Which image analysis software to use?

Hi,

I am using Confocal Laser Scanning Microscopy, ( Leica TCS SP8) to do 3D imaging of surfactant distributions on nonwoven polymer fiber. I treat my samples in a way that makes the surfactants fluoresce. It is this fluorescence, (associated with presence of surfactants) which I would like to quantify. My aim is to visualise the surfactants in 3D and also to quantify the amount of surfactants present in my samples for aging studies purposes. Please can any one recommend what is the best image analysis software for me to use? All the image analysis software tools freely available such as IFiji, Icy, CellProfiler, QuPath… have been developed for the analysis of biological samples. My samples are polymeric nonwoven fibers modified with surfactants. I have no programming knowledge and I am not sure how to justify using any of these softwares…Anyone knows of alternative image analysis softwares better suited for me please?

Hi Jona,

An image is an image, whether it’s biological or not is irrelevant. If it’s of good enough quality, the tools available for biologists should work for you. Fiji is a good start: it can open your confocal stacks and can quantify your fluorescence.

If you can share an image, we can even help you :slightly_smiling_face:

Cheers,

Matthieu

Hi MatthieuV,

Thank you for your response. I am happy to share an image but it seems my images(raw ome.tiff) are too big (739.505 KB) for the upload here :roll_eyes:
I am a phd student and will need to justify my choice of the analysis method, its suitability and how it all works and hence I’m seeking advice from experts in the field of imaging. I have used Fiji a little bit but I am not sure how to do 3D image analysis with it effectively and efficiently. I image on 2 different channels and capture fairly big z-stacks e.g. 500um & 5um steps. Any suggestions are greatly appreciated!

Best regards,
Jona

Here is an example of an Image I’ve constructed with Fiji, a z-projection, the red is reflection from fibers and the green is fluorescence which i would like to quantify, in 3D rather than 2D ideally!
15.3.19-CC-A-C2

Dear Jona,

It’s a bit complicated if we can’t access the original file but let’s start.

Can you open your file and do the following:

  1. For 3D visualisation:
  • Go to Image>Stack>3D project. You will get a 3D movie of your sample.
  • If you want to manipulate it in 3D, go to Plugin>3D viewer.
  1. For fluorescence quantification:
  • This really depends on your image -you’re imaging a 500um stack at 5um interval. The distance between slices is thick. What lens have you used? What is the size of your image (in pixels and um)? Have you reached saturation at any point? If you have, quantitation will be compromised.
    First, select your measurements of interest by going to Analyze>Set measurements. You will need Mean, Min&Max, Display Label.
  • Your Z-projection is a good start but which one have you used? A maximum intensity projection only keeps the brightest data and discards everything else while a Sum of slices keeps all the data. With a sum projection, you can measure how much fluorescence you have in your stack.
    If you want to measure overall fluorescence slice by slice, all you need to do is to go to Image>Stacks>Measure Stack… you should get a result table with measurements of fluorescence.

it’s a start

Cheers

Matthieu

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Images up to ~15GB can be shared through GoogleDrive links, and you can make a throw-away free Gmail account for that space as well. FirefoxSend is another option.

Thank you Research_Associate! I will try uploading my raw images again using your suggestions. :+1:

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Dear MatthieuV,

I will try out your suggestions. Also I think I’ve managed to find a solution for sharing my raw data;
Below is a link to a folder which contains 3 images, each is a z-stack, captured in 3 channels. One is a blank i.e. without any fluorescence, or very little background fluorescence, the other sample named 0.56% should show some fluorescence and the 1.25% should show a lot of fluorescence i.e.Here are the dimensions for the images; Dimensions|690x98 Objective used


Images are here…

Thanks a lot!
Jona

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I don’t really do much 3D analysis outside of Imaris, but I’m glad you were able to get the file shared and hopefully someone will be able to help you with your problem.