Biomedical Engineering Reference
In-Depth Information
FIgurE 14.4
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See color insert
.)
In vivo
3D images of (a) a zebrafish heart and (b)-(c) a zebrafish brain. The 3D
images are reconstructed from stacks of HGM images versus depth. (a) The 3D structures of the cardiac muscles
(SHG; green), cardiac cells (2PF; red), and red blood cells (THG; yellow); and (b) the 3D structures of the neural
tube (THG; purple), otic vesicles (arrows; THG; purple), and the nerve fibers (SHG; green) can be observed with a
submicron resolution. Image size: 240 × 240 μm
2
.
depth. For example, Figure 14.4a shows a 3D image of the zebrafish heart (Kung et al. 2007), including
the 3D structures of the cardiac muscle, cardiac cells, and red blood cells (RBC), while Figures 14.4b and
14.4c show the 3D structures of the nerve fibers, neural tubes, and otic vesicles (arrows) of the zebrafish
brain (Chen et al. 2006) with a submicron spatial resolution. Additionally, in another study (Tsai et al.
2006), the submicron spatial resolution in fixed human skin and its degradation versus depth beneath
the skin surface have been analyzed. Based on these submicron resolution results, HGM is shown to
have the capability of performing depth-resolved optical sectioning, providing cellular and subcellu-
lar information, and performing 3D imaging. Therefore, HGM has the potential for assisting or even
replacing physical biopsies and pathohistological analysis.
14.2.3 System Setup
Depending on the applications, the Cr:F-based SHG/THG microscopic system can be designed to use
either a forward-collection (Figure 14.5a) or a backward-collection (Figure 14.5b) geometry. Whatever
the geometry, an SHG/THG microscope is composed of four main parts—excitation laser, scanning
units, microscope, and detectors. For pixel-by-pixel imaging, the laser beam should be guided into a
scanning unit to achieve real-time 2D beam scanning. Before being guided into a scanning system, the
excitation beam has to be shaped and collimated by a pair of telescopes to avoid power loss at the scan-
ning mirrors and to fill the back aperture of the focusing objective. Depending on the applications, an
upright microscope or an inverted microscope is connected to the scanning system with an aperture
fitting tube lens. After passing through the tube lens and the optics in the microscope, the scanned laser
beam is focused onto the specimen by a high-NA objective. With the forward-collection geometry, the
generated SHG and THG signals are collected by a condenser and guided into photomultipliers (PMTs)
for detection. To obtain separate SHG (615 nm) and THG (410 nm) images, a dichroic beamsplitter with
a suitable cut-off wavelength of around 500 nm should be used to separate the different signals. Two
individual PMTs are used for detection. To increase the signal-to-noise ratio (SNR), bandpass filters
with different center wavelengths and bandwidths are inserted in front of the PMTs to filter out the
background noise. In addition, a color filter is used to filter out the laser radiation to avoid noise or dam-
age resulting from the relatively strong laser radiation. On the other hand, for the backward-collection
geometry, the excited epi-SHG and epi-THG signals are epi-collected by the same objective. The col-
lected signals are then reflected by a dichroic beamsplitter, which can let 1230 nm laser beams pass
through, reflect collected signals, and then direct them into PMTs for detection. The signal detection
geometry is the same as that used in the forward-collection imaging system. To obtain simultaneous
SHG and THG images, two PMTs are synchronized with the scanning system for intensity mapping.
