First post here. I scrolled and searched through the previous posts, but I did not come across a similar topic.
Description of the problem:
I have fungal spores expressing a fluorescent sensor that are germinating on a leaf surface. These are 4 channel, confocal z-stack images, taken at set intervals. The spores are made up of multiple cells, that are separated by cell walls. I am interested in segmenting the cells of the spores and then quantifying the intensity of the fluorescent reporter over the time series.
The 4 channels are blue, green, DIC and a ratio of the blue green channels output through Zen.
What I am struggling with is generating the ROIs for each of the cells.
Here is what I have done thus far:
- split channels --> for quantification I only care about 1 channel, however for figure generation I will need multiple channels overlaid.
- 0.5 Gaussian filter of the ratio channel to smooth the data
- Duplicate filtered image so I can generate a mask to then make ROIs
- convert one of the duplicated channels to binary
- watershed the images to split up any connected spores - this worked amazingly well.
That is where I get stuck.
How do I take the binary image and make masks to quantify the ratio image?
Please let me know if you need any additional information.
Looking forward to your replies!