J Neurophysiol 91: 1908 –1912, 2004.
First published December 10, 2003; 10.1152/jn.01007.2003.
Innovative Methodology
In Vivo Multiphoton Microscopy of Deep Brain Tissue
Michael J. Levene, Daniel A. Dombeck, Karl A. Kasischke, Raymond P. Molloy, and Watt W. Webb
School of Applied and Engineering Physics, Cornell University, Ithaca, New York 14853
Submitted 20 October 2003; accepted in final form 1 December 2003
INTRODUCTION
Noninvasive methods for imaging of deep structures in
intact animals include MRI and PET (Jacobs and Cherry 2001).
However, the resolution and speed of MRI is more than an
order of magnitude lower than that of fluorescence microscopy
and offers only a few functional contrast agents compared with
the tremendous library of available fluorescent indicators. Although PET accesses a variety of molecular probes, its resolution is on the scale of millimeters and it suffers from slow
image acquisition. Therefore the ability to perform minimally
invasive deep in vivo fluorescence microscopy represents a
breakthrough in intact-animal studies for biology and medicine.
Although routine use of multiphoton microscopy achieves
imaging depths in brain of typically ⬍500 ␮m, the ultimate
imaging depth is dependent on many parameters, including the
age of the animal and the optical geometry (Oheim et al. 2001).
Oheim et al. (2001) have extended the reach of multiphoton
microscopy by approximately 100 ␮m by appropriate use of a
low-magnification, high numerical aperture objective. Theer et
al. (2003) recently used regenerative amplification of 200-kHz
pulses to achieve high pulse peak powers while maintaining
reasonable average powers, enabling imaging depths of close
to 1 mm, although image quality suffered significantly at
greater depths. Gradient index (GRIN) lenses offer the opportunity to reach even deeper lying structures.
Address for reprint requests and other correspondence: W. W. Webb, Clark
Hall, Rm. 223, Cornell Univ., Ithaca, NY 14853 (E-mail: [email protected]).
1908
GRIN lenses use a negative gradient in the refractive index
of glass from the center of the lens to the outside edge to bend
and focus light. GRIN lenses are characterized by a length, or
pitch, and a numerical aperture (NA). The pitch of a GRIN lens
determines how many internal images are formed within the
lens. A 0.25-pitch lens focuses a parallel beam incident on the
front surface of the lens to a point on the back surface. A lens
of pitch 1 forms an upright image on the back surface, with an
internal, inverted image plane located at one-half the length of
the lens. GRIN lenses are commercially available in lengths of
up to several tens of centimeters, long enough, in principle, to
access deep brain structures in large animals and humans.
GRIN lenses have been used for fiber bundle– coupled confocal microscopy (Knittel et al. 2001) and in vivo epi-fluorescence microendoscopy (Jung and Schnitzer 2002). The use of
large (⬎1 mm diam) GRIN lenses for in vivo multiphoton
microscopy (Levene et al. 2002) and the characterization of
thin composite GRIN lenses for multiphoton microscopy (Jung
and Schnitzer 2003) have recently been reported. Multiphoton
microscopy (Denk et al. 1990) has several important advantages for in vivo imaging applications with GRIN lenses. It is
capable of resolving optical sections relatively far from the
surface of the lens, while tissue in close proximity to the lens
may suffer from mechanical damage or immune response. The
superior sectioning ability of multiphoton excitation is critical
for observing detailed structure in tissues in which fluorescence
sources are distributed throughout the region of interest. Multiphoton microscopy is able to excite intrinsic tissue fluorescence and UV absorbing dyes without the use of UV excitation, which has poor tissue penetration and produces a strong
fluorescent background both from tissue regions outside the
focal plane and from the high numerical aperture GRIN lenses
themselves.
METHODS
Microscope apparatus
The multiphoton microscope used here is the same as described
elsewhere (Kloppenburg et al. 2000) except that, for yellow fluorescent protein (YFP) imaging, the bialkali photomultiplier tube (PMT)
is replaced by a GaAsP PMT (H7422P, Hamamatsu, Bridgewater,
NJ), which exhibits a fivefold greater sensitivity to yellow wavelengths. The Ti:Sapphire laser source produced ⬃100-fs pulses at 80
MHz. The position of the GRIN lens relative to the objective lens is
controlled via hydraulic micropositioners (Narishige Scientific Instruments Lab, Tokyo, Japan) fixed to the nosepiece of the microscope.
The focus knob of the microscope moves the objective and GRIN lens
together as a fixed unit to the appropriate depth within the specimen.
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0022-3077/04 $5.00 Copyright © 2004 The American Physiological Society
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Levene, Michael J., Daniel A. Dombeck, Karl A. Kasischke, Raymond P. Molloy, and Watt W. Webb. In vivo multiphoton microscopy of deep brain tissue. J Neurophysiol 91: 1908 –1912, 2004. First
published December 10, 2003; 10.1152/jn.01007.2003. Although fluorescence microscopy has proven to be one of the most powerful tools
in biology, its application to the intact animal has been limited to
imaging several hundred micrometers below the surface. The rest of
the animal has eluded investigation at the microscopic level without
excising tissue or performing extensive surgery. However, the ability
to image with subcellular resolution in the intact animal enables a
contextual setting that may be critical for understanding proper function. Clinical applications such as disease diagnosis and optical biopsy
may benefit from minimally invasive in vivo approaches. Gradient
index (GRIN) lenses with needle-like dimensions can transfer highquality images many centimeters from the object plane. Here, we
show that multiphoton microscopy through GRIN lenses enables
minimally invasive, subcellular resolution several millimeters in the
anesthetized, intact animal, and we present in vivo images of cortical
layer V and hippocampus in the anesthetized Thy1-YFP line H mouse.
Microangiographies from deep capillaries and blood vessels containing fluorescein-dextran and quantum dot-labeled serum in wild-type
mouse brain are also demonstrated.
Innovative Methodology
IN VIVO MULTIPHOTON MICROSCOPY OF DEEP BRAIN TISSUE
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A piezoelectric focus controller (Physik Instrumente, Waldbronn,
Germany) adjusts the focus of the objective relative to the GRIN lens,
allowing z-series of the sample while holding the position of the
GRIN lens fixed.
Animal surgeries
GRIN lens and imaging parameters
The use of GRIN lenses for in vivo multiphoton microscopy is
shown in Fig. 1. One end of the GRIN lens is positioned close to the
focal plane of the objective lens of a standard multiphoton laser
scanning microscope, with the opposite end inserted inside the specimen under study. The GRIN lens refocuses the laser light tens of
micrometers from the opposite end of the lens, inside the specimen,
where the scanned beam excites fluorescence that is collected back
through the GRIN lens and the objective. Dispersion compensation is
not required because pulse dispersions in the GRIN lenses used here
are ⬃2,700 fs2, similar to typical high-NA objective lenses.
To image neurons and capillaries in mouse brain, we glued two
custom-fabricated 0.6-NA GRIN lenses of pitch 0.22 to either end of
a 0.1-NA GRIN lens of pitch 1 (NSG America, Somerset, NJ) using
a UV curable optical adhesive (Norland Products, Cranbury, NJ) as
shown in Fig. 2. The lenses were 350 ␮m in diameter, and the total
length of the glued lens was ⬃16 mm. All but the last 900 ␮m at either
end of the composite GRIN lens was protected from mechanical
damage by a metal sheath with an outside diameter of 600 ␮m. While
high NA end pieces determine the resolution of the composite lens, it
was necessary to use lower NA material for the central length of lens
to avoid focusing the excitation laser pulse inside the high NA
material, which can produce a fluorescent background and may result
in nonlinear optical effects such as self-phase modulation. This
choice, however, reduces the total field of view to a 58-␮m-diam
circle. Other configurations may be used when larger fields of view
are required. The pitch of the 0.6-NA material was chosen to give a
focal distance of 35 ␮m in air and 47 ␮m in water. Changing the
position of the focal plane of the objective results in a change in the
working distance (WD) from the end of the GRIN lens in the sample.
This allows z-scanning in the sample, from 0 to 95 ␮m from the
surface of the lens, without moving the GRIN lens.
Coupling to the GRIN lens was via a Zeiss Fluar 20⫻, 0.75 NA air
immersion objective (Carl Zeiss, Thornwood, NY). The NA-mismatch between the coupling objective and the GRIN lens resulted in
a 59% coupling efficiency. The transmission efficiency of the GRIN
lens was ⬃66%, resulting in an overall excitation efficiency of ⬃40%.
The full width half-maximum (FWHM) of the lateral point spread
function (PSF), measured using sub-resolution fluorescent particles,
was 825 nm, less than twice the diffraction limited value of 481 nm
and similar to previous reports (Jung and Schnitzer 2003). The axial
PSF was estimated to have a FWHM of 15 ␮m by measuring the
J Neurophysiol • VOL
FIG. 1. Experimental arrangement for using gradient index (GRIN) lenses
in conjunction with multiphoton microscopy. A hydraulic micropositioning
system locates the GRIN lens relative to the objective lens. A piezoelectric
focus control performs fine focusing without moving the GRIN lens inside the
animal.
fluorescence as a function of depth of penetration into a fluorescent
plastic slide. The large axial PSF is likely due to spherical aberration.
In all cases, the GRIN lens was inserted approximately perpendicularly to the cortical surface through the cranial opening in steps of a
few tens of micrometers. Pausing between steps allowed tissue to
physically readjust to the presence of the lens. It has been suggested
that rapid penetration by blunt tips in neural tissue minimizes tissue
damage, while slow penetration minimizes tissue compression (Edell
et al. 1992). Pausing between rapid steps should therefore minimize
both tissue damage and compression. Care was taken to avoid inserting the lens at locations with considerable blood from surgery on the
cortical surface. Once inserted, most trials produced useful images,
with little, if any, apparent accumulation of blood on the lens surface
during or after penetration.
For the images of blood vessels, the excitation wavelength was 830
nm in Fig. 3, A–C, and 780 nm in Fig. 3D, with ⬃50 mW of power
at the sample. Figure 3, A–C, shows 1-s scans taken at a WD of 50
␮m. Figure 3D is a projection of five images taken with WDs of
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Anesthesia and surgeries were performed in accordance with Cornell University–approved animal use protocols. The mice were maintained under ketamine (76 mg/kg)/xylazine (5 mg/kg) anesthesia
during the surgery and the following imaging session. To reveal the
cortex for GRIN rod penetration, we used a dental drill to create a
circular craniotomy (5 mm diam) centered above the dorsal parietal
cortex. The dura visible beneath was cut at the edge of the field and
carefully removed. For analgesia at the site of the craniotomy, lidocaine (2%) was applied topically. The body temperature was maintained at ⬃36° celsius using a feedback-controlled heating pad during
both surgery and imaging. Prior to the microangiography, the blood
serum was labeled by a tail-vein injection of a 100-␮l bolus of either
fluorescein-labeled dextran (20 mg/ml, 500 kDa, Molecular Probes) or
quantum dots (35 ␮M, 608 nm emission, Quantum Dot, Hayward,
CA) in physiological saline.
Innovative Methodology
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LEVENE, DOMBECK, KASISCHKE, MOLLOY, AND WEBB
13– 40 ␮m from the end of the GRIN lens. Each image in the stack is
the average of two scans lasting 3 s each.
For the images of neurons in Fig. 4, A–E, the excitation wavelength
was 920 nm with 20 mW of power at the sample. All scans were 3 s,
with no averaging. The WDs were 43 and 42 ␮m for 4A and 4B,
respectively. Figure 4C is a projection of four images with WDs from
FIG. 4. In vivo images of brain in anesthetized in THY1-YFP line H mice.
A–C: images of yellow fluorescent protein (YFP)-containing cell bodies of
layer V neurons 700 – 800 ␮m below the surface of cortex. D: image of axon
bundles in the external capsule ⬃1 mm below the surface of cortex. E: image
of neuropil in hippocampal region CA1, ⬃1.5 mm below the surface of cortex.
Scale bars are 10 ␮m.
46 to 62 ␮m. Figure 4, D and E, had WDs of 24 and 60 ␮m,
respectively.
RESULTS AND DISCUSSION
FIG. 3. In vivo microangiography in wild-type mouse. A: blood vessels
containing fluorescent quantum dots ⬃800 ␮m below the surface of cortex. B:
line scan of small capillary in A as indicated by dotted red line. C: zoom of
section in B; ⌬x is the spatial dimension of the line scan, ⌬t is the time
dimension, and the hypotenuse is the velocity. D: capillaries containing fluorescein-dextran more than 2 mm below the surface of cortex. Scale bars are 10
␮m.
J Neurophysiol • VOL
Microangiographies of capillaries and larger blood vessels
are shown in Fig. 3. Figure 3A shows a capillary along with
larger blood vessels ⬃800 ␮m below the surface of wild-type
mouse cortex after administration of fluorescent quantum dots
by tail vein injection. Fluorescent quantum dots have high
two-photon cross-sections and are excellent indicators for in
vivo angiography (Larson et al. 2003). Individual blood cells
do not take up the quantum dots and are apparent as dark spots
in the capillary. Figure 3B shows a line scan as indicated by the
horizontal red line in Fig. 3A. The horizontal coordinate in Fig.
3B represents space along the line, and the vertical coordinate
represents time as successive scans pass the same region. The
dark streaks are from the passage of blood cells and can be
used to estimate the velocity of blood flow, as indicated in the
portion of Fig. 3B shown in larger scale in Fig. 3C. Taking into
account the angle of the capillary with respect to the line scan,
the blood velocity was estimated to be ⬃0.6 mm/s, similar to
the velocity measured in rat cortex (Chaigneau et al. 2003;
Kleinfeld et al. 1998). Figure 3D shows blood vessels more
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FIG. 2. Composite GRIN lens used for in vivo neural imaging. A: schematic of composite lens with path of excitation light shown in red. B: image of
top portion of composite lens next to a penny.
Innovative Methodology
IN VIVO MULTIPHOTON MICROSCOPY OF DEEP BRAIN TISSUE
J Neurophysiol • VOL
within the WD of the GRIN lenses (Edell et al. 1992; Turner et
al. 1999) and independent of probe dimensions (Szarowski et
al. 2003). The feasibility of multiphoton microscopy in conjunction with cranial windows has been consistently demonstrated in chronic imaging studies (Bacskai et al. 2001; Christie
et al. 2001).
In addition to choosing lenses of the smallest practicable
diameter, the impact of tissue damage in the brain may be
minimized by working in larger animals and by using the
lenses to image in previously inaccessible sulci without actually penetrating brain tissue. Use of intrinsic signals (Dombeck
et al. 2003; Zipfel et al. 2003) or alternative labeling methods,
such as calcium indicators (Svoboda et al. 1997) or dyes that
stain ␤-amyloid plaques (Christie et al. 2001), with this deep
imaging technique could enable many new physiological experiments both in basic research and in animal models of
neurodegenerative diseases. This imaging modality may also
be easily combined with conventional in vivo electrophysiological techniques.
GRIN lenses used in conjunction with multiphoton microscopy open up the possibility of performing deep in vivo fluorescence imaging in brain. We have demonstrated the compatibility of GRIN lenses with in vivo imaging. Potential complications such as motion artifact and the accumulation of
damaged tissue at the front of the lens during penetration have
been shown to be negligible. Scanned fiber optic coupling to
long lenses may eventually enable work in large animals and in
awake behaving animals. Considerable progress in fiber optic
scanning for in vivo imaging of awake behaving animals has
already been made by Helmchen and co-workers (Helmchen et
al. 2001; Ouzounov et al. 2002). We anticipate that in vivo
multiphoton imaging with GRIN lenses will prove to be a
valuable tool for both biological research and clinical applications.
ACKNOWLEDGMENTS
We thank NSG America for their cooperation and for custom GRIN lens
fabrication and K. Hodgson for Fig. 1. We also thank Quantum Dot for the
quantum dots.
GRANTS
This work was supported by the facilities of the National Institutes of
Health (NIH) Biomedical Resource funded by the National Center for Research Resources-NIH/National Institute of Biomedical Imaging and Bioengineering. Additional support was received from the U.S. Department of Energy,
the National Science Foundation, and from an NIH Training Grant in Molecular Biophysics.
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interfere significantly with image acquisition.
It is likely that chronic implantation of GRIN lenses may
induce gliosis and inflammation; however, studies of gliosis in
response to electrode penetrations found responses limited to
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