SHG in Tumors: Scattering and Polarization - Second Harmonic Generation Imaging - page 398

FIgurE 17.4 Serial pinhole in vivo SHG images of collagen fibers in an intact rat tail. Images 1 through 5 are SHG
images of the same ROI when the size of the pinhole was varied from 60, 100, 150, 200, to 7000 μm, respectively.
The bright spots are blue fluorescent polystyrene beads for calibration. The images are 600 μm across. Research
was conducted in compliance with the guidelines of the Institutional Animal Care and Use Committee (IACUC).
(Reprinted from Han, X. and E. Brown. 2010. Measurement of the ratio of forward-propagating to back-propagat-
ing second harmonic signal using a single objective. Opt Express , 18 :10538-10550, Copyright 2010. With permission
of Optical Society of America.)

0.5

Bead TPEF fitting

0.4

0.3

0.2

0.1

Collagen SHG fitting

0

0 0.1 0.2 0.3 0.4 0.5

Relative pinhole size

0.6 0.7 0.8 0.9 1

FIgurE 17.5 Determining collagen SHG F i / B i from collagen SHG and bead pixel intensities, by the single-objec-
tive method. The epidetected total collagen SHG intensity (triangles), or total bead two-photon excitation fluores-
cence (TPEF) intensity (squares) versus pinhole size, fit to the model given by Equation 17.4 (lines). For each set
of images, these signal intensities were averaged from five ROIs around collagen fibers and three ROIs around the
calibration beads, respectively, and were normalized such that the maximum intensity with the largest pinhole was
set to one. The data plotted represent five image sets, that is, the mean SHG of 25 collagen ROIs, and the mean TPEF
of 15 bead ROIs. Fitting the plotted data to Equation 17.4 as shown provides the measured collagen SHG F / B and
bead TPEF F / B values, then the true (emitted) collagen SHG F / B value (i.e., F i / B i ) is calculated by Equation 17.5. The
horizontal axis is pinhole size in units of backward propagating SHG spot size on the pinhole plane, that is, frac-
tion of ω. The vertical axis is normalized SHG intensity. Note that the fitting curves identically equal 1 when the
pinhole size reaches R = 28, whose data point is not shown, and that the intensity information for R = 28 provides
the normalization value and hence is included in the overall fit. Research was conducted in compliance with the
guidelines of the Institutional Animal Care and Use Committee (IACUC). (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.)