Quantifying Angiogenesis in vivo
(Figure 4) (Autoquant, Media
Cybernetics). Vessel segments
were traced on composite
images between branch points
with ImageJ using the neuron
J plugin [5]. For quantification
of measurements, the total
number of branch points (as
a surrogate for total individual
segments), total vessel length,
and average vessel length were
determined.
Results
Using multi-photon intra-
vital imaging, we demonstrate
that it is possible to repeatedly
image, via a Z-series, the
same area over a period of
days or even weeks. Our
data demonstrate that
significant differences in
branching and total vessel
growth can be obtained from
Figure 4: (A) Representative composite image of vessels at Day 1, Day 6, and Day 9 visualized with FITC Dextran. Tracings
minimal numbers of research
of vessels performed in Neuron J. (C) Graph showing average vessel branching and total length on days 1 through 9. Scale
animals. Maximal increases bar = 100 µm
in total vessel length were
observed on day 6 with regression of vessel growth by day 9. observational bias, and expansion of our animal model to
The multi-photon excitation, among all imaging modalities, include the evaluation of more complex aspects of angiogenesis,
produces the lowest photon dose in the sample. This dose namely the incorporation of circulating endothelial progenitor
minimization enables us to follow angiogenesis in the same cells into neovessels.
animal over several days, greatly minimizing sample-to-sample
variation. Additionally, because we use a bolus of Matrigel
Acknowledgments
that is lower than the conventional Matrigel plug assays [6] we
This work was supported by the Wadsworth Center Advanced
can make efficient use of novel angiogenic compounds when
Light Microscopy & Image Analysis Core Facility and a grant
reagent quantities are limiting.
from the NIAMS grant # R01AR054828 and from NCRR grant
Analysis of the same region in a given animal over several
# 1S10RR023451.
time points resulted in the appearance of small deposits of
FITC-labeled material outside of the vessels. This may be a
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