measurement of axons diameter and area

[Yu, K.L.]

Dear all,

Does anyone of you have experience measuring the diameter and area of myelinated axons of the spinal ventral roots? They were ultrathin sections (1um), stained with Toluidine blue and observed in light microscope. Is there an efficient way to measure them in an automatic scale?  I have problems to adjust the background threshold. I appreciate very much if you could share your protocol or opinions in the processing via ImageJ.

Thanks in advance.

Yours sincerely
Carol Yu

[Martin Wessendorf]

Yu, K.L. wrote:

> Does anyone of you have experience measuring the diameter and area of myelinated axons of the spinal ventral roots? They were ultrathin sections (1um), stained with Toluidine blue and observed in light microscope. Is there an efficient way to measure them in an automatic scale?  I have problems to adjust the background threshold. I appreciate very much if you could share your protocol or opinions in the processing via ImageJ.

I won't address the image-analysis part of your question but you may
save yourself a lot of effort by not trying to measure all the
axons--just a random sample of them.  Check: CV Howard and Reed:
"Unbiased Stereology".  Bottom line: sampling is everything.  You get a
more accurate answer by counting a few (100-200) objects chosen in a
truly random fashion than a lot (thousands) of objects not chosen
randomly.

Good luck!

Martin Wessendorf


--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
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[Dimiter Prodanov]

Hi Carol,

I did mostly automatic measurements of axons. As far as I know
there is no satisfactory automatic routine for identification of the
whole fiber =
myelin sheath + axon. Few days ago someone proposed using snakes,
"active contrours"
so you can try that.
Sampling is indeed very important, but I dont agree that 100-200 fibers
is enough for large nerves.
In my experience per nerve section you should sample 25 - 40 % of the
area.
You can significantly reduce the brightness artifact by taking a mean
filter of say 25
and then subtracting the original image from the filtered one, just make
sure you tick
the 32-bit option of the Image Calculator, and then you can set the
Brightness automatically and
go back to 8-bit image.

best regards

Dimiter Prodanov

[Raad Nashmi]

Dear Carol,

You may want to overlay a grid over your image of axons.  Then you
would analyzed all the axons that touches the lines of the
grid.  This would be a non-biased sampling method of measuring axonal diameter.

You may also want to measure not only axonal diameter excluding
myelin (d) but also the diameter of the axonal fiber including the
myelin (D).  The myelination ratio (MR) also gives important
information (MR=D/d).
The methodology is published in Nashmi and Fehlings 2001 Neuroscience
104:235-251.

Regards,
Raad Nashmi

At 12:47 PM 11/28/2006, you wrote:

>Dear all,
>
>Does anyone of you have experience measuring the diameter and area
>of myelinated axons of the spinal ventral roots? They were ultrathin
>sections (1um), stained with Toluidine blue and observed in light
>microscope. Is there an efficient way to measure them in an
>automatic scale?  I have problems to adjust the background
>threshold. I appreciate very much if you could share your protocol
>or opinions in the processing via ImageJ.
>
>Thanks in advance.
>
>Yours sincerely
>Carol Yu

-------------------------------------------
Raad Nashmi   PhD
Senior Research Fellow - Lester Lab
California Institute of Technology
Division of Biology, M/C156-29
1200 E. California Blvd
Pasadena, CA, 91125
phone [phone]
fax [phone]