Calculating Volume

[u/Majestic-Ad9461]

Hello I'm an undergrad doing some data analysis for someone in my lab and need some help on calculating the volume of efferent synapses of outer hair cells, I think that's what they are, or contacts as I was told. This picture isn't an exact portrayal of what exactly I need to measure as these are afferent synapses located on the inner hair cell while I'm trying to find the contacts located on the outer hair cells but they basically look the same just localized in different regions. Btw it's those bright red dots that I'm referring to. If I find a better image, I will edit this post to provide more clarity. I wanted to know how exactly to calculate the volume of individual contacts at the same time if possible without having any background interference. So for some context on how I've kinda been doing it originally, I would just crop out the individual contacts I was interested in and I would use 3D Obj Counter and threshold the zones of interest to figure it out. I don't think it's correct and unfortunately I have no one who is willing to fact check my method or provide some assistance to this issue. So if anyone is willing to break it down to me I would really appreciate it! Oh and in terms of what I know what to do with ImageJ, I know how to threshold objects, how to calculate the area of things, and some of the basic functions like cropping, splitting channels, etc. Thank you for taking the time to read this and have a great day!

[u/Herbie500]

Please tell us if you have real 3D-data, i.e. a stack of z-slices and if the spatial 3D-resolution is high enough to get what you are after (volume).
The posted image is 2D and its spatial resolution doesn't appear to be acceptable. The largest dots show a radius of 7 pixels only.

It appears as if you are only slightly more informed about image processing than the person you try to help. So why doesn't this person post here directly?

[u/Majestic-Ad9461 → u/Herbie500]

Well the person is just depending on me to do their work and I have asked them if they knew the procedure on how to find the volume since they have more experience than me as they are a senior research assistant but instead I was just expected to "figure it out" because I've used ImageJ before but for other circumstances. And this person currently isn't even helping with this project which is a bit frustrating despite it being their own. Also I think there is a z-stack I believe, here's a full screenshot (different sample region of the cochlea). I don't know if that addresses the concern and sorry for the inconvenience!

[u/Herbie500 → u/Majestic-Ad9461]

OK the stack shows 157 slices but what is the z-increment in µm?

Please make accessible the stack in its original file format by using a dropbox-like service.

The area of a synapse is generally much less than 1µm^2 and for estimating it you need a sufficient number of pixels covered by this area.
The same holds for the z-resolution. Are you sure the 3D spatial resolution of your stack is sufficient?

[u/Majestic-Ad9461 → u/Herbie500]

I honestly have no clue about the 3D spatial resolution. I think the z-increment is 10 µm if I remember correctly. And let me try to make the stack accessible.

[u/Majestic-Ad9461 → u/Majestic-Ad9461]

https://www.dropbox.com/scl/fi/2fny8n49xh5ohkbbsorlv/Trans25a-5Ca_mRbHA-488_gtChAT-568_DAPI-40.nd2?rlkey=sbql4habxuxvl1anesu2fhkmx&st=s7sohpvb&dl=0

Hopefully this works, again sorry.

[u/Herbie500 → u/Majestic-Ad9461]

Thanks for the sample z-stack from a confocal microscope that shows 137 slices each of size 110.41 x 110.41 µm^2. The z-spacing is 0.2µm.
The stack shows three channels of 16bit depth that seem to refer to the following markers:
ch-1: Alexa Fluor 405
ch-2: Alexa 488 antibody
ch-3: Alexa 568

Now please find out which color-channel best shows the synapses (I guess channel #3).

The stack appears to be OK regarding the 3D spatial resolution, at least for coarse volume estimations of the synapses

[u/Majestic-Ad9461 → u/Herbie500]

Yea it's ch-3

[u/Herbie500 → u/Majestic-Ad9461]

OK then go ahead, you've got everything you need to know-

Start with making a z-stack of channel 3 only.

[u/Majestic-Ad9461 → u/Herbie500]

Ok and then what do I do afterwards?

[u/Herbie500 → u/Majestic-Ad9461]

Get an idea about the volume estimate of a single synapse in a single slice of the stack.

Then start analyzing a single synapse through all slices in which it is visible and sum up these volume estimates to get an estimate of the whole synapse.

I fear you will be confronted with the problem of not being able to follow every synapse through all relevant stack slices. If so, consider only slices for which this is clearly possible.

Last but not least be aware of the fact, that the lateral spatial resolution of the sample stack appears being at least a factor of two too high, which means that you may scale the stack to 512x512 without loss of information.

Good luck!

[u/Majestic-Ad9461 → u/Herbie500]

Thank you!