Fluorescence image analysis introduction


Update 06/10/2016


I’ve just released QuPath, which is entirely new, open source software for bioimage analysis.

If you are interested, you can find out more and download QuPath at https://qupath.github.io


If you are a biologist working with microscopy images, you might be interested in the following introduction to image analysis:

Analyzing Fluorescence Images with Fiji PDF link (Warning: This is about 40 MB)

The optimistic aim in creating this was to help demystify light microscopy data, by outlining from (more or less) first principles the main ideas, concepts and techniques encountered when working with digital images.  While the main emphasis is on fluorescence microscopy data, much of the content is applicable to all kinds of images.

Various questions, practicals and puzzles are strewn throughout the text, for which the following additional data is needed:

Extra data for practicals (~5 MB)

The text was created while I worked at the Nikon Imaging Center at Heidelberg University, and many of the real example images included are thanks to my marvellous colleagues there.

I’ve put this online in the hope it might be useful to someone.  If you happen to read it and have any comments, corrections or suggestions, please let me know!


If you wish to link to this, please use http://go.qub.ac.uk/imagej-intro – linking directly to the PDF may stop working when it is updated, and doesn’t include the practical data .  Thanks!


Would you be interested in an online course (MOOC) for bioimage analysis based loosely on 'Analyzing fluorescence microscopy images with ImageJ'?

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Would it be worthwhile to make a print copy of the ImageJ handbook available (priced to cover printing costs, in the manner of www.openintro.org)?

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§ 22 Responses to Fluorescence image analysis introduction"

  • Kenneth Cheng says:

    Hi, I’m a graduate student at medical school and I perform ICC or IHC (immunofluorescence) experiments as a regularity so this content you offer indeed interests me. Somehow I could not open the link above, could you generously send a copy to me via e-mail? Thanks for the time:)

    • Peter Bankhead says:

      Hi Kenneth,
      I’m glad you’re interested in the introduction! I’ve just checked the link and it should be ok, but because the file is so large if you simply click on it there may be a long delay during which nothing seems to happen… can you right-click and choose ‘Save target as…’ / ‘Download linked file As…’ and get it that way? Unfortunately the file size exceeds what my email can handle, although if it still doesn’t work let me know and I can try sending another link.

      • Kenneth Cheng says:

        Hi Pete, thank you for your response! However I still could not get to the file. I assume that it may be due to network problem of our college, so I’ll just try again later using another network. Thanks again!

        • Arturo Mancera R says:

          Have you tried programs like EZ downloader? all you need to do is copy and paste de web adress, and no matter how large can be a file. There are some other options like bit torrent.

          • Peter Bankhead says:

            Hi, thanks for the suggestion. I’ll look into that if new problems arise – for now, hopefully one link or the other is working for everyone (if not, just let me know).

  • Mai Haoshan says:

    Hi, I’m a postgraduate working at photodynamic diagnosis. Some people call it fluorescence diagnosis of cancer. Fluorescence image analysis is a big part in my research. So I think your introduction may help me a lot. But I can not open the link. And the right-click way can not work too. Is there any other way to get your introduction?

  • bioyujun says:

    Hi,I’m a graduate student majored in biology,and I am trying to learn fluorescence image analysis now,I think your introduction may help me a lot,however I found I can not open the link,right-click way failed too,so could you send me a copy of this document or give me another link?

  • Peter Bankhead says:

    Hi, sorry about that and thanks for your patience – I’ve emailed each of you an updated link to try. Here it is for anyone else who needs it:

    (Link changed 26-05-2014)

  • Eric Olson says:

    Dear Peter,

    Thank you for this terrific Introduction. You have the gift of clarity!
    I am spreading the word (and the pdf).



    • Peter Bankhead says:

      Thanks Eric, I really appreciate your encouragement – and hope the pdf can somehow be useful!

  • Walid says:

    Dear Peter,

    I would like to thank you so much for your precious efforts , really I was waiting such a brilliant work like this . greetings from UNSW Australia.

    Walid, PhD student

  • Jeol says:

    I’m a graduated student in USTC, and my interesting focuses on the medical engineering, and medical image process. Hopefully have the course of the Image J.

  • Chris says:


    I’m interested in your tutorial, but I’m not able to view it either. I can download it, but the file appears as corrupt. Are you able to send me a copy? Thanks!

    • Hi Chris,
      Thanks for letting me know – not sure what the problem is, but I can’t download it either today. I’ll send you a link in the meantime, which should work until the problem gets fixed.

  • Chase says:

    Hi Peter,

    I was able to successfully download your PDF from the updated link provided above. Thank you for making it an available resource to those of us interested in expanding our understanding of fluorescence imaging and the quantitative principles contained therein.


    Chase, medical student

  • Jenna Meyer says:

    I’m trying to measure the area of innervation in a z-stack image using thresholding using a max project image. is there a way to apply a threshold established using the max project “sum of images” to the z-stack of that composite image. so the threshold established using the max project image of slices 1-32 to each of the 1-32 slices (using the measure stack plugin)? I’m finding the resulting threshold units are different.

    Thank you!

    • Hi Jenna,
      From your description, I think you’re using the ‘Image -> Stacks -> Z project…’ command with projection type ‘Sum Slices’. This will give a sum (not a max) projection. The easiest way to do what you want may be to use ‘Average Intensity’ as the projection type instead.
      If that doesn’t do it, you could ask on the ImageJ mailing list for more help – http://imagej.nih.gov/ij/list.html
      I haven’t used the measure stack plugin myself.
      Best wishes,

  • Wossen says:

    Dear Pete…this is really helpful. thank you so much…from Tasmania

  • Yu Wang says:

    That’s greatly helpful

  • Jeni says:

    Hi Peter, how would you suggest I referenced this document? Has it been published anywhere other than this blog?

    Your guide has massively improved my understanding and thesis image analysis, thank you so much! I’ve emailed it around my research group!

    • Hi Jeni,
      Thanks for your comment – I’m glad it has helped!
      Regarding referencing, the main source is here. The only other place I’ve added the document is on ResearchGate – you might find some links there to how others have cited it.

  • Jenny Sackin says:

    Dear Peter,
    I am just returning to research after a 13 year break and am finding your tutorial invaluable as image analysis has really moved on! Thank you for access to the PDF.


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