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Jim Henthorn says:
>. I put a pressure gauge, low accuracy, on the liquid side to find a
> difference of approx. 0.5 psi. (1L v.s. 5L) using the standard 5L tank.
This
> problem became more apparent when I went to my 20L sheath tank
(a used soda can
> works great) for sorting with the larger nozzles. It had a pressure
difference
> of approx. 2 psi. (1L v.s. 20L). This amount of pressure difference
should have
> very little effect on short sorts (say THAT three times fast!) under 2
hours
> using nozzles under 100 microns, but what about the long sorts
where < 1 % of
> cells are sorted, where everything matters?
>
At Nottingham, I use 200um tips and sort for 4-8 hours (yes, a whole
afternoon !!) using an EPICS 541 at sheath pressures of 4, 5 or 6 psi.
Like you, I use a 12 litre home made sheath tank (an autoclavable
polypropylene carboy) and noticed significant differences in droplet
breakoff as the tank emptied.
I now constantly monitor sheath pressure; I have connected a
pressure sensor between the 0.2 um sheath filter and the flow cell by
inserting a T-piece. The sensor is a low cost ($30 approx) 16PC15DF
from Honeywell (UK agents Scientific Electro Systems Ltd, 451 London
Road, Westcliff-on-Sea, Essex, UK, tel +702 335174). This is excited
from a standard laboratory regulated power supply, 10 V dc, and
produces 100 mv output fsd for 15 psi, so I connect it up to a hand held
digital multi-meter. To callibrate this output, I connected the input to
the sensor to the sheath pressure line, in place of the sheath tank. I
selected various sheath pressures, and noted the reading.
For normal use, I usually keep a small air 'buffer' between the sensor
membrane and the sheath fluid. Rapid pressure changes will cause
the membrane to rupture, so I now use a four way tap rather than a
T-piece and only allowpressurisation of the sensor once the system
has stabilised.
This system is very sensitive, openning and closing of pinch valves on
the sheath line (down-stream) can be detected, as can partial
blockages of the flow cell tip.
>
> P.S. Do I need to go to BDIS and show them how to make a
"constant
> flow" cytometer or what?
>
Coulter were very helpful when I first noticed this problem, about 5
years ago. They now incorporate a similar sensor and feedback loop
into their ELITE sorter, this seems very good, I tried pinching the
sheath line and the droplet breakoff remained constant, I heard the
system filling the tank with more pressure!!
Please contact me for further info if helpfull
Happy sorting ... May the flow be with you
Nigel Blackhall
Plant Genetic Manipulation Group, Dept Life Science,
University of Nottingham,
University Park, Nottingham. NG7 2RD
Fax +602 513251 E-mail plznb@pln1.life.nott.ac.uk
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