I'm relatively new to microscopy imaging analysis so I'm seeking some help! I have z-stack images (.czi files) from zebrafish using a Zeiss LSM 880 confocal microscope at 40x water immersion objective. My advisor has suggested using the ZEN software to do a maximum intensity projection and then using orthogonal view. The images still look "messy" after conducting these steps in the ZEN Blue v3.1 software, so I'm wondering if you have any suggestions or protocols to analyze images. Ultimately, I would like to compare fluorescent intensities, myelin sheaths/olig, and/or internode length across my samples. (also- should I implement a deconvolution step?)
Thank you in advance!
You can always convert the czi z-stack (or any z-stack data) into individual frames and then work with ImageJ File ->Inport->Image sequence to try and get better results. Having the full stack exported into frames gives you the flexibility to include or exclude the frames that might be off-target or contain the artifacts. You can also get orthogonal projection from your stack using ImageJ using Image -> Stacks->Reslise command. It might take some experimenting to figure out what output you exactly need. In terms, if you want to use deconvolution it entirely depends if the phenomena you want to demonstrate are clearly visible without deconvolution you most probably don't need to do it. If not, deconvolution may increase the image sharpness, but if the original sample is messy or with the artifacts, there is no magic software or processing algorithm to make it look good using z-stacking or not.
As suggested by the others, ImageJ/ Fiji is a very nice tool to analyse microscopy images. If you're new to bioimage analysis and would like to get a better understanding, I can really recommend you the following channel:
https://www.youtube.com/channel/UC-hlwQ9Q4GS3rtv2EwSStAQ
Concerning comparing you samples: it it not be the best idea to compare fluorescent intensities! Samples bleach due to storage, antibody performance varies and different laser intensities/ illumination times can bias your results. Try to find a more reliable way, like quantifying cell numbers or marker-positive area.
This paper could be interesting for you: DOI:10.1038/s41598-017-16797-1, however, the there used ImageJ plugin is currently broken. But it can give you some ideas about possible acquisition and evaluation steps and perhaps the issues get fixed soon.
If you are having troubles with certain analysis steps, you can find help here: https://forum.image.sc/
Happy imaging!
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