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We have the lasers configured as you suggest with the UV closest to the
flow cell as this allows adjustment of the second beam without having to
wrap you fingers around the UV beam! Currently we are using a half mirror
for the UV and fire the 457.9 over the top but this is quite tricky to set
up as the separation between the two beams is so small. The UV laser is
set on the optical bench to the lower alignment pinhole settings and the
457.9 is set to the central pinhole settings. We use the UV beam expander
but do not use any beam translators, however all we do is sort chromosomes
so you might need the flexibility of the beam translators in your system.
We have an anti-reflection coated dichroic mirror on order thom Chroma
which will handle the laser powers to replace the half mirror (we found
that the UV beam was degrading the filters supplied by Coulter). We use
300 mW of both UV and 457.9.
We get best resolution using the 40 x 80 Confocal lens PN6857630 with the
high speed quartz nozzle. All signals are collected as peak. To use the
pulse pile up mode in the instrument we have found that it is essential to
use a UV only specific signal to trigger the gated amp and the system
discriminator. Scatter is not very useful as a trigger for chromosome
suspensions but after a lot of testing Coulter have supplied us with a 430
dichroic long pass and a 420 band pass filter set which allows UV excited
Hoechst to be collected on PMT2 without any signal from the 457.9. This is
used to trigger the gated amp which is set up with the gated amp enabled
and lower beam lockout disabled. Hoechst and Chromomycin are collected
using a full mirror and a 490 long pass filter with PMT3. The output
signal is also fed into the PMT4 signal processing board to allow
Chromomycin to be collected in peak mode. The data is first gated on PMT2
upper (Y) ploted against PMT2 lower accepting data hard up against the Y
axis (this excludes events where two chromsomes are simultaneously in both
beams). We also gate on UV forward scatter plotted against PMT2 upper to
exclude some small debris. The flow karyotype is collected by plotting
PMT3 upper against PMT4 lower.
We align the instrument with Coulter DNA check and usually get full width
CVs of well under 2% (normally 1.6%) for both UV and 457.9 excitation.
With this configuration we are able to maintain very good resolution up to
4000 - 5000 events per second on human chromosome preps with sorting
purities as assessed by in situ hybridisation better than 95% and most
usually 98%. The instrument has proved very stable when sorting - so much
so that we have been able to leave it sorting without attention for periods
of over one hour! This has not been my experience with other commercial
sorters.
We use the latest version of WinMDI from Joe Trotter which has 256 x 256
density plot resolution to produce hard copy onto a HP Deskjet 560c. This
produces very nice output.
With best wishes
Nigel Carter
Cordelia Langford
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