---------------------- Forwarded by Ian DIMMICK/Europe on 09/01/2002 17:29
---------------------------
Ian DIMMICK
08/01/2002 20:14
To: cyto-inbox
cc:
Subject: Re: meaning of FL2-area and FL-2 width (Document link: Ian
DIMMICK)
Dear Janet,
(Embedded image moved to file: pic00041.pcx)(Embedded image moved
to file: pic18467.pcx)
>From the above two diagrams you can see the generation of a pulse in
diagram 1 as a cell passes the interrogation point where the laser is
focussed on your cell nuclei, in the second diagram the pulse can be
processed in various ways
The height of the pulse is proportional to the intensity of the
fluorescence signal (in your case PI) and in part due to the shape of the
laser interrogation spot.
The width of the pulse is proportional to the time taken for the cell
nuclei to traverse the interrogation point of the laser (so called time of
flight)
The area of the pulse is calculates from the integral fluorescence (area)
that is cross hatched in diagram 2
The reason for using these two parameters(area v width) is primarily
to discriminate single cells passing through the flow cell from cells that
may have no spacial separation when passing through the flow cell ie
doublets.Doublets will give an increased width measurement (26% increase
for every doubling of volume of a cell based on a spherical arithmetic
model), The area measurement will also increase however when analysed the
histogram of width v Area will enable you to gate out any
doublets(disproportionate width to Area) and therefore avoid mis
interpereting a Go doublet for a G2m event
Hope this helps
Ian
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