Figure 1.
AngioTool's GUI and analysis flow.
(A) AngioTool GUI for analysis of angiogenesis networks. A detailed quick analysis guide can be accessed through the help button. (B) Representation of the logical analysis flow performed by AngioTool which is based on identification of vessels using multiscale Hessian analysis and skeletonization. The analysis process is mostly automated (yellow boxes) with minimal user intervention required to select the test image and during the visual identification of vessels (green squares).
Figure 2.
Angiogenesis in the embryonic hindbrain.
Hindbrains were dissected, stained and microscopic photographs were taken and assembled as described. (A) Representative E11 hindbrain. (B) Result image after analysis of the hindbrain shown in (A). (C) Enlarged area from skeleton shown in (B). The outline of the vasculature is shown in yellow, the skeleton representation of vasculature in red and branching points are blue. (D) Graphical representation of the analysis performed on three individual hindbrains.
Figure 3.
Post-natal angiogenesis in the murine retina.
Retinas were dissected, stained, flat mounted and microscopic photographs were taken and assembled as described. (A) Representative P6 retina and (B) the resulting image after analysis (C) Graphical representation of the analysis performed on four individual retinas. (D) Enlarged area from (B). Vessels outlines are shown in yellow, the skeleton in green and branching points in blue.
Figure 4.
Analysis of angiogenesis in LY294002 treated allantois explants.
Allantois explants were cultured in the presence of inhibitor or its vehicle, fixed, stained and microscopic photographs taken as described. (A) Representative images of a control (top) and a LY294002-treated (bottom) allantois explant (left), the resulting images after analysis (middle panels) and a representative, enlarged part of the result images shown in the middle panels. The vessel outlines are shown in yellow, the skeletons in red and the branching points in blue (B) Graphical representations of the analysis with AngioTool performed on eight control and eight LY294002-treated explants. Statistical analysis was by T-test (Mann Whitney) ***, p<0.001, **, p<0.01.
Figure 5.
Analysis of angiogenesis in Y-27632 treated allantois explants.
Allantois explants were cultured in the presence of inhibitor or its vehicle, fixed, stained and microscopic photographs taken as described. (A) Representative images of a control (top) and a Y-27632 treated (bottom) allantois explant (left), their skeletonised images (middle panels) and a representative, enlarged part of the skeletonised images shown in the middle panels. The vessels outlines are represented in yellow, the skeleton in red and the branching points in blue. (B) Graphical representations of the analysis with AngioTool performed on seven control and ten Y-27632 treated explants. Statistical analysis was by T-test (Mann Whitney) *, p<0.05; **, p<0.01.
Figure 6.
Reproducibility of image analysis using AngioTool.
The eight images of control DMSO- and LY294002-treated allantois explants (see Fig. 4) were analysed independently by two investigators. The results obtained are plotted. White bars, analysis performed by person 1, grey bars, analysis performed by person 2. Results obtained were subjected to statistical analysis by 2-way ANOVA. This showed highly significant differences for LY294002 treatment under all conditions tested (p<0.001) whilst there were no significant differences between the results obtained by the two investigators (p>0.66).
Figure 7.
Optimising skeletons for a thorough analysis.
The control allantois explants for the experiment shown in Fig. 5 were overlaid according to the pre-set parameters (skeletons not optimised) or after careful optimisation. This lead to significantly different branching indices (B; p<0.001, Mann Whitney T-test). (A) shows an enlarged area of a stained explant; the same area is shown after skeletonisation using the pre-set parameters (C) or after optimisation (C'. A few examples of missed vessels and junctions in the non-optimised skeleton are highlighted by ringed areas and arrowheads.
Table 1.
Summary of angiogenesis related parameters computed by AngioTool.