Biomedical Engineering Reference
In-Depth Information
(a)
XY scanning mirror
Pupil lens
Silver mirror
Excitation beam
Quarter
Waveplate
810 nm 100 fs
Tube lens
Dichroic
Variable pinhole
Objective
PMT
Silver mirror
Animal
Collector lens
Band pass filter
(b)
Pinhole
Pinhole
Objective
Objective
Air
Tissue
Air
Tissue
FIgurE 17.2
Collagen
F
/
B
SHG measured with a single objective. Using the system illustrated in panel (a),
both the forward- and backward-directed SHG signals can be captured with a single objective, and
F
/
B
ratios can
be calculated via serial scans of the same region of interest (ROI) through different sized pinholes. This is pos-
sible because as illustrated in panel (b), at shallow imaging depths, the backward-propagating SHG signal exits
the two-photon focal volume with minimal subsequent scatter and will produce a sharp peak in an image plane,
which will pass through a confocal pinhole (b, left image). On the other hand, a fraction of the initially forward-
propagating SHG signal will ultimately backscatter toward the objective lens, passing through the object plane
at multiple locations, and resulting in a weak diffuse signal at an image plane. By repeatedly imaging the sample
through a series of different sized pinholes, the shape of this total SHG distribution can be measured and, with
suitable calibration, the underlying
F
/
B
ratio can be determined, as described [26]. (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
.
)
