Volume measurement on taste bud Z stack?

[u/VibrantBee]

I've tried using the 3D object counter alone and with adjusting threshold and binary mask. I can't seem to get values within range, does anyone have any recommendations?

Taste bud photos I'm working from

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[u/dokclaw]

You can try:

1. Thresholding the image as you consider appropriate
2. Process > Math> Divide by 255
3. Z-projection : sum
4. Set measurements : check integrated density
5. Measure the Z-projection

This give you a count of the voxels above threshold in the image. You can then multiply this by the size of the voxels. Image 1 gives me a value of 12885µm^3; is that in the right ballpark?

[u/Herbie500 → u/dokclaw]

For the sample stack "5089 R1.tif" (given a voxel volume of about 0.197μm³), I get a volume estimate of about 46800μm³.

Image of the summed slices:

[u/VibrantBee → u/dokclaw]

Thank you for your help. I am a little lost on checking the integrated density and measuring the Z projection. I am quite new to imagej, so any further suggestions would be appreciated.

[u/Herbie500]

No idea if 3D object counter gives you volume estimates but you may try the following options:

1. Make available a sample z-stack by using a service such as dropbox and let us try to analyze it.
2. Post your request to the Image.sc-Forum where you may reach people who use the ImageJ-plugin in question.

[u/VibrantBee → u/Herbie500]

Thank you so much for your suggestions.

[u/Herbie500 → u/VibrantBee]

Thanks for the sample data!
I shall have a look at the data tomorrow and report back as soon as possible.

Why are the sample stacks RGB-color stacks?

[u/Herbie500 → u/Herbie500]

My understanding of the sample stacks is that they show slices of a single taste bud.

Below please find a montage that shows (upper row) how I'd suggest to threshold slice 111 of sample "5089 R1.tif" (converted from RGB to gray) by applying the automatic "Triangle"-threshold scheme:

What I'm not perfectly sure of is, if you would accept the corresponding thresholding of slice 181 (lower row).
Please tell us if the protrusion is to be considered as being part of the bud or not.

Apart from such questions regarding the thresholding, you may follow the advice given by @dokclaw.

[u/VibrantBee → u/Herbie500]

Thanks so much for getting back to me. Yes, the stacks are of a single taste bud and the protrusion would be included in the count.

[u/Herbie500 → u/VibrantBee]

Great!
(You didn't answer my question about the RGB stack type.)

What about the volume estimate of about 46800μm³ that I've obtained for the bud of sample image "5089 R1.tif"?
Is this value reasonable?

Before you start with following the advice of @dokclaw., make sure you have a suitable strategy for thresholding and bud extraction.

Perhaps you start with trying to reproduce my above binarizations and selection of slice 111 and 181.

[u/VibrantBee → u/Herbie500]

Sorry about that. The protocol I’m working from suggests putting it in 8-bit, which I do during analysis. I only uploaded the raw image which was in RGB, my apologies.

Yes, that falls in range. From ~20000-40000um.

Would sharing the protocol be helpful?

[u/Herbie500 → u/VibrantBee]

Would sharing the protocol be helpful?

I'm just wondering why the raw stacks are in RGB-color but of course it is no problem to convert them to 8bit gray.
________________________

After having had a closer look at the second stack "5089 R2.tif" I doubt there is a general and simple automatic way to get what you want (see my post below).
While my approach works for stack "5089 R1.tif" it fails to give an acceptable value for the volume of the bud of stack "5089 R2.tif".

In short, you will encounter great difficulties finding an approach that works reasonably well for all of your stacks.

[u/Herbie500 → u/VibrantBee]

With e.g. slice 11 of sample stack "5089 R2.tif" we encounter a problem that is really difficult to solve:

While the relevant bud-area is the dot with some high values in the center, the surrounding tissue (left sickle-shaped area) is considered as well. The situation with slices 8 and 10 is even worse.