RE: 6 side streams in ARIA?!?

Gene & Vinko,

For what it's worth, I routinely use the 100um nozzle to sort.	Usually
on "medium" at ~30psi, but sometimes on low at ~20psi.	I agree with
Gene that your picture shows fanning, and that you need to optimize both
pressure and drop drive frequency (and I've found that each nozzle has
it's own pressure/frequency sweet spot).  In addition, Gene is correct
in that at low pressures small changes in drop drive frequency do make
big differences.  

However, I disagree with Gene about voltage and 2nd/3rd/4th drop
adjustments.  

I routinely use 5000 volts with a 100um nozzle.  When I sort with a
100um nozzle on low pressure (20psi) with 5000v I find that the outer
side streams are easy to push too hard.  You can't move the slider all
the way out.  If you do, you can actually put the stream onto the face
of the high voltage plate (not a good idea).  However, if you're
judicious in how far you move the slider, you will find that you can get
nice stable streams (as in your picture).  If you drop the voltage to
2500v, you may need to push those sliders all the way out just to get
the streams to hit the far right/left collection tubes.  
 
As for the 2nd/3rd/4th drop adjustments, on low pressure I find that
10/5/0 usually works best for me.  On low pressure sorts, these
adjustments actually can make a BIG difference in center stream fanning.
I've attached a picture of just how big a difference it can make.  Only
difference between these three images is the 2nd/3rd/4th drop adjustment
(all using 100um nozzle @ 20psi & 5000v).

And Vinko, if you think you've got it bad, one of my pictures shows at
least 8 "side streams"!   ACHHH!!

- Greg
 
Greg A. Perry, Ph.D.
Technical Director
Flow Cytometry Core Facility
Creighton University
Omaha, NE  68178
(402) 280-1841

From: Pizzo,Eugene
Sent: Thu 12/6/2007 18:37
To: cyto-inbox
Subject: RE: 6 side streams in ARIA?!?


Hi Vinko,
That's a really cool picture and in some sense it shows the future of
flow cytometry as the engineers learn to add more streams AND more
colors to sorting but what you have is not six streams but four streams
with center stream fanning. It is the result of a drop drive frequency
that is "inadequate" to clarify drop separation. 
On the ARIA large nozzle sizes are somewhat unstable and require a fine
tuning of nozzle position, drop drive frequency, amplitude, and
pressure. Use the built-in "low" setting only to ballpark. Then begin by
adjusting pressure and drop drive frequency to be certain you're getting
good separation of drops and merging satellites.
Because the separation of your streams looks otherwise so good I'd guess
it's just a minor frequency change to tighten your center stream. You're
plate voltage - at 5000 volts - is too high for a large nozzle however,
that could be "stretching" your streams.
I only use such a high voltage for higher pressures/smaller nozzles. I
run the 100um nozzle closer to 2,500 volts. Don't bother with the 2nd,
3rd, and 4th drop adjustment - strangely I've never seen this have any
effect since the old Vantage/SE days - since we've gone digital if you
need to use the drops adjustment it's a good bet you're in the wrong
frequency. Keep it at 20,10,5.
Gene Pizzo, M.S
Manager, Flow Cytometry Facility
UCONN Health
Farmington, Ct. 06030
860 679-7567
http://flowcytometry.uchc.edu <http://flowcytometry.uchc.edu/> 

________________________________

From: Vinko Tosevski [mailto:vinko.tosevski@medri.hr]
Sent: Wed 12/5/2007 17:11
To: cyto-inbox
Subject: 6 side streams in ARIA?!?



Hi,

My department recently bought an Aria and I was among the first ones to
go through operator's training for it. So, although I have previous flow
experience, I am largely a beginner in the sort world.

Today I tried to sort some cells using 100u nozzle and LOW sort mode
(for the first time; most of the time we use 70u nozzle and HIGH sort
mode). The stream looked OK but, when I applied the voltage and used
TEST SORT button, I got 6 side streams instead of 4!??! (I will try to
attach a picture but I've never seen any attachment going through) Is
there a simple explanation to this phenomenon and how can I resolve
this? When I move the sliders used to adjust the position of side
streams, the outer two side streams on each side respond. But, the
center stream splits/gets 2 side streams of its own after applying a
voltage, and they are inresposive to the movement of any sliders. These
two are not too much apart but are clearly visible as separate streams
in camera window...

Any comment is more than welcome.

Sincerely,
Vinko

----
Vinko Tosevski, MMolBiol

Research fellow/Teaching assistant,
Department of physiology and immunology, Rijeka Medical School, Ul. b.
Branchetta 20, HR-51000 Rijeka, Croatia

Tel: +385 51 651 192
Fax: +385 51 675 699






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