Biomedical Engineering Reference
In-Depth Information
12.4 imaging and characterization of Pathological corneas
An example of the application of multiphoton microscopy in the diagnosing of corneal diseases is the
detection of infectious keratitis. Shown in Figure 12.4a is the large-area, multiphoton autofluores-
cence (green) and BWSHG (blue) images of the
ex vivo
human cornea that have been infected with
Acanthamoeba castellinii
and
Pseudomonas aeruginosa
. Intrinsic autofluorescence shows the presence
of
Acanthamoeba
cysts (yellow arrowheads) and
Pseudomonas
bacteria (red arrowhead), and the devia-
tion of the BWSHG image pattern from its normal counterpart indicates the degradation of corneal col-
lagen. An increase in autofluorescence was also observed in the infected cornea. Magnified images from
the selected regions of interests (ROIs) in Figure 12.4a are shown in Figures 12.4b through 12.4d. The
presence of pathogens can be clearly visualized from autofluorescence. For the purpose of comparison,
multiphoton autofluorescence image of isolated
Pseudomonas aeruginosa
in Figure 12.4e shows that the
spots in Figure 12.4b are individual bacteria (red arrowheads), which does not resemble the appearance
of the fluorescent
Acanthamoeba
cysts (yellow arrowheads) in Figures 12.4b and 12.4c. Besides, it was
found that the less affected region with preserved collagen structure as demonstrated by the SHG signal
(Figure 12.4d) is less likely to be infiltrated by inflammatory cells (yellow arrowheads). This observa-
tion implies the presence of quiescent
Acanthamoeba
cysts within the clinically clear area. These results
show that cornea infected with pathogens may be imaged, and delineation of infectious pathogens and
corneal collagen can be achieved by the use of label-free multiphoton microscopy [64].
The other example we presented here is the
ex vivo
multiphoton imaging of fungal keratitis in human
cornea [64]. The large-scale multiphoton image (Figure 12.5a) taken at the surface of the ulcerated area
was found to be composed of fluorescent signals and irregularly distributed SHG signals remnants.
The parallel fluorescence pattern and residual SHG generating collagen can be observed in the mag-
nified images (Figures 12.5b and 12.5c). Meanwhile, the laboratory examination represented that the
responsible pathogen of this infection was the
Alternaria
sp. Compared with the multiphoton image
of purified
Alternaria
(Figure 12.5e), the tube-like structure found in Figures 12.5a and 12.5d suggests
that these structures are most likely the hyphae of the infecting fungus. This result again demonstrates
the advantage of multiphoton microscopy in identifying some infecting pathogens without additional
histological processing.
Another important corneal pathology that has been examined with multiphoton imaging is kerato-
conus. For reasons that are not completely understood, corneas of patients with keratoconus undergo
structural transformation in the corneal lamella and result in significant vision degradation. In recent
years, researchers have realized the potentials of multiphoton imaging in diagnosing this condition
[63,65], and a demonstration of the use of multiphoton microscopy in visualizing structural changes
associated with this pathology is shown in Figure 12.6. The large-area, multiphoton autofluorescence
and BWSHG images of the
ex vivo
human keratoconical cornea markedly identify the global architec-
tural change in such a specimen. Intrinsic autofluorescence shows the presence of the keratoconical apex
while BWSHG signal identified the global organization of collagen fibers around the apex. Corneal
topography in Figure 12.6b shows that the location of the keratoconical apex is consistent with that
found from multiphoton imaging. To visualize detailed and localized changes of the pathological tis-
sue, magnified regions of selected ROIs are shown in Figure 12.6c. Specifically, I and III are enlarged
images from the selected ROIs indicated in Figure 12.6a. Further enlargement of these regions shows
that the fluorescent mass near that apex is composed of epithelial cells with elongated and spindle-like
shape (I-1). Furthermore, BWSHG patterns near the apex are aligned in parallel, suggesting that the
altered corneal collagen structure can affect the morphological appearance of the nearby epithelial cells.
Moreover, from SHG images shown in III and III-1, the patterns of centripetal and thickened stromal
collagen directed toward the apical domain were found. The reorganization of collagenous stroma may
be due to pathological structural modification in response to intraocular pressure. This observation
demonstrates the ability of multiphoton microscopy not only in detecting the detailed collagen struc-
tural alteration but also in examining changes in corneal global morphology.
