During cell cycle analysis assay by Flow Cytometry, user must choose log or linear format for FSC, SSC, and PI. The relationship is not as usual (such as power function). I want to know what is the formula which determine the relationship between log and linear in this case.
The linear scale merely means the absolute number unit of fluorescence intensity generated from the flow cytometer. It is like counting how bright a signal each detector detects. Thus the scale merely goes like this 1, 2, 3, 4, ... and since the numbers goes to 20 000 or more it is displayed probably like 0, 1000, 2000, ... but it is still linear as in the number runs like 1,2,3,4 within 0-1000, 1000-2000 etc.
The log scale means that a logarithm function has been applied to the x axis and thus the data is expressed in a logarithmic scale and it is usually in base 10 like this 10^1, 10^2, 10^3, 10^4, 10^5. This way, if the data is a signal that varies over a huge number of x values the scale will spread them out nicely for you to visualise it.
For FSC ans SSC, you must use linear scale as the size of a cell is linear from x micron to y micron depending on what cell you are using and reflected in the FSC (between 200 to 400 for example) and SSC (affected mainly by granularity). In cell cycle analysis you will actually see 2 peaks in the FSC probably as the cell size would nearly double and you will see a population in the location equivalent to 2x micron to 2y micron on the FSC linear scale (between 400 to 700).
For PI, the dead cell can have 100 times the brightness as live cells thus a linear scale is not good for visualisation and thus you should use log scale.
Dear Vincent
Thanks for your answer. I used linear format for FSC and SSC and log for PI. But the values regarding to PI-A and PI-W are meaningless. For instance the range of values for PI-A is (-56.80 ,3875.18).
in attachment you can see the histogram corresponding to PI-A. What do you think?
The linear mode is not suitable for depicting data that spreads
over a wide range, as is often the case with the fluorescence signals associated with cell surface markers. On the other hand the resolution in the linear mode is better over a single order of magnitude (here the 1,000
to 10,000 range). Generally, the linear scale is only used to depict Forward
Scatter, Side Scatter and DNA content
(Propidium Iodide staining), while the log
scale is used for most other applications (including all fluorescent markers).
Best,
Hamadi
Thank you very much Hamadi,
But the main question is that:
Is there any mathematical relationship between Linear and Log format. Meaning that, if one have data regarding Log scale, is it possible to have data in linear format?
Bests
Amin
Yes, you can. The flow acquisition/analysis softwares allow you to switch from log to linear scale or vice versa.
For cell cycle analysis, linear scale for the PI channel. DNA content of live cells: 2X~4X
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