Endomicroscopy - Biomedical Optical Imaging Technologies: Design and Applications - page 284

8.3.4 Nonlinear Endomicroscopy

The endomicroscopy platforms described thus far are all based on imaging of
single-photon fluorescence or backscattered light. Nonlinear microscopy techniques
which have been successfully implemented in benchtop systems have also begun to
be transitioned into compact and miniaturized packages compatible with endomi-
croscopy. These techniques all achieve optical sectioning, not through the use of a
pinhole or slit mask, but through a quadratic dependence on illumination intensity
which all but confines the generation of nonlinear signals to the focal plane [ 21 ].
Similar to the use of single rigid GRIN lens assemblies for confocal fluorescence
described above, Jung and Schnitzer presented two-photon fluorescence images of
fluorescently labeled neurons in brain slices, acquired with submillimeter diameter
triplet GRIN objectives [ 94 ]. The short length GRIN objective eliminated the need to
prechirp the excitation pulses to compensate for dispersive pulse broadening within
the optical path to the specimen. The same group extended this work to in vivo
animal models in 2004 [ 50 ]. Kim et al. described a benchtop microscope built in-
house with a 1-mm-diameter, 15-mm-long GRIN lens which permits imaging at
NA 0.45 in confocal fluorescence, multiphoton fluorescence, and second-harmonic
modes [ 22 ].
Development of endomicroscope systems for nonlinear microscopy with fiber-
optic instead of free-space beam delivery is made more difficult by broadening of
the excitation pulse within the fiber itself. Gobel et al. presented a fiber-optic two-
photon imaging system using a coherent fiber bundle and GRIN lens objective
[ 55 ]. The 100 fs pulses from the Ti:Sapphire laser source were prechirped by a
grating pair to compensate for group velocity dispersion within the fiber bundle;
this approach was successful in restoring the original pulse width for average
output powers below 5 mW. However, at the higher intensities typically used in
multiphoton microscopy, severe pulse broadening remained which led to suboptimal
fluorescence excitation at these intensities. Lelek et al. described an alternative
method to both temporally and spectrally shaped femtosecond pulses prior to deliv-
ery through a fiber bundle [ 30 ]. This approach provided improved compensation for
dispersive effects arising from nonlinear processes such as self-phase modulation,
which are increasingly present at higher pulse energies but cannot be compensated
with a grating or prism pair to introduce negative prechirp before launching.
As with the confocal fluorescence endomicroscopy systems described earlier, the
use of a fiber-optic bundle inherently results in images with the discrete core pattern
superimposed. This effect can be mitigated by image post-processing methods, but
an underlying loss of information cannot be avoided. Again, application of single
optical fibers with distal tip scanning enables a continuous image to be formed.
Double-clad fibers offer improved efficiency in collection of the nonlinear signal
generated at the sample by using a small single-mode core surrounded by a larger
inner cladding to deliver and collect light, respectively. Myaing et al. used a double-
clad fiber with 3:6-mcore,90-m inner cladding, and 125-m outer cladding
mounted within a piezoelectric tube actuator to generate a spiral scan pattern at 2.6