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Author Topic: Analysis? (Help!)  (Read 8088 times)

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Offline formalanne

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Analysis? (Help!)
« on: January 23, 2007, 05:44:49 PM »
Does anyone have any information/ advice about morphometric analysis of neurons stained with cresyl violet?
« Last Edit: January 24, 2007, 10:14:26 PM by formalanne »

Analysis? (Help!)
« on: January 23, 2007, 05:44:49 PM »

Offline richard03

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Re: Analysis? (Help!)
« Reply #1 on: January 24, 2007, 11:11:55 PM »
After staining with cresyl violet, you can use an image analysis software to do morphometric analysis (neuron size, density, total number, etc).

Use this page to find a suitable software.

http://www.ihcworld.com/imageanalysis.htm

Offline formalanne

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Re: Analysis? (Help!)
« Reply #2 on: February 08, 2007, 06:33:26 PM »
thank you! but unfortunately the software is not available to us, so we have to do it manually, do you know anything about how to count effectively without the software?

Offline ImmunoNYC

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Re: Analysis? (Help!)
« Reply #3 on: February 09, 2007, 12:39:18 AM »
You can take photos and print them and just count by marking with "x" the ones you have already counted. But that, in my opinion, is archaic and painfully time consuming. So why not download free software:

http://rsb.info.nih.gov/ij/
http://ddsdx.uthscsa.edu/dig/itdesc.html

You can also use programs such as Adobe Photoshop to quantitate purple pixels and use this as an "index" of positive cells.

Offline formalanne

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Re: Analysis? (Help!)
« Reply #4 on: February 09, 2007, 01:35:08 AM »
well, I certainly feel like I have crawled out of the dark ages...thank you so much

Offline hokie

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Re: Analysis? (Help!)
« Reply #5 on: February 21, 2007, 08:35:56 AM »
There is a serious problem here --

This does NOT obtain the correct answer.

The problem is that the things you see in a section are profiles, not cells. What you want to do is to count cells and counting profiles from a single image cannot work. That is a well known problem. Solutions have been posted since at least the 1890s. A solution that works and can be implemented in the lab was published in 1984 under the pseudonym DC Sterio.

Look at any of a number of books before the 1984 solution is published to see good discussions about the problem. Books by DeHoff, Elias, and Weibel all go into lengthy discussions about the problems of counting cells. Post 1984 books all push proper counting methods.

The use of image analysis packages to count pixels or to count segmented profiles speeds up the process of getting the wrong answer faster. The number of pixels counted in a section is an indicator of the total volume of the cells, not the number of cells. The mean area of a profile is not related to the mean volume of a cell.
Do more, less well. ~Ewald Weibel

Offline ImmunoNYC

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Re: Analysis? (Help!)
« Reply #6 on: February 22, 2007, 12:26:25 AM »
Interesting points, however if she counts absolute number of events purely without counting pixels or area ina  2D image then doesn't the disector principle not apply here? Also, if she is counting cultured cells in a monolayer then the disector principle also does not apply.

Offline hokie

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Re: Analysis? (Help!)
« Reply #7 on: February 22, 2007, 07:12:11 AM »
This is not a question of whether or not the disector principle applies. It is a question of whether or not the disector principle should be applied. Sorry for my silly semantics here.

The question really is about "counts absolute number of events purely without counting pixels or area in a  2D image". If the events can only occur in a single section, then sure a section is sufficient. Counting profiles is counting an event. Unfortunately cells may not appear in a single section. The event in this case is a particular cell appearing in a section. Counting nucleoli is counting an event. The chance of a nucleoli appearing in multiple sections is lower since this object is smaller than th cell. This works as long as each cell has a single nucleoli. The bias is reduced, but not eliminated. The event being counted has to be 0-dimensional.

Remember that cameras are sampling devices. The smallest object that is visible is a pixel in size. The best that can be done with an image is to count pixels. Objects that are larger than pixels run into the problem that they need to be counted in an unbiased manner. That requires a counting frame.

Image analysis packages often use a technique in which objects on the edge of the screen are counted if they touch 2 of the edges and not counted if they touch the other 2 edges. This sounds reasonable, but leads to a bias.

If "counting cultured cells in a monolayer" then sure the disector principle canbe avoided. Why? Because nothing is sectioned and objects being couonted are not being split. But, a counting frame is required. The only way around this is to count everything. Counting everything is the same as placing a very large counting frame around the entire culture.

I happened to read an interesting article last night in Brain Research about alcoholics and neuron loss. According to the article the claim that neurons are lost is a mistake due to counting in single sections. Proper stereological counting showed that there is no loss of neurons. The changes seen included changes in compartment volume and the volume of neurons. These factors do not affect correct counting. These factors do affect counting profiles.

Do more, less well. ~Ewald Weibel

Offline ImmunoNYC

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Re: Analysis? (Help!)
« Reply #8 on: February 28, 2007, 12:31:55 AM »
Same thread with different answers here. I am closing this thread for simplicity and keeping this one open:

http://www.ihcworld.com/smf/index.php/topic,1910.0.html

Re: Analysis? (Help!)
« Reply #8 on: February 28, 2007, 12:31:55 AM »