btilton@caregroup.harvard.edu wrote:
> Is there anyone who has upgraded their FACStar Plus with the turbo sort
> option and is happy with it? My FACStar Plus was upgraded with the TSO
> last year. The most noticeable improvement was fewer clogs with the
> macro sort head. Hi-speed is a different story though.
> I am unimpressed with the TSO when it comes to running hi-speed.
>
> my first problem is with severe noise in the Side scatter PMT. I can
> minimize this noise with the SSC obscuration bar, but It is not a
> complete fix and I take a big hit in the signal itself.
>
> second, making decent side streams is a challenge. Even if I can make
> good side streams, often the distance to the media in the tube is great,
> and I suspect a substantial number of cells end up drying and dying on
> the side of the tube.
>
> Between these two problems, I seldom use the TSO. Unfortunately, I have
> been getting a lot of sorting requests that require sorting at Hi speeds.
>
> My request is two-fold.
>
> 1) any suggestions for the problems stated above.
>
> 2) I would like to get a general idea of how many users are satisfied
> with the performance of there Turbo Sort Option installed on the FACStar Plus.
>
> Brian Tilton
> Flow Cytometry
> Beth Israel Deaconess
> Medical Center
> Boston, MA 02215
>
> tel# 617-667-4901
Brian,
We have a TSO option on our FACStar Plus, and 2 Vantage SEs with it as well.
It sounds like
your TSO issues are mostly with SSC noise and clean droplet formation. Ours works
as expected,
but it is important to not over drive the sort head. SSC noise creeps in whenever
the drops
break off too short for any given jet setup. It is important to keep the drop
wavelength at ~ 4.5
stream diameters, and the energy to the head reasonable. For example, at 33PSI we
usually
have the system at ~66,000Hz and a breakoff at 33 - 35 drops. The pulse shape
controls for
the drop deflection pulse usually allow for a nice side stream tuning during
setup. So, we're
pretty happy with it - mostly because it gives 1/10 drop clock resolution at any
speed and is a
siginificant improvement over the standard FACStar Plus configuration. We use
ours for
a wide range of applications at several general speed ranges, it is a work horse
in our facility.
The FACStar Plus with TSO will never be as fast as the Vantage SE (with
digital
circuits). The Plus/TSO sorting efficiency at higher speeds in dual laser mode is
less than that
of the Vantage SE. Still, it sorts very well for us. We rarely push it beyond 1
cell every
6 drops with any nozzle. This gives us about 10,000/sec at 66,000Hz, and
8,000/sec at 40,000Hz
as "speed limits" we seldom break. When losses are an issue, we try not to exceed
1 cell evey
8 drops. We have not had to use a drop deflection envelope > 1.5 drops in any
configuration with
any of the nozzles (60um -> 100um).
It sounds to me like you're applying too much energy to the Macro Sort head.
If your drop
breakoffs are < 18 drops or so at 10 - 12 PSI you might reduce the drive level to
get the breakoff
at 16 to 20 drops. At > 20PSI, I would expect the breakoff to be 20 - 30 drops,
and 30 - 40 drops
at > 30PSI. Use the drop drive attenuation switch to your advantage - attenutate
unless you
must have it off to get reasonable drops. This will help performance. Also,
double check the
tightness of the head to its mount. Any loosness there will also cause these
problems. Check the
pressures and ddf combinations you're using to be sure the distance between drops
is
about 4.5 stream diameters. This is fairly critical to maintaining good TSO
stream stability.
Try using drop deflection pulse amplitudes near maximal, and only enough plate
voltage to get the
side streams to clear the waste by 3/16 inch or so. We have found that having the
catch tubes lower
is not a problem. It is noiser to have a short distance and a large charge field
between the plates
to force the side streams where you want them to go. We find that we need the
ddf attenuation off
only at > 30 PSI, and we then keep the breakoffs > 30 drops. "Be gentle" is the
rule of thumb.
Be sure to clean with dH20 to insure there is no salt anywhere around the
area - salt tracking
is always a potential problem, especially at higher speeds/voltages. We usually
tune
the nozzle to laser intersect distance to minimize SSC noise. You may have the
laser placed
where the early drop curvature makes noise worse than it need be. Sometimes, a
fraction of a
drop up or down removes a good deal of the SSC noise.
Hope this helps - good luck,
Joe
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