Biomedical Engineering Reference
In-Depth Information
bovine corneas that have been injected with
Pseudomonas aeruginosa.
Images acquired at different
time-points (0, 6, 12, and 24 h) following injection show the effects of simulated infection on corneal
stromal collagen as a function of time. At 0 h, injected
Pseudomonas aeruginosa
in radiated pattern can
be observed by autofluorescence imaging. At this time, the stromal collagen is largely intact with no
signs of degradation. With progression of the simulated infection process, a decrease in BWSHG signal
and increase in autofluorescence are observed. In 24 h, the corneal stroma is almost filled entirely with
intense autofluorescence, whereas little SHG signal was observed. Therefore, qualitatively, increased
damage of cornea is accompanied with increased autofluorescence and decrease in the SHG signal.
To visualize our observation in greater details, selected regions of interest at 0, 6, and 24 h follow-
ing pathogen injection were selected from Figure 12.8. The respective magnified images are shown in
Figures 12.9a through 12.9c, where injected
Pseudomonas aeruginosa
are clearly visible (arrows). With
increase in infection time, there is a decrease in BWSHG signal and corresponding increase in autofluo-
rescence. Therefore, similar to the images acquired from excised human corneas infected with fungous
amoeba and bacteria, severely infected corneas containing weak SHG signals and significantly increased
autofluorescence were observed [64].
Multiphoton microscopy has also been employed in experiments imaging and characterizing the extent
of bovine cornea glycation. The glycation process is thought to contribute to many of the complications
as seen in aging and diabetes mellitus [66]. Hence, early diagnostics of the influence of glycation-induced
ophthalmological pathologies is of primary importance for their therapeutics.
Cornea glycation was induced by incubating cornea in 0.5 M ribose solution to mimic physiologi-
cal hyperglycemia [67]. The control group was treated with the same chemical composition as the
glycation solution with ribose being excluded. All corneas were incubated at 37°C with condition
of 5% CO
2
for different periods of 2, 4, 6, 8, and 10 days. Time-lapsed multiphoton imaging on gly-
cated and control samples of bovine cornea is illustrated in Figure 12.10, in which autofluorescence
and BWSHG intensities extracted from glycated bovine cornea increase and decrease, respectively, as
glycation prolonged in number of days. In contrast, for control group, no significant variance of the
BWSHG intensity was observed. The analytic result of MPM images, shown in Figure 12.11, delineates
the trends of autofluorescence and SHG intensities as a function of duration of glycation. The rise
of autofluorescence accompanied with the formation of the autofluorescent advanced glycation end
products (AGEs), and the decline of BWSHG indicates glycation-induced disorder in the collagen
structure [68]. The consequences of MPM images and its analysis are twofold: indication of the rate
of glycation in bovine cornea and elucidation of the drastic responsiveness of collagen over elastic
(a)
(b)
(c)
20 μm
FIgurE 12.9
(a), (b), and (c) are the respective magnified images of selected regions of interest at 0, 6, and
24 h following simulated infection images in Figure 12.7. (a,b) Injected
Pseudomonas aeruginosa
are clearly visible
(arrows). With increase in infection time, there is a decrease in backward SHG signal and corresponding increase
in autofluorescence. (c) The corneal stroma is almost filled entirely with strong autofluroescence. (Adapted from
Chang, Y. L. et al. 2010.
Appl Phys Lett
97:183703.)
