Biomedical Engineering Reference
In-Depth Information
0.9
F
/
B
= 0.1
F
/
B
= 0.5
F
/
B
= 1
F
/
B
= 5
F
/
B
= 10
F
/
B
= 50
F
/
B
= 100
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
0.5
1
1.5
2
2.5
3
Relative pinhole size
FIgurE 17.6
Relationship between pixel intensity, pinhole size, and
F
/
B
ratios, by the single-objective method.
The relative SHG intensity versus relative pinhole size curves at different collagen SHG
F
/
B
ratios were based upon
Equation 17.4 and
C
= 0.001. The relative SHG intensities equal 1 at a pinhole size of
r
=
R
max
= 28. Note the sharp
curvature due to the Gaussian distribution of direct backward-propagating SHG (
B
i
) (most evident in the steep
early rise of the curves with lower
F
/
B
), and the slow and steady curve rise due to the subsequently backscattered
forward-propagating SHG (
F
i
) that produces a diffuse signal that does not vary with radius (most evident in the
slow rise of the curves with large values of
F
/
B
). Also note that given the typical variation in experimental data (e.g.,
as seen in Figure 17.5 above), when
F
/
B
тиа~ 5, the curves may not be statistically distinguishable, and thus, this
method may be best suited for samples with collagen fiber
F
/
B
ratios <~ 5 (see the text for further detail). (Reprinted
from Han, X. and E. Brown. 2010. Measurement of the ratio of forward-propagating to back-propagating second
harmonic signal using a single objective.
Opt Express
,
18
:10538-10550, Copyright 2010. With permission of Optical
Society of America.)
intensity will vary with relative pinhole size for different
F
/
B
ratios, as fit by Equation 17.4. The correc-
tion factor
C
can be eliminated and the collagen fiber
F
i
/
B
i
SHG ratio can be determined by the equation:
MeasuredcollagenSHG /
F Br
F Br
atio
real c
ollagenSHG /
F Br
F Br
atio
=
(17.5)
MeasuredbeadsTPEF /
atio
real beadsTPEF /
atio
In this manner, this method was utilized to extract collagen
F
i
/
B
i
values from intact (not sectioned)
rat tail as well as from muscle fascia using the SHG signals captured from a single objective, and these
F
i
/
B
i
values were not significantly different from
F
i
/
B
i
values for collagen obtained using both forward
and backward detectors (i.e., the traditional approach using two objectives, one on either side of the
sample) [26].
Overall, this technique demonstrates that by imaging the sample repeatedly through different sized
pinholes to measure the shape of the total SHG distribution, and extrapolating the
F
i
/
B
i
from the
F
d
/
B
d
by a bead calibration method as described above,
F
i
/
B
i
measurements can thus be obtained
in vivo
from intact biologic specimens from a single objective. This represents an important advancement that
should improve our ability to conveniently obtain
F
/
B
measurements from biologic tissue under
in vivo
conditions, which may in turn ultimately yield real-time clinical imaging diagnostics for cancer or other
diseases.
