Need help with linear density measurement using binary image


#1

Hi,
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.


#2

@Kibrom_Alula

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:

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!

eta :slight_smile:


#3

Thank you so much etarena.

Please see uploaded image.


#4

@Kibrom_Alula

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.

eta :slight_smile:


#5

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.


#6

@Kibrom_Alula

You can read up on the different measurements here.

Hope this helps!

eta :slight_smile: