>Hallo Flowbies, (I hope that's not a rude word)
>
>2 Questions:FACS Vantage
>
>1. When sorting with dual laser does one calculate the drop delay from the
>2nd laser spot or from the first?. I've been using the 2nd and sorting
>seems to be o.k (haven't checked recovery) but someone else I know
>calculates from the 1st.
>
>2. When doing FRET analysis should the laser with the lower wavelength
>always be in position 1?
>
>Haven't been able to find anything regarding the above anywhere.
>
>
>slainte
>
>Ann
>EMBL
>Heidelberg
Ann,
The drop delay should be calculated from the 1st laser spot.
A simplified explaination of the FACS Vantage sort control would be: (1)
see 1st laser signal, (2) wait for a period of time defined by the drop
delay setting, and (3) send signal to charge the droplet(s) for sorting.
Let me explain why calculating the drop delay from the 2nd laser still
acheives a satisfactory sort. The FACS Vantage normally produces a stream
that flows at 10 m/s. When using a 70 um (here u = mu = micro) nozzle and
10-15 psi sheath pressure, the manual recommends that a drop drive
frequency (ddf) between 22 and 27 kHz should be used. For this example,
let's use a 25 kHz ddf. 10 m/s divided by 25 kHz yields 400 um; this is the
distance occupied by one drop. The distance between the 1st and 2nd laser
should be 175 um, according to the manual, which is less than half a drop.
Now, as long as you deflect more than one drop per sort packet, an error of
175/400 drop is not catasrophic, and you should still enjoy a good
recovery. (Because a drop delay measured from the 2nd laser is shorter, the
cell to be sorted may appear in the drop before the target drop.)
3xxx2xxx1xxx based on 1st laser
N--*--*------------------------------@-----o o o o o o o o
3xxx2xxx1xxx based on 2nd laser
(Use monospace font like Courier or Monaco to properly view above diagram.)
Stream flows from left to right: N=nozzle, -=stream, *=laser spot, @=cell
to sort, o=droplets, Nxxx=drop N of a 3-drop sort packet. According to this
diagram, when the drop delay is based on the 1st laser, the target cell
always falls in the middle drop of a 3-drop packet. When based on the 2nd
laser, the target cell falls in either the leading or middle drop.
Eric
/\/\/\_ Eric Van Buren, aa9080@wayne.edu
\ \ \ Karmanos Cancer Institute and Immunology & Microbiology
\_^_/ Wayne State University, Detroit, Michigan, USA
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:53:42 EST