Large Scale Time Series Microscopy of Neovessel
Growth During Angiogenesis
Urs Utzinger1, Brenda Baggett1, Jeffrey A. Weiss2, James B. Hoying3, Lowell T. Edgar2
1
Department of Biomedical Engineering
University of Arizona
2
Department of Bioengineering, and
Scientific Computing and Imaging Institute
University of Utah
3
Division of Cardiovascular Therapeutics
Cardiovascular Innovation Institute
University of Louisville
ONLINE SUPPLEMENTARY MATERIALS FIGURE AND MOVIE LEGENDS
Supplementary Figure 1: Top) Cross section of the onstage incubation chamber. Tow photon
microscopy at 780 nm excitation produced collagen related SHG and EGFP related fluorescence.
A mixture of 5% CO2 and air flowed into the chamber and was preheated in the water bath.
While the microscope stage moved, the skirt attached to the objective lens slid along the top
stage glass plate maintaining a sealed chamber. Medium was replaced daily using stainless steel
tubes without the need to move the sample dish. Middle) Sample dish with PTFE molds and
stainless steel anchor mesh is illustrated prior to pouring the microvascular cultures. Bottom)
Microvascular cultures and their anchor frames were held in place using stainless steel wires
attached to the side of the petri dish
Supplementary Figure 2: The image processing pipeline of 4D imaging is illustrated. We
transferred individual files from the imaging session to an external hard drive (eHD) and
compressed them into a single archive file. The archive file was created for each day of imaging
data and then transferred to the iRODS storage framework. We assembled the individual files
into a matrix for each measurement channel and time point and stored it as HDF file. We
preprocessed those files and stored them in a tagged image file format for further analysis and
visualization. A custom program allowed for spatial co-registration and region of interest (ROI)
selection. We rendered full fields of view with down sampled data while ROIs were visualized
at full resolution.
Supplementary Figure 3: There was minimal difference between the onstage incubation and
incubation in a regular CO2 incubator as shown in this phase contrast and contrast enhanced
image of two micro vessel cultures.
Supplementary Movie 4: Episodic growth and retraction is illustrated in this movie. We
rendered signal from EGFP in green. The movie visualizes parent vessels (one o’clock position),
sprouts, as well as individual cells. We rendered collagen related SHG in black. The scale bar is
50 microns long. Each frame is 2 hours apart. The movie shows advancement, retraction and
reorientation of a sprout. The sprout tip often bifurcates briefly and the sprout diameter changes
considerably over time. Two parent vessels connect early in the movie as they are located close
to each other. Considerable migration of some single cells can be observed.
Supplementary Movie 5: Time laps evolvement of the vascular network from Fragment 4 in Fig.
4 shows the dynamics of neovessel regression and direction change
Supplementary Movie 6: Collagen interaction with sprout tips is illustrated in this time series
movie of the region of interest depicted in Fig. 6B. We rendered SHG white and EGFP green.
For rendering of the collagen, we identified the depth location of the sprout tip (yellow circle)
and rendered the data over a depth of only 10 microns, therefore following the depth location of
the tip. We reduced the fluorescence intensity from the sprout overlaid it with the SHG image.
The scale bar is 50 microns long. During sprout advancement the matrix is deformed which is
most noticeable between sprout tip and parent vessel at the beginning of the sprouting and also
during inosculation. The sprout tip appears to recruit collagen fibrils and form a mesh around the
older sections of the sprout. Fan like rearrangement of the collagen fibrils in front of the sprout
tip is most noticeable before inosculation. Increased condensation of collagen can be observed
unilaterally at the inner side of the curved neovessel.
Supplementary Figure 7: Several neovessel inosculation patterns were observed. A) Neovessels
approaching each other with subsequent tip to tip inosculation, B) Tip to parent vessel
inosculation shown here with multiple connections between two parent vessels, C) Neovessel
branching and both branches inosculating shown here with the formation of a 5 way intersection
with multiple inosculations, D) Tip to stalk inosculation shown here with the stalk forming a
branch before inosculating. Numbers indicate hours after start of imaging.
Supplementary Movie8: Inosculation of the sprouts depicted in Fig. 6A is illustrated in this time
series movie from a three o’clock view. The largest extent of the field of view is about 480 by
200 microns. The time interval between each frame is 2 hours. We rendered signal from EGFP
green and SHG from collagen black. Two sprout tips grow towards each other over some
considerable distance while there is both advancement and retraction. Inosculation occurs in
“handshake” like manner where the two tips overlap on their side and then merge together.
Significant variations in sprout diameter can be observed over time.
Supplementary Movie 9: A) The full time series data from Fig. 2 and Fig. 8 (left) is illustrated in
this movie. The micro vessel culture is circularly constraint and imaged every 2 hours for 5
days. We down sampled data 5 times before rendering. We show fluorescence from EGFP from
a top down view with vessels further away rendered darker than the ones closer to the observer.
The largest extent of the field of view is 2 by 2 mm. Almost all parent vessels form multiple
sprouts usually originating at the parent vessel ends. Large sprouts form over a distance of
several 100 microns. Growth occurs in an episodic fashion of advancement and retraction
B) The region of interest of the circular constraint culture in A) is depicted in this movie.
Fluorescence signal from EGFP and SHG from collagen is rendered. The image at the bottom
illustrates a minimum projection of the transmitted laser power in black and white and the z
projection of the EGFP fluorescence in blue. One to two sprout form at the ends of the parent
vessels and grow through the field of view. Advancement, retraction and reorientation as well as
inosculation occur over the 5 day period.
Supplementary Movie 10: A) The full time series data of a long axis constraint culture is shown
using same rendering parameters as in Supplementary Movie 9. Significant contraction occurs in
the unconstraint direction. Sprouts form in a similar fashion as in Supplementary Movie 9,
however sprouts are oriented in constraint direction which is caused by the contraction, but also
growth, primarily oriented towards the culture’s anchor.
B) The region of interest of the long axis constraint culture from A) is shown. While parent
vessels are preferentially oriented towards the culture’s anchor already at beginning of the time
laps, the neovessels exclusively orient towards the constraint boundary.