Figure 1.
The extent of apoptosis in the Drosophila embryonic VNC.
(A) A Drosophila embryonic VNC. The Region Of Interest (ROI) box in red indicates the areas analysed in (B,C). (B) 3D rendering of confocal stacks of sections through the VNC to show the abundant number of embryonic apoptotic cells stained with anti-cleaved-Caspase-3. (C) Cross section view of the image in (B).
Figure 2.
How DeadEasy software works.
Figure 3.
DeadEasy Caspase: the mathematical algorithm.
DeadEasy Caspase detects apoptotic cells in embryos stained with anti-cleaved-Caspase-3, and are characterised by low cytoplasmic signal, high background and volume ≥1.56 µm3. (A) Diagram showing the region of the embryonic VNC (blue) scanned for counting. (B) Caspase histogram. (C) Enlarged images to compare a raw image vs. the result (single confocal 0.25 µm slices shown). (D) Diagram of the algorithm. (E) Images showing the different steps of processing, correlating with each step in the diagram in (D). (F) Examples of bad quality stainings or images that must not be used for cell counting as they will produce false positives.
Figure 4.
Qualities of Caspase stained cells identified and counted by DeadEasy.
(A) Images showing a 3D rendering of the whole stack stained with Caspase, a single 0.25 µm section from this stack and the same section after object recognition by DeadEasy. (B) Enlarged image to show what Caspase stained cells look like, revealing the characteristic amorphous shape, low signal intensity and high background resulting from these antibodies. The accurate cellular parameters are given in Table 1. (C) User-friendly pop-window prompted by the programme to enable the user to modify the parameters easily, following the guidelines indicated in Table 3.
Table 1.
Properties of cells counted by DeadEasy Caspase.
Figure 5.
Validation of DeadEasy Caspase.
(A) During processing, DeadEasy creates a second stack of confocal images reproducing the identified objects in locations that correspond to those of cells in the original raw stack. By placing the mouse over each of the objects, an identifying number is revealed (as shown in Figure 2), showing whether a cell is counted appropriately. We compared one cell at a time in this way through multiple stacks. (B) Using a second validation method, a merged stack can be created from the raw images (green) and the processed images with the identified objects (red). Colocalising cells in the merged stack (yellow) indicate the identified cells, green cells are false negatives and red cells are false positives.
Table 2.
Validation of DeadEasy Caspase programme.
Table 3.
Parameters that can be altered by the user and the consequences.
Figure 6.
Examples of application of DeadEasy Caspase to address biological questions.
DeadEasy Caspase is used to count the number of apoptotic cells stained with Caspase in wild-type embryos at different stages and in H99 mutants lacking apoptosis. Numbers over box-plots indicate number of embryos analysed per genotype.