- I'm recently working on "nuclear DNA content ( or genome size, 2C-value )" estimation, staining with PI. Two questions :
- I am looking forward for your answer, thank you all.
A sharper peak indicates less variation, which is represented by a lower CV. A wider peak indicates more variation, resulting in a higher CV.
The width of your gate should match the width of your peak. If you have too wide a gate, outliers will be included and affect your CV. If the gate is to narrow, it will cut off a portion of your peak. While this will likely result in a lower CV, it is not representative of your data, as part is being excluded.
Thank you, Andrew Real. But I checked the picture attached, I found I gotta a mistake. I'm very sorry, the new and correct picture has been uploaded.
In fact, peak a, the sharper peak, have a higher CV (about 12.2%), while the wider peak have a lower CV (about 6.5%). Isn't it strange ?
I could email you the .fcs data if you are interested. That would be really help.
Thank you again. Have a good day !
I fully agree with Eva Temsch's explanation: The peak width is not proportional to the CV, but it depends also on the peak position. Peaks at higher position (e.g.by increasing the PMT voltage) become wider. The reason is that the CV gives the peak width as percentage of the peak position.
On your question how to set the gates: There are programs which do it automatically. But in most case (and it seems, also in your case) you have to set the gates yourself. Of course, there is the danger to set the gates too narrow in order do obtain a good CV. But if you set them to wide you will include a lot of debris (background) which will distort the CV in the opposite dircection.
My recommendation: Look for the points of strongest curvature in the histogram and set the limits of gates there. I have tried to show it in the attached file.
An additional comment to my answer after discuccing the problem with my old colleague Joerg Fuchs: A program which allows automatic gating of histogram peaks is FlowMax from the PARTEC Company (Germany). According to Joerg it gives reasonable results. A problem my be that it doesn't accept your data file format. But in this case you may write a small program for transforming your data file on the base of the FCS format description (see e.g. the attached file)
Dear Xiong Yanshi, I think that your question was very clearly explained by Eva Maria Temsch. Subsequently Armin Meister gave you some practical hints, including the link to a specific software FloMax by Partec that I know very well. As we faced some limitations of this software, we developed our own free cytometry software called simply Cyto_SW that you can download and try (see the link below).
Our software has the automatic peak fit (automatic gating) providing similar results as FloMax. Our software uses the standards coming from the laser field, where also peaks of Gaussian type are often analyzed. This means that we set the gates for the automatic peak fit at the 13.5% intensity levels (see the lower line in the attached picture). You can also set manual gates to see, what is the effect of narrower range on peak's CV. For details see the Quick Start Manual.
Main advantage for you is the fact that this is a free software, ready to use (Windows platform) without any need to write a conversion program. In case you have any difficulties with loading your FCS files, send them by email to: [email protected] and we will update the software.
http://www.wolf-danniel.com/flow-cytometry-software.php
Thank you very much, Eva Maria Temsch, and Andrew Rael, Armin Meister and T. Mates as well. That's very kind of you, and I am really grateful for that.
Have a good day !
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