This protocol automatically quantifies the fluorescence in preimplantation mouse embryos.
Tutorial to use the plugin :
- 1 : In Extract channel box, select the channel of interest which will be used to define 3D ROIs.
- 2 : In Wavelet Spot Detector Block, Scales 3 to 5 are able to detect 3D ROIs depending on the number of z-stacks, image quality and embryo stages. Select one scale and modulate the number in the corresponding window. Repeat this point until you obtain relevant ROIs.
You can combine two scales at the same time, ie scale 3 and scale 5 to define the best 3D ROIs.
- 3 : Click on "RUN" on Icy software. In "Workbook to file" box, all ROIs statistics will be added. ROIs are exported in a CSV file.
In order to facilitate your analysis, you can delete all non significant ROIs and add manually new ones using Icy facilities.
-> On Icy, select "ROI" tabs (right part of Icy) and delete all non-interesting ROIs.
-> To add ROIs manually, select "Region of Interest" tab ( top part of Icy) and use 2D ROis and merge several 2D ROIs into one 3D ROI corresponding to one nucleus.
When you have done, go back to "ROI" tab of Icy and export your data in a CSV file.