In vivo imaging of unstained tissues
using a compact and flexible
Christopher M. Brown
David R. Rivera
Dimitre G. Ouzounov
Wendy O. Williams
Watt W. Webb
In vivo imaging of unstained
tissues using a compact
and flexible multiphoton
Christopher M. Brown,a,*David R. Rivera,a,*
Ina Pavlova,bDimitre G. Ouzounov,a
Wendy O. Williams,cSunish Mohanan,c
Watt W. Webb,aand Chris Xua
aCornell University, School of Applied and Engineering Physics,
271 Clark Hall, Ithaca, New York 14853-2501
bRice University, Department of Bioengineering, 6500 Main Street
Suite 135, Houston, Texas 77030
cCornell University, College of Veterinary Medicine, Ithaca,
New York 14853-6401
endoscope (MPME) to acquire in vivo images of unstained
liver, kidney,andcolonfrom ananesthetized rat. Thedevice
delivers femtosecond pulsed 800 nm light from the core of a
raster-scanned dual-clad fiber (DCF), which is focused by a
miniaturized gradient-index lens assembly into tissue. Intrin-
the tissue is epi-collected through the core and inner clad of
diameter and 4 cm in length. The image field-of-view mea-
sures 115 μm by 115 μm and was acquired at 4.1 frames/s
with 75 mW illumination power at the sample. Organs
were imaged after anesthetizing Sprague-Dawley rats with
isofluorane gas, accessing tissues via a ventral-midline
abdominal incision, and isolating the organs with tongue
depressors. In vivo multiphoton images acquired from liver,
kidney, and colon using this device show features similar
to that of conventional histology slides, without motion arti-
this is the first demonstration of multiphoton imaging of
unstained tissue from a live subject using a compact and
flexible MPME device. © 2012 Society of Photo-Optical Instrumentation
Engineers (SPIE). [DOI: 10.1117/1.JBO.17.4.040505]
Keywords: endoscopy; fiber optic applications; imaging; medical
imaging; microscopy; multiphoton processes.
Paper 12043L received Jan. 20, 2012; revised manuscript received
Feb. 28, 2012; accepted for publication Mar. 1, 2012; published
online Apr. 9, 2012.
minimize patient discomfort by providing a diagnosis without
tissue removal, speed diagnosis, reduce cost by allowing for
real-time determination of tissue disease state, and generate a diag-
mallyinvasive diagnostic techniques, multiphoton microscopy is
promising because it is capable of acquiring label-free tissue
images at high resolution that provide diagnostic information
similar to that obtained from tissue biopsies processed into his-
tology slides.1–3For multiphoton microscopy to successfully
multiphoton microendoscope (MPME) must be developed that
combines small size to access a wide variety of organs in vivo,
ing capability to produce images that are useful for disease diag-
nosis (i.e., capable of subcellular resolution allowing for image
acquisition of cellular details such as nucleus size and nucleus/
images in vivo using GRIN lenses in combination with table-top
two-photon laser scanning microscopes.4,5In this paper, we
and flexible MPME. The fiber delivery and the small size (3 mm
a similar fashion to a number of clinical endoscopic devices.
Though there have been significant efforts to develop
compact and flexible endoscopic devices based upon micro-
electromechanical systems6–8and devices that operate nonreso-
nantly,9–12to date, the devices that have the best combination of
small size, high frame rate, and high-quality image acquisition
are distally resonant-scanned optical-fiber systems.13–19In these
devices, illumination light is delivered through an optical fiber
into an objective lens that focuses and epi-collects light from
tissue. In distally scanned systems, the optical fiber is mounted
to one or more actuators that deflect the fiber tip in a two-
dimensional resonant spiral scan,13–16
scan,18,19or a combined resonant-nonresonant raster scan
pattern.17As described previously, an advantage of the resonant-
nonresonant raster scanner compared with the resonant spiral or
Lissajous scanner is increased uniformity of pixel-dwell-time,
and thus increased uniformity of illumination and light collec-
tion across the image field-of-view (FOV).17
Research groups that have developed these miniature in vivo
imaging devices use them for either neural research6,13,18or
for clinical microendoscopy.10,11Groups that have developed
devices for in vivo imaging of the brain function have sought
to conduct multiphoton imaging of rat cortical blood flow and
neural activity using fluorescently labeled dextran and Ca indi-
imaging have sought to conduct multiphoton imaging of kidney
tissue stained using Fluorescein11and to conduct narrow-band
imaging of various fluorescently labeled organs throughout the
GI tract.10Although, dyes and stains that improve the contrast
of imaged tissue are acceptable for research purposes, it is highly
desirable that clinical imaging of human tissue be independent
of exogenous contrast agents for acquisition of quality images.
To date, few contrast agents have been clinically approved for
use in human patients (e.g., Fluorescein, Indocyanine Green, and
Acriflavine in certain parts of the world) due to dye toxicity.
Multiphoton microscopy has demonstrated the capability to
image intrinsic fluorescence and second-harmonic generation
(SHG) signal in tissue without requiring exogenous stains or
dyes. Here, we describe the use of an MPME device to acquire
in vivo images from unstained kidney, liver, and colon of an
anesthetized rat. To the best of our knowledge, this is the first
demonstration of in vivo multiphoton image acquisition from
unstained tissue using a compact and flexible MPME device.
*These authors contributed equally.
Address all correspondence to: Christopher M. Brown, Cornell University, School
of Applied and Engineering Physics, 271 Clark Hall, Ithaca, New York 14853-
2501; Tel: (607)255-8034; E-mail: firstname.lastname@example.org
0091-3286/2012/$25.00 © 2012 SPIE
Journal of Biomedical Optics040505-1April 2012 • Vol. 17(4)
The MPME uses a femtosecond source (Mai Tai HP, Spectra
Physics) to deliver 800 nm light through the core of a raster-
scanned dual-clad optical fiber (DCF SM-9/105/125-20A,
NuFern). The focusing lens is a Gradient Refractive Index
(GRIN) objective (GT-MO-080-018-810, GRINTECH). Intrin-
sic fluorescence and SHG signal are epi-collected through the
core and inner clad of the dual-clad fiber, proximally split
into two collection channels using a dichroic beam splitter
(Di01-R405-25 × 36, Semrock), and detected using two PMTs
(R7600U-200, Hamamatsu) as shown in Fig. 1. The MPME
acquires images at 4.1 frames∕s with a FOVXY of 115 ×
115 μm. Lateral and axial two-photon resolutions measured
0.8 and 10 μm (full width at half maximum), respectively, in
the image plane. For in vivo imaging, illumination power at
the sample is approximately 75 mW, below the threshold for
mutagenicity.20Additional details regarding the MPME, dis-
persion compensation system, and pulse characterization of
the illumination light are described in previous publications.17,21
light-tight enclosuretoreduce backgroundsignal detectedbythe
device (Fig. 1). Inside the enclosure, MPME position was con-
BAR Positioning Arm, Flexbar Machine Corp.) allowing for six
degrees of freedom of gross movement. The flexible arm was
mounted on a precision motorized micromanipulator (MP-285,
Sutter Instrument Co.) allowing for submicron XYZ axis motion
control of the endoscope. An infrared (IR) imaging system was
used to assist with navigation of the endoscope and monitor the
during the imaging procedure. The imaging system used two
to illuminate the animal while acquiring images from two CCD
camera systems. Illumination light from the IR LEDs was
blocked from the MPME PMTs using a short-pass filter
(FF01-720/SP-25, Semrock). The IR filter, combined with the
poor quantum efficiency of the PMTs at the IR illumination
wavelength, allowed for simultaneous MPME and IR wide-
field imaging during the experimental procedure. Together, the
two IR imaging systems allowed for 10× and 2× magnification
IR imaging of the device and anesthetized rat (Fig. 2).
Laboratories International, Inc.) model was used in this experi-
with a gas anesthetic (∼5% isofluorane-oxygen mixture). After
reaching the appropriate level of sedation for surgery, the animal
was fitted with a nose cone to maintain the sedation (∼2 to 3%
cally restrained in a dorsal recumbent position. A small ventral-
midline abdominal incision was made to expose the internal
organs to the MPME. After imaging the kidney and the liver, a
second incisionwas madeinthe colon toexposeits innersurface
then elevated with tongue depressors to reduce motion artifact
(Fig. 2). The endoscope was then maneuvered into position for
image acquisition using the flexible arm and micromanipulator.
A total of 10 rats were imaged using these procedures. After
All animal procedures were conducted in accordance with a
approved protocol and relevant standard operating procedures.
During the in vivo imaging sessions, the image plane of the
MPME was positioned ∼20 to 30 μm below the tissue surface to
obtain the en face unstained, unaveraged in vivo MPME images
shown in Figs. 3 and 4. Due to axial motion of the live tissue
relative to the MPME image plane, images acquired may be
above or below this depth. The recognizable tissue features
were highly consistent in all imaged rats. These images show
many features that are recognizable in histological tissue sam-
ples.22Figure 3(a) shows an image of intrinsic fluorescent signal
in the rat kidney. In this image, optical cross-sections of tubular
nephrons are visible in the periphery of the organ along with
features such as cells, renal tubules, renal interstitium, and
renal lumen. Figure 3(b) shows an image of intrinsic fluorescent
signal from the interior wall of the rat colon. This image shows
an optical cross-section of a colon crypt. Figure 4(a), 4(b), and
Video 1 show intrinsic fluorescence (pseudocolored green) and
SHG (gray) images ∼20 to 30 μm below the surface of the rat
liver. These images show hepatocytes (i.e., functional liver cells)
Fig. 1 Invivoimagingexperimentallayout.Notethatthe800nmexcita-
Fig. 2 Infrared camera images: (a) 10× view of 3 mm OD endoscope
Fig. 3 Unaveraged, unstained multiphoton endoscope images of rat
kidney and colon. Images show intrinsic fluorescence emission
20 μm below the surface of the organ showing epithelial cellular nuclei
the kidney, and renal lumen (L) inside the renal tubules; (b) image 20 to
30 μm below the surface of the interior colon showing a cross-sectional
view of a crypt (C) and a variety of enterocyte cells lining the intestine
(E). Note that dark cellular nuclei are viewable in many of these cells.
Journal of Biomedical Optics040505-2 April 2012 • Vol. 17(4)
surrounded by a collagenous tissue capsule. The hepatocytes are Download full-text
arranged in cords, forming structural units. The blood-filled
spaces between the cords are sinusoids. Since blood is a strong
light absorber, we see an absence of intrinsic signal in the sinu-
soid. Strong SHG signal can be seen in the septa of the liver.
Images in Figs. 3 and 4 were interpreted with the assistance
of a certified pathologist. When imaging kidney, liver, and
colon tissue with this device, over ∼75% of recorded images
were free of streaking or warping of features within the image
frame eventhough the organ movesrelative to the MPME due to
respiration and heart beat, as shown in Video 1. This can be
credited to rigid mounting of the endoscope during image acqui-
sition, isolating tissue while imaging, the 4.1 frame∕s image
acquisition speed, and the high uniformity of the resonant-
nonresonant fiber scanner. The demonstrated device can be
further improved by achieving faster frame rates with high
signal-to-noise ratio, distal axial sectioning, larger image FOVs
while maintaining high-image resolution, and decreasing the
device size. Several recent developments are designed to address
these issues. For example, by incorporating lensed fibers a larger
FOV can be achieved in a miniature endoscope.23Furthermore,
a higher frame rate and axial sectioning can be achieved by
incorporating a multifocal approach in the MPME.24To the
best of our knowledge, this research demonstrates the first
multiphoton images from unstained tissue in a live animal using
a compact and flexibleMPME device. These images show many
of the features that are commonly seen in biopsied histopathol-
ogy slides from these tissues, indicating the potential of the
MPME device for in vivo diagnostics of tissue health.
R01-EB006736 “Development of Medical Multiphoton Micro-
scopic Endoscopy” and the NIH/NCI Grant R01-CA133148.
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Fig. 4 Unaveraged, unstainedmultiphoton endoscope images of rat liver
located approximately 20 μm below organ surface. The pseudo-color
images show grey SHG signal (<405 nm) and green intrinsic fluorescent
emission (420 to 690 nm). (a) Liver image shows 20 to 30 μm diameter
hepatocytes with a dark 5–10 μm diameter nucleus (N) and bright sur-
rounding cytoplasm (C), hepatic chords (HC) composed of chains of
hepatocytes, and a hepatic sinusoid (HS)—the blood filled space
between hepatic chords. (b) Liver image (also shown in Video 1)
shows features including: bile ductile (BD), bile salts (BS), septa (S)—
a finefibrillar connectivetissuethatcoversthesurfaceofthehepatocytes,
and a hepatic venule (HV). (Video 1, QuickTime, MOV, 3.8 MB). [URL:
Journal of Biomedical Optics040505-3April 2012 • Vol. 17(4)