Biomedical Engineering Reference
In-Depth Information
such as biopsy examination and electron micrographs. However, potential artifacts associated with the
process of fixation and labeling are always concerns. In addition, examination of fixed specimens can-
not provide dynamic information of the ophthalmological systems under study.
Since the cornea is transparent and exteriorly located, optical microscopy is an appropriate tech-
nique for direct imaging and visualization of its structures. In the last few decades, a number of optical
imaging modalities have been developed for these purposes. Techniques such as slit-lamp examina-
tion, optical coherence tomography (OCT), reflective confocal microscopy, and harmonic generation
microscopy have been successfully developed for improving the understanding of corneal physiol-
ogy and diagnosis of pathological conditions for potential clinical applications [4-14]. In addition to
the widely used slit-lamp technique, the interferometry-based OCT methodology has been used in a
variety of medical applications, including those in ophthalmology [7,15-19]. It was shown that OCT
is capable of providing cross-sectional images at micron-level resolution [6,20]. In this manner, the
layered structure from the anterior to the posterior segments of the eye, including that of the cornea,
can be visualized [21,22]. The application of OCT upon the human cornea was first reported in 1994,
which mapped the contours of the epithelium and endothelium, and quantitatively analyzed corneal
thickness [22]. This technique has also been used in characterizing corneal pathologies, such as bullous
keratopathy [17], corneal edema [23], and macular diseases [24]. In one study, the potential of OCT, as
a medical diagnosis tool, was investigated by imaging cornea morphology before and after photothera-
peutic keratectomy [25].
Confocal microscopy is another optical imaging technique that has been applied to cornea imaging.
Since its initial introduction by Marvin Minsky demonstrating the improvement in image quality by the
use of confocal aperture [26], subsequent developments have led to tandem scanning reflective confocal
microscopy with multiple scanning points [27] elucidating the full thickness of human cornea ex vivo
and of rabbit cornea in situ [28]. In the reflection mode, this noninvasive imaging modality has been
applied clinically in ophthalmology. In previous reports, in vivo and ex vitro confocal images of corneas
have proven to be effective in the visualization of epithelia, endothelial cells, and the stromal keratocytes
[9,29-31]. Wound healing processes following refractive surgeries were also visualized, which helped
to determine the potential complications arising from surgeries [32-34]. Nowadays, reflective confo-
cal microscopy is being routinely applied in clinical ophthalmological observation and the detection
of corneal diseases [9,10,35,36], such as infectious diseases, keratitis [37-39], and keratoconus [40,41].
While OCT and confocal imaging are effective in the visualization of corneal microstructures, imag-
ing of the stromal collagen, the main component of the cornea, has been challenging with these two
techniques. In confocal imaging, reflection is not an effective image contrast mechanism for visualizing
the collagen fibers in the transparent cornea. On the other hand, although OCT has been demonstrated
to be capable of imaging corneal stroma [23,42], it cannot provide structural information at submicron-
level resolution. Finally, since both OCT and reflected confocal microscopy use reflection as the contrast
mechanism, intrinsic molecular changes, which may be important for basic studies and disease diagno-
sis, cannot be investigated. In recent years, the development of nonlinear optical microscopic imaging
modalities such as multiphoton fluorescence or second harmonic generation (SHG) microscopy has
contributed to the repertoire of tools that researchers can use in addressing biomedical questions. Like
confocal imaging, the intrinsic optical sectioning capabilities of nonlinear optical interaction can result
in images with excellent axial depth discrimination. Furthermore, by limiting specimen excitation to
the focal volume, specimen longevity is greatly prolonged. Finally, the near-infrared wavelengths used
in multiphoton and SHG microscopy are less absorbed and scattered by tissues, thus allowing greater
imaging depth to be achieved [43,44]. The development of multiphoton fluorescence and harmonic
generation microscopy has led to tissue imaging applications in a number of areas, including derma-
tology [45,46], hepatology [47,48], neurology [49-51], and ophthalmology [52,53]. In ophthalmologi-
cal imaging, multiphoton microscopy holds particular promise for the diagnosis of corneal diseases.
Unlike other tissue types, the cornea cannot be routinely removed for histological examination and the
development of a high-resolution, in vivo monitoring technique will be of significant value for clinical
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