This is my first post on here, I am hoping to get some comments on the technique I am using in Fiji / ImageJ so far. I have stacks of fluorescent confocal microscopy images. Each stack is a single time point and contains several slices.
So far I have done the following:
- Generate PSF using PSF generator (Richard & Wolf method)
- Deconvolute in Deconvlution lab (Richardson-Lucy algorithm)
- Aligment/Registration using StackReg (Affine transformation)
- Hysteresis connection thresholding
- Particle analyzer… to detect spots/specks/points
So here are a few questions:
-Where do smoothing and denoising fit in and how do I obtain parameters to quantify them.
-How do I quantify Hysteresis thresholding? (Otsu, Renyi Entropy, adaptive hysteresis…?)
-How do I align two stacks from different time points if they are also miss-aligned on the z plane by a slice or so
I don’t have a dark image that I can use to calculate SNR.
If you have any remarks about the techniques I am using, please let me know as it would be very helpful to me.
Sample screenshot from stacks: i.imgur.com/men7R8N.png