Janet Parameter height, area, and width can be visualized by looking at the curve (pulse) one sees if looking at (or envision) an oscilloscope tracing of a particle transiting the laser beam. The height is the height of the pulse, the area is the area under the curve and the width is the width of the pulse at some threshold level (for linear signals only). The area always provides the total fluorescence of the cell so for DNA measurement this is what we want. If the laser beam is wider than the cell the height and area are proportional. If the laser beam width is less than the cell diameter (slit scanning) then this is not true and you need to use area to get the total fluorescence. Envision 2 identical cells with the same number of fluorescent molecules attached. In one cell the fluorochrome is concentrated in one small area whereas with the other cell the fluorochrome is evenly distributed over the entire cell. In the first cell (using a narrow laser beam and disregarding any fluorochrome interactions which may quench some fluorescence) the height of the fluorescence signal will be very large but the width will be very narrow. In the second cell the height will be much less but the width will be greater. Using height one would conclude that the first cell has more fluorescence which of course is untrue as we set up the experiment. Using the area both cells will be evaluated as having the same fluorescence. Now back to your situation where the laser beam is almost certainly wider than the cell (nucleus) diameter. Here width for a single cell is actually the width of the laser beam. Envision a single G0 cell transiting the laser compared to a G0 doublet (with the doublet longest axis perpendicular to the laser beam). The height of the doublet (in a FACScan) will be slightly greater than that of a singlet (if the laser beam diameter was the size of a nucleus then the height would be exactly the same since no more than one nucleus worth of PI could be in the laser beam at any one time). Note that these conditions change as the size of the cell (nucleus in the case of DNA) changes since the laser beam diameter is fixed. However the width of the doublet compared to the singlet will be much greater (i.e. up to twice). The area of the G0 doublet will also be twice that of the singlet. Without any doublet detection/correction you will measure this G0 doublet as a single G2/M cell - a significant error. By plotting the width versus the area you can visualize the G0 doublets (and higher order complexes) from the singlet G2/M cells and gate them out. (You can also use height vs area but width vs area on the FACScan/Calibur seems more sensitive). This is hardware doublet discrimination. Software methods (e.g. ModFit) calculate (and then subtract) the number of G0 doublets by counting the triplets, quadruplets etc. However, this often can not be used depending on the cell populations present (for example with endoreduplicating cells). Note that the doublet G0 population width is a smear (on a histogram) rather than a tight population. This is because not all doublets will transit the laser beam with their longest axis perpendicular to the laser. As the angle between the longest axis and the laser beam becomes less than 90 degrees then the width decreases. If the doublet long axis is parallel (or close to parallel) to the laser beam the doublet is not detected. Thus, the fluidics system needs to be operating properly - i.e on the FACScan running on the low fluidic setting (this is critical for the best CV also as well as separating independent doublets (2 cells not connected but in the laser at the same time)). Hope this helps. Larry At 01:41 PM 1/7/2002, you wrote: >I know this is going to sound like a stupid questions but we(me and several >post docs) are currently involved in a heated debate as to the nature and >meaning of FL2-area and FL2-width in connection with the measurement of >DNA content(PI stained) of ethanol fixed cat lymphocytes. Can someone give >us a good definition of each and an explanation of why we use these for DNA >measurement instead of FL2-Height? We are using a BD FACSCalibur for our >work. I have done DNA work for years and I have never really thought about >this before-this is how I was taught to do it. Again I am sure this is >result of never having taken a formal course on flow and just picking it up >from the person who previously held my job and other investigators. > >I have searched the archive and can not find a good explanation. Maybe I >need a course in using the search engine for the archive!!! > > >Thanks in advance. > >Janet Dow > >Janet Dow >Research Technician and Manager >Flow Cytometry Facility >North Carolina State College of Veterinary Medicine >Room C-314 >Raleigh, NC 27606 >(919)513-6364 Larry W. Arnold, Ph.D. Associate Professor Director, Flow Cytometry Facility Department of Microbiology and Immunology Lineberger Comprehensive Cancer Center CB# 7290 University of North Carolina at Chapel Hill Chapel Hill, NC 27599 Phone: 919-966-1530 FAX: 919-962-8103
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