I have to be careful about my math as well after the last debacle but I remember from my days in Heidelberg that someone used a construction similar to a sorter to get sufficient acceleration to fragment chromosomes, but I do not remember the pressures they applied. I also seem to remember that there is an orifice based device to break up bacteria. Having had a look at some ancient literature from the last century (Lindmo et al p 145-169 in Flow Cytometry and Sorting) I got jet flow speeds between approx. 8 and 50m/s. For simplification assuming a round 200u flow channel in my cuvette and an 80u orifice / 65 u jet diameter, the constant product of VxF results in approx. 10 fold speed difference before and after the orifice. For high speed sorting at 14kg/cm^2 and 220 kHz (50m/sec jet velocity) and assuming an acceleration distance of 1mm at an average speed 27.5m/sec the acceleration is achieved in 36usec. This calculates as an acceleration of 45/36^-6 = 1.25*10^6m/sec^2 = 127421g. For the 80u orifice at 1kg/cm^2 (general sort) resulting in 13m/sec jet speed the 1mm takes 140usec resulting in acceleration is 8.4*10^4m/s^2 =8519g. As at the lower pressure the jet diameter is actually slightly wider the acceleration would be a bit higher. This is all oversimplifying things a bit thinking of a cuvette and not considering velocity profiles, turbulence ..., but I just couldn't cope with the half page of indices and Greek letters used in the article of Kachel et al. that late at night / morning. However, I am sure the people who built those beasts have done far more detailed calculations and would now be quite happy to give us some better calculations. Apart from pressure / jet velocity the geometry of the design must have a great impact on the acceleration and I assume the high speed systems must have a much longer acceleration distance and diameter ratios. Regards Gerhard -----Original Message----- From: Howard Shapiro [SMTP:hms@shapirolab.com] Sent: Thursday, March 09, 2000 1:38 PM To: Cytometry Mailing List Subject: Pressure Tactics (High Speed/Pressure Sorting) Although I do as little sorting as possible, I have been following this thread with a great deal of interest. It is inspiring to see people abandoning the convenient "my sorter can sort faster than your sorter" mantra and asking which of the differing physical parameters of "high speed" and "low speed" sorting regimes may be responsible for loss of cell viability or function. Pressure is certainly a candidate. It seems intuitively unlikely that exposure to loud noise at 60 or 100 kHz should be harder on cells than exposure to loud noise at 10 to 40 kHz. And I don't think that traveling 30 m/sec is worse than traveling 10 m/sec; the g-force coming out the nozzle shouldn't be that high (although I haven't done the calculation - it's been a long time since physics class). People used to talk about shear stress affecting cells, but with sheath flow, the cells shouldn't be subjected to big-time shear stress. So it's probably pressure, or changes in pressure. I remember that when the first of the Livermore high-speed sorters went on the air, their users were concerned primarily with sorting chromosomes, which were unaffected by the high speed and pressure, and not cells, which didn't survive a trip through the sorter. There was talk of cells "blowing up", experiencing a severe case of decompression sickness or "the bends" at the cellular level. That is still probably a good model for cell damage; maybe the sublethal effects reflect "rapture of the drops". Physiologists have learned a lot about how to keep the large cell aggregates which comprise divers and astronauts alive after exposure to extremes of pressure; it's usually rapid decompression which causes problems, and the solution typically involves slowing down decompression and/or modifying the ambient gas mixture to minimize the formation of microbubbles. It might be a good idea to look at cells subjected to high pressure in bulk, and compare the effects of rapid and slow decompression on viability and function. If cells decompressed slowly survive and function better than those decompressed rapidly, it would be advisable to look at different gas mixtures, since it is probably impractical to slow decompression as cells go through the nozzle into air at high pressure - and speed. Dredging up memories again, I remember Linus Pauling extolling the virtues of xenon as an anesthetic - perhaps it would make a good sheath drive gas. That would certainly force changes in the rate structure of sorting facilities. But, as I said, I sort as little as possible, so I'm not under pressure... -Howard
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