Quantifying Angiogenesis in vivo
Figure 2: Comparison of 2D projections of vessels (processed in 3D—see text) imaged from mice anesthetized with either isoflurane (left image) or ketamine (right
image). The apparent larger vessel diameter in the isoflurane image is due to movement during the imaging, which could not be fully compensated with image
processing. Scale bar = 100 µm
detectors are located as close to the objective lens as possible Cybernetics) to reduce noise and to smooth motion effects
and outside the confocal scan head, thereby maximizing the introduced into individual sections by the animal’s breathing
signal at the lowest possible photon dose [4]. A 20X (0.7 NA) and heartbeat. Image stacks collected from ketamine/xylazine-
water objective lens was used to collect all images. For spectral anesthetized mice were deconvolved to further improve the
emission determination, an internal PMT was used. The spectra signal-to-noise ratio and to facilitate tracing of the vasculature
were collected from 450 to 600 nm
with 5 nm resolution. To capture
three-dimensional (3D) information,
Z stacks (1.2 µm/slice) were collected.
Mice anesthetized with ketamine/
xylazine had significantly less
movement and muscle twitching than
did mice anesthetized with isoflurane
(Figure 2). Although it is possible to
reduce the effects of such movement
in the resulting stack projection via
3D digital image processing, less
image processing is always more
desirable. Additionally, the isoflurane
caused a higher background level in
the surrounding interstitial spaces,
especially after the second and third
injections of the dextran. Because the
emission spectrum of the background
structures was very similar to the
spectrum for the vessels, we suspect
that dextran/FITC leaks out of the
vessels over time (Figure 3).
Image processing and analysis.
Figure 3: Spectra emitted from labeled vessels (FITC/dextran) and from the interstitial spaces. Inset photo
Image stacks were processed with a
shows the sites of collection of the spectra. The vessels and the interstitial spaces have similar spectral output
indicating the presences of FITC/dextran in both compartments. Scale bar = 100 µm
3D Gaussian filter (ImagePro, Media
26
www.microscopy-today.com • 2009 September
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