Biomedical Engineering Reference
In-Depth Information
Oim
2.4
WT
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0
10
20
30
40
50
60
Depth (microns)
FIgurE 6.6
Ratio of experimentally measured forward and backward collected SHG as a function of depth into
oim and WT skin. These photon propagation data are consistent with a multiple scattering process. (Reprinted
from
Biophys. J
. 94, Lacomb, R., O. Nadiarnykh, and P. J. Campagnola, Quantitative SHG imaging of the diseased
state osteogenesis imperfecta: Experiment and simulation, 4504-4514, Copyright (2008), with permission from
Elsevier.)
can attribute to having a smaller scattering coefficient than the WT at the SHG wavelength, that is, forward
directed photons have a higher probability of remaining forward directed in the less scattering tissues. This
is consistent with the bulk scattering measurements shown in Table 6.1, where these values at 450 nm were
~300 and 175 cm
−1
for the WT and oim, respectively. To validate the distinction between the oim and WT
F/B
data, we have performed a
t
-test at every depth and obtained
p
values in the range of 0.06-0.10, show-
ing that they are statistically distinct at the 10% level. We note that while the dermis is <50-60 microns
thick in murine skin, the tissue biopsies were in the range of 100-200 microns in total thickness, being
composed of the epidermis, dermis, and adipose layers. While only the dermis (and only from the collagen
component) provides SHG contrast, the entire thickness represents a scattering medium.
We also observe that for both tissues the
F/B
increases with increasing depth into the tissue. This result
is consistent in the framework of photon diffusion theory, where at least one MFP is required between the
location of the emitted photon and the forward boundary of the specimen for efficient multiple scattering
to occur [30]. Thus, at increasing focal depths, the probability of multiple scattering events decreases as
the forward pathlength to the tissue boundary shortens, and subsequently the
F/B
ratio must increase.
Owing to the fibrillar morphology in these tissues, the SHG has an initial emission directionality
composed of both forward and backward components, and we incorporate this factor into the simu-
lations through the creation ratio
F
SHG
/B
SHG
. The trends of how this emission ratio is determined by
fibril size and packing into SH producing domains were described in Section 6.3 on phasematching.
Representative simulated curves of the depth-dependent
F/B
assuming 100% and 50% forward for oim
and WT skin are shown in Figure 6.7a. The simulations used a four-layer model comprising the dermis
and adipose layers. If we first only consider the 100% forward emission curves, we observe that these
simulations reproduce the overall experimental trend in that the oim is characterized by a larger
F/B
than the WT at all depths because of the lower scattering coefficient. Despite providing qualitative dis-
tinction between the tissues, we note that this simulation largely overestimates the magnitude of the
ratio (approximately by a few fold) for each tissue. This result indicates that it is inappropriate to assume
that all SHG photons are emitted in the forward direction, and additionally that bulk optical parameters
alone constitute an insufficient description. The case of 100% forward emission in tissue is not physically
