure of the tissue [28-30]. 2PAF is
generated by excitation of endogenous fluorophores such as
the cofactors nicotinamide adenine dinucleotide phosphate
(NAD(P)H) and flavin adenine dinucleotide (FAD) or
melanin. SHG, on the other hand, is generated by non-
centrosymmetric molecules or macro-molecular structures,
such as collagen. Both SHG and 2PAF signals are arise from
a two-photon process and therefore, the signal is produced
selectively at the focus of the excitation laser where the
intensity is greatest. This intrinsic axial sectioning property
makes MPM ideal for three-dimensional imaging. In
comparison with confocal microscopy, MPM can image at
some depth in tissue because of reduced scattering of the
infrared excitation light and greater detection efficiency of the
emitted signal due to the elimination of the confocal pinhole
and relay optics in the detection path. In addition, excitation
with infrared light causes less thermal damage to the sample
than visible wavelength excitation for the same intensities
[31].
In previous results, we demonstrated the use of MPM to
image the TM and nearby outflow structures within the intact,
enucleated, unfixed mouse eye [15]. The mouse eye is ideal
for these preliminary studies of MPM imaging due to its small
size, thin sclera allowing for increased penetration depth of
light for imaging and due to the well described anatomy of the
outflow system in past research. The mouse eye anterior
drainage system anatomy is very similar to that of the human
Figure 1. A schematic of the custom multiphoton microscope illustrating the beam path of the excitation laser and the emitted signals. The
pulsed laser light is first sent through a prism compressor (to ensure the shortest pulse duration at the sample) and passed through two
galvanometric scanning mirrors for raster scanning at the sample. The excitation laser beam travels through a scan lens (SL) and tube lens
(TL) before being sent into the microscope through the side port. A custom made lens relay system is used to convert our inverted microscope
(Olympus IX71) into an upright microscope for in situ imaging. The multiphoton signal from the sample is collected back through the
microscope objective and separated from the excitation laser light with a dichroic mirror (DM1). An additional dichroic mirror (DM2) is used
to separate the SHG and 2PAF signals, which are subsequently focused on separate photo-detectors. Abbreviations: prism (Pr), half-wave
plate (HW), polarizer (PP), telescoping optics (T), objective lens (Obj). Inset shows a photograph of the custom stereotaxic device that holds
the mouse and orients the eye position and angle with respect to the objective lens. A reservoir is held over the mouse eye to keep the eye
moist during imaging sessions.
Molecular Vision 2012; 18:1840-1848 <http://www.molvis.org/molvis/v18/a190> © 2012 Molecular Vision
1841
eye and has been studied extensively, providing an excellent
understanding of this model for future research in diseases of
the eye. In the present study, we demonstrate MPM for in situ
imaging of the mouse eye, to obtain the full three-dimensional
images of the anterior chamber without any disruption to the
tissue as occurs during the enucleation process. We describe
our custom built multiphoton microscope specifically
designed for ophthalmic imaging and the method for
alignment of the mouse eye using a custom stereotaxic device.
Our findings provide the foundation for achieving in vivo
imaging of the mouse eye.
METHODS
Animal protocol: The animal protocol for multiphoton
imaging was approved by the University of Colorado
Anschutz Medical Campus Institutional Animal Care and Use
Committee (IACUC). The mouse strain C57BL/6 (Jackson
Laboratory; Bar Harbor, ME) was selected for imaging
because it has no documented ocular phenotype. C57BL/6
mice were obtained from retired breeder stock used in
unrelated research projects. All mice were between 4 and 6
months of age. A total of 4 mouse eyes were imaged, all with
qualitatively similar results. Representative images from one
mouse eye are presented. For each experiment, the mouse was
imaged immediately after euthanasia and each imaging
session was around one hour in duration. Only one eye of each
mouse was imaged during a given session after mounting and
alignment of the eye with respect to the objective lens.
Histology: After the multiphoton imaging experiments, the
imaged mouse eye was enucleated and then preserved in 4%
paraformaldehyde in PBS overnight at 4 °C. The eyes were
embedded in paraffin and sectioned at a thickness of 6 µm.
Tissue sections were stained with Mayer’s hematoxylin and
eosin Y (H&E; Richard-Allan Scientific, Kalamazoo, MI).
Bright-field imaging was performed using a Nikon Eclipse 80i
microscope (Nikon, Melville, NY) equipped with a Nikon D5-
Fi1 color camera and a Nikon 20X/0.30 Plan Fluor objective
lens.
Multiphoton microscopy: The images shown in this work
were taken on a custom-built multiphoton microscope system.
The system is built around an Olympus IX-71 (Olympus,
Center Valley, PA) inverted microscope with several
components that were custom designed and integrated for
ophthalmic imaging capabilities (Figure 1). The excitation
laser source is a pulsed infrared laser (Mai Tai HP; Spectra
Physics, Santa Clara, CA) with a center wavelength of 810 nm
emitting a train of pulses at an 80 MHz repetition rate. To
optimize the multiphoton signal, the laser pulses are
temporally compressed to the shortest pulse duration at the
back aperture of the microscope objective by first passing
through a prism compressor to compensate for the material
dispersion due to the optical components in the beam path.
Near transform-limited pulses of ~100 fs (the shortest pulse
duration for the spectral bandwidth of our laser) were
measured by a custom-built ultrafast pulse characterization
apparatus based on the principle of frequency resolved optical
gating [32]. The laser passes through a pair of telecentric
lenses to increase the beam diameter to overfill the back
aperture of the microscope objective to achieve a diffraction
limited focal spot. Raster scanning of the laser focus across
the sample is achieved by use of two non-resonant
galvanometric mirrors (Cambridge Technology, Watertown,
MA). A combination of scanning lens (SL) and tube lens (TL)
is used to transform the angular translation of the laser beam
at the scan mirrors into a lateral displacement of the laser focus
in the focal plane of the objective lens. The pulse train with
an average power of 15 mW (incident at the sample) is focused
with an Olympus LUMPlanFL 40X/0.80 NA water immersion
objective (Olympus) with a working distance of 3.3 mm.
The multiphoton signals generated by intense laser light
interaction with the sample include second harmonic
generation (SHG) and two-photon autofluorescence (2PAF).
The emitted signals are collected in the epi-direction and
separated from the excitation laser by a dichroic mirror (long
pass at 685 nm, FF685-Di02; Semrock, Inc., Rochester, NY).
Any residual excitation laser light is blocked by an additional
sputter-coated, high-throughput shortpass 2-photon emission
filter (ET700sp-2p ; Chroma Technology, Bellows Falls, VT).
The SHG and 2PAF signals are spectrally separated with a
dichroic mirror (T425lpxr; Chroma Technology) and
bandpass filters (HQ400/20m-2p for SHG and
HQ575/250m-2p 2PH; Chroma Technology) and detected by
two separate high-sensitivity, photon counting detectors
(H7422P-s40; Hamamatsu, Inc., Bridgewater, NJ). The signal
from each detector is amplified (Model ACA-4–35-N, 35dB
gain, 1.8GHz bandwidth, non-inverting amplifier; Becker and
Hickl GmbH, Berlin, Germany) and converted to TTL
(Transistor-transistor logic) pulses using a constant fraction
discriminator (Model 6915; Phillips, Mahwah, NJ). The TTL
pulses are sent to the counter inputs of a data acquisition card
(PCIe-6259; National Instruments, Austin TX) with a
maximum count rate of 80 MHz. Custom software was
developed in Labview (National Instruments) running on a
personal computer (Dell Inspiron, Round Rock, TX) to
control the galvo-mirrors and read in the signal from the
counters.
Image analysis: Acquired multiphoton images were post-
processed using Matlab (MathWorks, Natick, MA) and
ImageJ software. Except when noted, all image dimensions
are 256×256 pixels with a 13 μs pixel dwell time and frame
averaging of two. The maximum image area is limited by the
field-of-view of the objective lens and the scanning range of
the galvanometric mirrors to ~140×140 μm2. Calibration of
the image dimensions was performed by imaging a standard
mesh with a calibrated grid size (Copper 400 Size Mesh; Ted
Pella, Redding, CA).
To image over larger regions of the eye, tiling of many
individual scans was performed. This was accomplished by
Molecular Vision 2012; 18:1840-1848 <http://www.molvis.org/molvis/v18/a190> © 2012 Molecular Vision
1842
translating the sample with a motorized stage (MS-2000; ASI,
Eugene, OR) in the x and y directions with a step size of
140 µm to acquire a series of images. The images were then
combined in software into one composite image (tiling). The
effects of tiling can be seen in the reconstructed images as
stripes with reduced intensity due to variations in the signal
intensity across the field-of-view. This artifact can be
eliminated by scanning a smaller region for each individual
image, or by implementing more sophisticated image
reconstruction software to correct for the intensity variations
currently under development. A series of two-dimensional
images was acquired at different depths and then projected in
three-dimensions (3D) using ImageJ for 3D visualization. 3D
animations were prepared using Zen 2009 software package
(Carl Zeiss MicroImaging, Inc., Göttingen, Germany). In the
3D image reconstructions, the 2PAF and SHG signals are
superimposed onto the same image for co-localized
visualization. Appropriate noise reduction filtering (6-point
smoothing function) and thresholding were applied to
eliminate the noise from detector dark counts and to optimize
visualization.
Mouse holder: A custom stereotaxic device is used to position
the mouse for multiphoton imaging of the eye. The holder is
mounted on to the motorized stage that translates for tiling.
The mouse holder incorporates two manual rotation stages to
allow angular adjustments along two axes of rotation, to
optimally position the mouse eye with respect to the objective
lens. Acquisition of images at different depths is
accomplished by translating the microscope objective
vertically with a computer controlled stepper motor
(MFC-2000; ASI, Eugene, OR) to adjust the focus knob on
the Olympus IX-71 microscope platform. During the imaging
experiments, it was important to keep the exterior surface of
the eye moist. This was accomplished by a custom reservoir
(Figure 1) positioned on top of the mouse eye to hold balanced
salt solution (BSS) between the eye and the objective. The
fluid in this reservoir also serves as the immersion liquid for
the objective lens.
RESULTS
The superior quadrant of the mouse eye was imaged with our
custom multiphoton microscope from the surface of the
cornea to the iris and anterior capsule of the lens. To visualize
the different regions of the eye, we performed three-
dimensional reconstructions of separate sections, illustrated
schematically in Figure 2. Section A-A represents a solid cut
through the top of the cornea where the image volume consists
of the stroma and extends to the epithelium layer. Section B-
B shows a solid cut through the cornea and into the anterior
chamber. The ring appearance is noted due to the lack of signal
generation from the aqueous humor located in the center of
the volumetric section. Section C-C is the section deepest
inside the anterior chamber capturing part of the iris, which
generates a strong 2PAF signal (red) in the center while the
outer ring consists of the cornea which predominately emits
SHG (green).
Three dimensional image reconstructions of the data
taken in the three sections of the anterior chamber of the eye
are displayed in Figure 3. The images (Figure 3A-C) are
shown starting from the exterior surface of the cornea and
progressing deeper into the anterior chamber. False colors
represent the SHG (green) and 2PAF (red) signals. Figure 3A
shows the 2PAF signals from the epithelial cells at the surface
of the cornea and SHG signal from the collagen-rich stroma
further into the cornea, similar to our previously reported data
in the enucleated eye [15]. The reconstructed three-
dimensional volume is composed of 17 stacks in z (depth) of
4×5 tiled images (1 tile=136×136 μm2), each stack is separated
in z by 10 μm. In Figure 3B, taken further into the eye, the
signal is predominantly SHG (green) from the stroma of the
cornea. The endothelium layer is distinct from the stroma by
its strong 2PAF signal (inner red circle), while additionally in
the same section, the epithelium layer is visible at the edge by
its 2PAF signal (the outer red region). The reconstructed data
consists of 12 z stacks (separated by 10 μm in depth) of 5×10
tiled images (1 tile=122×122 µm2). In section 3C, the iris
becomes visible at a depth of 250 μm below the cornea. The
nonlinear signal from the iris was exclusively 2PAF (red) from
the pigment granules. The 3D image reconstruction consists
Figure 2. Diagram showing the different
regions of the mouse eye that were
measured with the multiphoton
microscope, results shown in Figure 3.
A-A represents a solid cut of the cornea
from the top surface into the stroma. B-
B is a cross-section of the anterior
chamber filled with aqueous humor.
Since the aqueous humor does not emit
any signal, the sectioned image appears
in the shape of a ring. C-C is a section
deep inside the anterior chamber that
includes the front of the iris.
Molecular Vision 2012; 18:1840-1848 <http://www.molvis.org/molvis/v18/a190> © 2012 Molecular Vision
1843
of 16 z stacks (separated by 20 μm) of 12×6 tiled images
(1tile=122×122 μm2). Figure 4 show a two-dimensional tiled
view inside of the eye at different depths extending (from left
to right) from the center of the pupil out to the outer edge of
the iris and into the conjunctiva. The data are represented by
5×16 tiled images (1 tile=68×68 µm2). Four different depths
are displayed in 20 μm steps starting at a depth of ~500 µm
from the epithelium layer of the cornea. From left to right, one
can identify the iris (I), trabecular meshwork region (TM),
sclera (S) and conjunctiva (C). A higher resolution image of
the conjunctiva is shown in Figure 5 where individual cell
bodies are visible. The cells can be visualized by 2PAF
predominately from endogenous fluorescent cofactors such as
NAD(P)H and FAD [28].
Histology was performed to screen for potential damage
to the tissue from the infrared excitation laser used in
multiphoton imaging. After completion of the imaging
experiment, blue dye was placed on the region of the mouse
eye that was exposed to the laser light to identify the location
for histology. Results from histology showed no visible
Figure 3. Three dimensional
reconstructions of the images of the
mouse eye taken with our multiphoton
microscope. Below each 3-D
reconstruction is a two-dimensional
projection showing the side view of the
image composite. The cornea (C) and
iris (I) are indicated on the images. A:
Image reconstruction of one quarter of
the cornea section (indicated as section
A-A in Figure 2). The epithelium layer
(EP) is visible by the strong 2-photon
autofluorescence signal (red; 2PAF)
clearly delineating it from the stroma of
the cornea that emits predominately
second-harmonic generation from
collagen (green;SHG). B: Image
reconstruction of a cross section through
the anterior chamber (AC; section B-B
in Figure 2). The 2PAF signal on the
outer rim of the image is from the
epithelial layer on top of the stroma
while the SHG signal is from the
collagen in the stroma. C: Image
reconstruction of the measured region
deep into the anterior chamber that
includes the iris (I) which emits 2PAF
signal predominantly from the pigment
granules.
Molecular Vision 2012; 18:1840-1848 <http://www.molvis.org/molvis/v18/a190> © 2012 Molecular Vision
1844
damage to the tissue (Figure 6), indicating that we are able to
image the eye structures at light intensities below the
photodamage threshold.
DISCUSSION
In the current study, we demonstrate a multiphoton system
capable of imaging deep inside the anterior chamber of a
mouse eye without need for enucleation. Although imaging
of the mouse cornea in vivo has recently been demonstrated
[33], the current study is the first, to the best of our knowledge,
in which multiphoton imaging was used to image the
structures deep within the anterior chamber of a non-
enucleated mouse eye. We have demonstrated the capability
of imaging in the angle of the eye at depths of 600 μm and the
ability to visualize the anatomy without the use of any external
dyes or fluorescent labeling. Our multiphoton microscope is
optimized for deep tissue imaging and is integrated with a
custom stereotaxic device capable of rotation and translation
with five degrees of freedom. The capability of both
translational and rotational alignment is key to accessing
different locations inside the anterior chamber. A fluid
reservoir is designed to keep the eye moist for an extended
period of time, necessary to obtain images of the tissue in its
native state.
At our demonstrated penetration depth of 600 μm inside
the eye, we can image a section through the entire anterior
chamber, visualizing it in its native state. Due to light
scattering and optical aberrations from inhomogeneities in the
ocular tissue, the resolution of the microscope when imaging
Figure 5. Two-dimensional tiled image showing a section through
the sclera to the conjunctiva. The sclera (S) emits predominately
SHG signal (green) while the conjunctiva (C) is visible by 2PAF
(red). In this image, individual cells within the conjunctiva (see
arrow) can be clearly resolved by their intrinsic autofluorescence
from endogenous cofactors such as NAD(P)H and FAD.
Figure 4. Two-dimensional tiled
multiphoton images of the angle of the
eye taken at different depths. Distinct
features of the anatomy of the eye are
visible. Images from top (A) to bottom
(D) represent sections taken at different
depths below the surface of the eye from
(A) 580 μm, (B) 540 μm, (C) 520 μm,
and (D) 500 μm. From left to right, we
can see the iris (I), trabecular meshwork
region (TM), sclera (S) and conjunctiva
(C).
Molecular Vision 2012; 18:1840-1848 <http://www.molvis.org/molvis/v18/a190> © 2012 Molecular Vision
1845
at-depth is reduced from the theoretical diffraction limited
resolution of ~225 nm [34]. We are currently working on
improvements to image resolution through several avenues
including better aberration correction of the laser focus [35].
When imaging deep in tissue, care is taken to keep the
excitation laser power as low as possible to avoid thermal
damage. By using sensitive photon-counting detection, we
were able to successfully image deep into the eye without any
visible damage to the tissue, i.e., when repeating the scan over
the same region, we did not observe any alteration in the
imaged structures. Histology performed on the region of the
eye that was imaged also showed no evidence of tissue
damage. As an added safeguard for future in vivo studies, we
can implement feedback controls to continuously monitor the
multiphoton signals to detect any sudden increase that may
indicate potential tissue damage, such as strong absorption by
pigment-containing tissues. Upon detection of a rapid increase
in signal, the laser power can be reduced during the scan to
prevent any damage. From experiments at different laser
Figure 6. Histological section of C57BL/6 mouse eye imaged with
our multiphoton microscope. Eye structures appear normal after
imaging. No distortion or photocoagulation is noted in the tissues
near the drainage angle of the eye. Blue dye was used to mark the
orientation of the eye before enucleation (*). S=sclera. R=retina.
I=iris. C=cornea. L=lens. The scale bar represents 50 µm.
excitation powers, our results indicated the pigmentation of
the iris is the most susceptible to tissue damage most likely a
result of the light absorbing properties of melanin. The cornea
and sclera were more resilient to higher laser powers.
Currently, MPM imaging is much slower than OCT when
imaging over the same volume. In spite of this disadvantage,
for future clinical applications, MPM may have an advantage
for high resolution “optical biopsy” of specific locations.
MPM as an add-on to a standard OCT clinical instrument
would potentially give both the fast scanning ability combined
with high-resolution functional imaging in localized areas.
Another limitation of MPM is the depth of penetration and
decreased resolution at-depth due to scattering and tissue
inhomogeneities. We are currently pursuing methods to
address these limitations to higher resolutions in a way that
will allow for practical use.
The study presented in this paper shows promising
opportunities toward in vivo multiphoton microscopy for
ocular imaging. The inherent sectioning capabilities and
resolution of MPM along with its noninvasiveness are key to
future clinical studies of live subjects and could add a wealth
of information toward understanding diseases of the eye. For
glaucoma, MPM imaging for studying the dynamic nature of
TM cell survival in vivo has great potential. Recently, Tan et
al. demonstrated that MPM imaging is capable of measuring
the presence or lack thereof of TM cells on excised human
corneal rims [36], however, in this study, they used exogenous
markers and did not image through tissue to access the TM
region. In our recent publication, we demonstrated use of
coherent anti-Stokes Raman scattering (CARS) imaging for
quantifying TM cells on excised human tissue without use of
exogenous markers [16] and believe this is an additional
multiphoton imaging method that could lead to clinical use in
studying TM cells in live specimens when combined with our
multiphoton imaging platform.
ACKNOWLEDGMENTS
The authors thank Justin Brantley for his assistance with the
design of the mouse holder and Mark Petrash for providing
the mice for this study. Supported in part by a Bioscience
Discovery Evaluation Grant from the State of Colorado and
the University of Colorado Technology Transfer Office
(Grant No. 11BGF-30).
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Articles are provided courtesy of Emory University and the Zhongshan Ophthalmic Center, Sun Yat-sen University, P.R. China.
The print version of this article was created on 8 July 2012. This reflects all typographical corrections and errata to the article
through that date. Details of any changes may be found in the online version of the article.
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