[Cytometry] Problems with low pressure sorting on Aria I

Vinko Tosevski vinko.tosevski at gmail.com
Mon Jul 27 05:36:33 EDT 2009


Dear all,

Thank you very much for your numerous suggestions and comments... In
my case, I found a solution in briefly sonicating the nozzle (1min),
rinsing it with DI water, drying it with compressed air and fitting a
new O-ring (just in case :) The stream looked very nice afterward,
with "Attenuation OFF", default sheath pressure (30 PSI) and custom
frequency needed to have a nice satellite merge. I am definitely
thinking of doing the same washing procedure after each usage and
storing it dry, cause 100u is seldom used...
Also, I found that just a tiny amount of air in the bubble filter can
make a perfect stream to start behaving erratically.

Anyway, thank you once more for swift and invaluable help...

Regards,
Vinko

P.S. Below are some of the answers I've received that might be helpful.

Original question:
Dear all,

I would like to ask for your suggestions on problems that I'm having
lately with my 100u nozzle/low pressure sorting (Aria I). I browsed
the PUCL archives and I saw a good tread on that subject from 2005. I
was aware of most of the suggestions mentioned there, but still, I
can't make my 100u nozzle to work lately.

The problem I'm seeing is, at least for the most of the time, stream
without any drop formation - as if you had high pressure stream and
then turned on the attenuation. Sometimes, turning the attenuation off
(it's ON by default) at low pressure actually restores the normal
appearance of the stream, but sometimes even that leaves the stream
without any drops, just uniform flow of sheath... I tried moving a
nozzle a bit, finding the "better" position, and noticed that I get
some elements of the normal stream when I press the nozzle downward a
little bit (I guess the point is in pressing the o-ring on the other
side of the nozzle upward). The o-ring was new, by the way...

Can someone tell me what could I try? Does having a nozzle being able
to move like a little lever means that my "nozzle bay" is somewhat
damaged? High pressure setup works like a charm, without any
problems... What do others do when they are having problems with low
pressure sorting?

Any suggestions or comments would be greatly appreciated!

Thanks

Vinko

---
Vinko Tosevski
Zurich University Hospital
Department of Pathology
Institute of Experimental Immunology
Building 44, Room J42
Winterthurerstrasse 190
CH-8057 Zurich
Tel: +41 44 6353706


Answers (some):

Hi Vinko

Have you tried adjusting the canula (the screw on top of the nozzle)?
I would try adjusting in 1/8th of a turn increments in either
direction until you do see decent drop formation.  I have had to
adjust this when getting a new nozzle...  But at one point you had
decent drop formation with the same 100 um nozzle?
-------------------
Have you tried changing the frequency?  It was once explained to me that
although there are default values set for each sort set up, each
instrument is a bit different.  I find that when using lower pressure
sorts, I have to adjust the frequency a bit to get decent looking drops.
They're still not as pretty as the high pressure set up, but it does
make a difference.
---------------------
Hi Vinko,

First questions - how old is your nozzle, and do you sonicate it on a
regular basis (as in, after every time you use it).  We had an Aria I
set up - which was upgraded to Aria II - until September 2008.  For
the nozzles, I would sonicate these (and the new ones, too) for 10
minutes in 20% contrad, and store them dry (you could also store them
in water or contrad).  Age can also be a factor - if you are using the
100um nozzle on a regular basis, your nozzle may just be wearing out.
I'd suggest checking the nozzle under the microscope - if you don't
sonicate regularly, you may find that the nozzle is "gunked up" (for
lack of a better word).
 Some other suggestions - check the cam (round bit with the arrow on
it) and see if moving this changes anything.  Check the sheath
pressure to make sure it's actually 20psi.  If I remember correctly
with the 100um, we worked with the attenuation off when our Aria was a
"I" configuration, but each Aria is a little different.

 It might just be that the nozzle is going bad.  Nozzle life various
nozzle to nozzle as well.
-------------------------------
Hi-

How old is your 100um nozzle?!  How about trying to decrease the
Frequency value and adjust the amplitude to get a good drop off?!  The
o-ring actually seals off the gauge so that sheath fluid or cell
solution will not leak from within.  Every Aria systems differ and
some may have to adjust the physical nozzle (like you've done) to get
a drop-off whereas some may not need to do so to attain such task.  I
personally never have such problem like you've encountered after the
mentioned resolutions.  If adjusting the Frequency value doesn't help,
a new 100um nozzle will be needed.
----------------------------
Vinko,

In my experience, different nozzles behave differently, as do different
flow cells.   Whenever I get a new flow cell, I have to find the sweet
spot again.  I do this by varying the sheath pressure, frequency and
amplitude until I find a spot that I consider perfect.  And I have to do
this for both my 70um and my 100um nozzles.

My experience has been that USUALLY things don't change a whole lot.
However, once in a while they change significantly.  I've probably gone
through 10 flow cells on our instrument over the past 5 years, and I
think twice I've had to make significant changes to the sheath
pressure/frequency/amplitude combinations to get good stable droplet
breakoffs again.  (For me on the 100um nozzle I want merging satellites
within 2 drops, and stable side streams for at least an hour.) I'm
usually in the 28-30psi/40Hz/30 range, but a couple of times I've had to
make SIGNIFICANT changes to this to get (and keep) things stable.
O-Rings can be a cause (I've seen this), but it's usually short term and
changes if I change the o-ring.  Flow cell variations are long term and
more nozzle independent.

I would muck around with the pressure/frequency/amplitude settings until
you find a sweet spot.  You may need to change your settings
significantly to get to a good spot.
----------------------------
Hi Vinko,

We use low pressure and the 100um nozzle in our Aria quite routinely.

The frequency we use is usually around 29-30 and the amplitude around
8-10 (quite low).

If the droplets do not form, I have found it is usually something very
simple like an air bubble in the flow cell. A clean of the flow cell
with 70% ethanol usually solves the problem.
---------------------------
Hi Vinko,
We sort 80% of the time with either our 100.  The 100 is at 20psi with
a Freq of 25-28 and amplitude from 2-10 (att on).  The sort head of
the Aria I does indeed always have some "wiggle" room and I find that
pushing all the way to the left seems the best spot in my instrument.
The Aria II has a new sort head and nozzle configuration that removes
the wiggle and is very much improved over the original (we upgraded
one of ours to the "II").  It is possible that you need to try and
make a small adjustment of the cam screw on the nozzle.  That is a
trial and error procedure...have you ever done it?  If not, just give
it a minute turn in each direction and see if it helps.  That's really
all I can suggest...other than the obvious...sonicating the nozzle!
------------------------------
Vinko,

To my opinion you have to adjust your nozzle, but you might try the
following first:

Increase your amplitude
Try to find the optimal frequency.
When this doesn't help:
Switch of the stream, take out the nozzle and check whether it is
moistened. If so, it is leaking and the rubber O-ring is not properly
fitted. You can loose energy generated by the transducer through a
leaking nozzle. Dry it, fit the O-ring and replace the nozzle.
Retry adjusting the amplitude and/or frequency.

If this doesn't help and when you have to move your nozzle while in
position to generate droplets it is likely out of proper alignment.

Turn of the stream.
take out the nozzle
Take a small screw driver and turn the screw on top of the nozzle.
First try 180 degrees. When you have to pull out the nozzle to
generate droplets, turn the screw clockwise. When you have to push the
nozzle inwards to generate droplets, turn the screw anti-clockwise
Refit the nozzle and turn on the stream.
Try different amplitudes and or frequencies.
Repeat this procedure using small increments/ decrements with the
screw driver to close in upon droplet formation with optimal amplitude
and frequency.

This should work. (see also the FACSAria manual page 198).

We even forced our 100 µm nozzle to work at 40 psi, without
satellites. Stable and no problems.
------------------------
Vinko,

Have you looked at your 100um nozzle under a microscope? Do you have a
different 100um nozzle to try? It sounds like this one may not be
perfect.

If your 70um fits, I’d guess that the flow cell is fine. I had a flow
cell that needed to be replaced, but it made an ugly stream with any
nozzle/pressure.

Also, are you adjusting the drop drive frequency to find a spot with a
stable, fairly high breakoff? It can take some messing with to find
settings that will work on a particular instrument. You also may find
that small adjustments to the sheath pressure (cytometer menu) will
make a big difference. I’ve never found that the BD default values
work as anything better than a guideline.
--------------------------
Hi,
I recently had problems running the ARIA with the 100 micron nozzle. What
solved my problem was the O ring size. BD told me that the O ring size
should be .0075  -  .0095 , for 100 micron or higher nozzles. On the O-
ring packaging look for the C/S number. When I replaced a .0073 O ring
with a .0082 size O ring, all was well.
Also here are the starting point setting Bd gave me

amplitude   20
frequency   30
drop 1      330
gap         6
drop delay  29.00

Hope this is some help.
--------------------------------
Hi Vinko,

Sounds like pressure to me, the BD software version 6.1.1 has a bug
where by it doesn't automatically change the pressures over when you
select between 70um low, or custom to 100um low or custom. We normally
have to manually go into Cytometer - Sheath Pressure - and change it
to 20 psi.

Try it and see, if you can't then you need to check the over all
pressure of your instrument, which I can tell you how to but would
rather wait and see if its the first problem.
------------------------
Hi Vinko,

Haven´t read the thread from 2005, I will briefly describe, what I do in
such cases. Maybe you find a point you didn´t tried so far.

1) I remove the nozzle and dry everything. So I have cotton swabs, with
which I can dry inside the cuvette and nozzle locker. With a paper tissue, I
clean/ dry upside where the jet stream is coming out and between the
cameras. I also dry the deflection plates.

2) Next I sonicate my nozzle in RINSE solution in a plastic beaker. After
that I clean it with deionized water and dry it with air pressure.

3) Than I take a fresh o-ring (and here I had once a bad stock, I needed 3
different fresh o-rings, till I got a nice droplet break off), rinse it with
water, dry it and put it into place. I control the position with a magnifier
glass. Then I switch on the pressure and observe, if everything stay dry and
if the stream is coming out properly.
If the breakoff (the satellite droplets have to be quicker than the normal
drops and should fuse within the window) is not looking fine, I try to push
the nozzle to a better fitting. Rarely I just change the frequency about .x
to get a better picture and adjust the drop delay if necessary.

I know it is sometimes hard, but one time, it will work.
-----------------
Hi Vinko,

I ordinarily operate my Aria with a 100um nozzle, sheath pressure at
20psi, frequency at 30mHz and attenuation off. I generally find the
failure to make drops is either the wrong sheath pressure, lots of air
in the system (check your fluidics cart bubble filters) or problems
with nozzle placement. Sometimes the nozzle screw needs to be turned
slightly to set it in a more optimal position. I also have a little
play in my nozzle position which doesn't cause me too many headaches
but I prefer to take the nozzle out entirely and reinsert it rather
than tweak its position in the flow cell. The overwheIming majority of
problems I have with low pressure sorting (I also run the 130um at 12
psi) is due to air. I think the low pressures don't move trapped air
through the system very efficiently. Sometimes I will elect to do a
another fluidics start up to really purge the system or shut the
stream on and off quickly 3 times in a row to hopefully remove air. I
hope this helps.
--------------------
Hi Vinko,

    I have somehow lost touch of my AriaI experience. Having a nozzle
being able to move a bit within the nozzle bay does not signify any
damage to the slot. Don't overdo it or else you might crack your flow
cell. If my memory does not fail me, let me ask you a few questions
first.

    If I understand you correctly, you were saying that the same nozzle
works like a charm under high pressure setup, but not when it's under
low pressure setup. If I am right, it could be a leak somewhere (I can't
tell for sure), thus the low psi can't make the hydrodynamic focusing
works.

    Have you checked under the microscope to see if there's a clog
within the nozzle? Will it help to sonicate for 30s to 1 minute?

    Oh ya, have you bleed your air bubble filter too?


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