Drop Deflection Modifications
- Stream charging limit increased to approximately 150 volts to maintain side stream deflection angles at higher pressures.
- Add-on fluidic/electronics cabinet has three user adjustable knobs that help control drop formation and deflection:
- Charge plate voltage (adjustable 6KV to 8KV)
- Pulse shape A (Leading edge of deflection pulse)
- Pulse shape B (Trailing edge of deflection pulse)
The Tubro Sort Option kit we received needed some careful tuning after installation.
We found that a couple of minor modifications we required (a capacitor and resistor were added
to the system) to tune up the side streams, charge plates were modified (lowered), and careful adjustment of the drop drive frequency/amplitude is
essential to avoid problems with satellite drops at higher drop rates. Properly tuned and optimized, however,
Turbo Sort really works well for us. The additional pulse shaping adjustments and charge plate
voltage control are used often to tune it properly when changing nozzles, pressures, and drop packet sizes.
TSO is not as simple to setup as the standard Vantage in terms of drop stability, etc., but my guess is most operators would probably pick it up quickly.
What we think - so far
We really like it. So far, we have been able to at least double the flow rate for most sorting experiments.
Many sorts that would usually need 8 to 10 hours of Vantage time are being done in 3 to 4 hours without any loss in sorting efficiency.
Most sort samples we see here at TSRI are usually hard enough to obtain that we need to be rather conservative in terms of what a tolerable sample loss is, so we like to
account for at least 90% of what is really there and not lose more than about 20% of any sorted subset due to coincidence aborts.
If you can tolerate higher losses, you can sort very, very fast with TSO. At moderately fast speeds, TSO allows a 10% subset to be sorted at nearly 2.2 times the rate than on a standard FACS Vantage.
To illustrate, compare sort recovery values for standard and TSO Vantage parameters.
The relative yields and purity remain essentially the same (reasonable recovery and 98%+ purity). The pressures used vary from 18 to 35 psi or more to handle the TSO "sweet spots" that appear to be around 38,000, 48,000, 61,000, and 66,000 drops/second using the 70 micron nozzle. Care is taken when adjusting the dead time and timing the deflection packets.
We have done test sorts at up to 94,000 drops/second with the 50 micron nozzle, and it seems that the ability of various cell types to survive the higher pressures and velocities is probably going to be the limiting factor. We are currently working our way up the pressure/speed scale (as time permits us to test various cell preparations), but initially it seems that most common cells will behave fairly well after sorting at pressures up to around 28 or 30 psi,
where some of the more fragile cell types begin to show some negative effects (increased numbers of "dead" upon analysis). We hope to have results soon from some proliferation assays being done on cells sorted at high velocity. Stay tuned.... it looks like two and three laser sorting will work just fine too (even though BD won't support it yet with TSO), as long as you are sober about
We've already sorted mouse spleen cells at 10,000+ cells/sec (61,000 drops/sec) in dual laser mode (FITC, PE, APC) with excellent results
in terms of yield, viability, and purity.
We don't have the BD Sort Enhancement Module, so I cannot comment on how TSO may work with it (probably not well - according to the BD manual)... In summary, our sorter logs for August and September
are telling the story - the older FACStar instruments were fired up only twice during a month instead of used daily, and I'm having trouble
getting "test bed" time on the Vantge with TSO now that our users are seeing how quickly we can sort their samples.
- fine tuning the instrumental dead time
- the delay time between beams
- sample flow rate/coincidence aborts
It looks like the additional losses to the delay circuit in dual lasr mode amount to an additional 1 -> 2 percent more than in single laser more under our standard sorting conditions.
January 1998 - recplaced the peak sense and hold board (#13) with a new redesigned circuit board (Vantege SE version) developed to eliminate the losses that occured in dual laser mode. The improvement is significant, no huge differences in sorting efficiency when in multi-laser mode. The new board allows for up to 3 laser beams in
3 different spatial positions, although we are still using two spatial positions.
Joe Trotter, Director TSRI Flow Cytometry
Turbo Sort - ® Becton Dickinson Immunocytometry Systems 1995
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