Endomicroscopy - Biomedical Optical Imaging Technologies: Design and Applications - page 285

frames per second over a 220-micron-diameter FOV [ 39 ]. A NA 0.46/0.7 GRIN
lens was used to image fluorescent beads and fluorescently labeled cells in culture.
Later, Bao et al. described a handheld two-photon microscope again using dual-clad
fiber, but mounted in a compact raster scanner similar to that developed by Optiscan
for confocal endomicroscopy. This device produced images over a 475 m 2 FOV
at 1.7 frames per second, with images shown of intact (thick) rat kidney following
staining with fluorescein [ 14 ].
Distinct from fiber-optic bundles and double-clad fibers with conventional single-
mode cores, several novel types of optical fiber have been developed which can
eliminate or compensate for spectral pulse broadening (see Sect. 8.2 ), each of which
has been integrated into nonlinear endomicroscopy systems. In 2008, two-photon
imaging systems were demonstrated with hollow-core photonic crystal fiber for
excitation pulse delivery by both Engelbrecht et al. and Le Harzic et al. [ 28 , 29 ].
Piezoelectric tube scanners translated the PCF tip in spiral or raster scan patterns,
with a GRIN lens assembly as the objective. A miniature dichroic beamsplitter
directed emitted fluorescence to a multimode fiber for delivery to a photomultiplier
tube or avalanche photodiode. Both groups demonstrated imaging in biological
tissues [ 28 ] and in cultured cells [ 29 ], with the former using a 25-Hz frame rate over
a 200-micron-diameter FOV to observe dynamic fluorescent intensity fluctuations in
the rat cerebellum following labeling with a fluorescent calcium indicator. Le Harzic
et al. demonstrated imaging over a larger FOV (420 420 um), albeit at a lower
frame rate of 0.8 Hz.
In addition to multiphoton fluorescence imaging, second- and third-harmonic
imaging had been demonstrated with compact devices compatible with endomi-
croscopy. Second-harmonic generation (SHG) can be detected in regularly ordered
tissues, such as collagen, using the same femtosecond Ti:Sapphire laser source
as used for two-photon imaging. Researchers who had previously demonstrated
two-photon endomicroscopy also reported second-harmonic imaging with similar
optical setups; no fundamental modifications to the endomicroscopy probe itself
are required, although a narrow band-pass filter centered at the SHG wavelength
is added in front of the detector to block residual excitation light. Wu et al. [ 54 ]
and Bao et al. [ 27 ] both used miniaturized systems based upon double-clad fiber to
image second-harmonic emission from rodent tail tendon. The former used a piezo
tube spiral scanner with GRIN lens, while the latter used the electromagnetically
driven raster scanner with a miniature multielement objective lens developed by
Optiscan. Both groups demonstrated combined SHG/two-photon imaging using
a single platform, highlighting the feasibility of imaging in both these nonlinear
modes. Less work has been reported on third-harmonic endomicroscopy, but
Chia et al. reported a handheld probe with large mode area fiber (35-um core
diameter) for beam delivery, multimode fiber for THG collection, MEMS scanner,
and macroscopic optics [ 95 ]. To generate THG signals in the visible wavelength
range, where many detectors have optimum sensitivity, it is necessary to use a
femtosecond source emitting in the farther into the near infrared. These authors
used a Cr:forsterite laser emitting in the 1,200-1,300-nm range and demonstrated
its use in recording THG images from the skin of normal human volunteers.