I’m new here and I need tips for measuring “linear density” by converting my sympathetic nerve fibers immunofluorescent image to binary format (black & white). I was able to convert my image to binary, but I need help with setting the threshold. I mean, how do I pick what minimum (lower limit) and upper limit threshold values are? Do I need to draw a line around my image border in the binary image? Do I need to mask (I don’t know what that means)? And under the “set measurements” there is no choice for density? Do I need background subtraction? Thank you for your help.
Welcome to the Forum!
So … you have a ton of questions and that’s great! Glad you posted on the Forum…
Let’s get you started first with some helpful links that might answer some of those questions:
- ImageJ wiki - the best place to learn everything about ImageJ/Fiji!!
- “Introduction to Fiji” workshop and corresponding slides - worth the time to get a solid intro
- Principles page - collection of principles for the entire image analysis process, from acquisition to processing to analysis
- Segmentation page
- “Segmentation in Fiji” workshop and corresponding slides
- Trainable Weka Segmentation plugin - a great tool for segmentation that comes directly with Fiji. NOTE: Fiji is Just ImageJ - it is simply a distribution of ImageJ that comes with a bunch of plugins bundled - ready for you to use out-of-the-box. If you are just getting started, we recommend downloading/using Fiji.
So… for your particular ‘workflow’ that you need to develop to segment your nerve fibers… it would be best if you can post an original image here on the Forum - then we have a better idea of what is needed to help you.
But some ‘quick’ answers to your questions:
It is best to set thresholds using automatic methods… that gives you reproducibility and keeps things unbiased. In general - stay away from manual settings…
I don’t think you’ll need to draw this border… but again - having the original image posted will help us see why you think you need this.
A mask is essentially a binary image… you set the ‘background’ pixels to ‘0’ and then the object pixels to ‘1’ - this is based on the threshold you set.
You might have to do those calculations yourself… using those measurements that are available in Set Measurements.
Background subtraction - will depend on your original image… we need to see it to advise you on that.
Hope this helps a bit!
You could also look into CurveAlign… it’s not in ImageJ itself - but a good tool especially for what you need it seems… Might be worth a look-see.
Thank you again Eta. I have one question. Under the set measurements, I don’t see Intensity. My results show “Area” with different values for different images. Could this be representing intensity? Thank you.