Hi Vinko, What you're seeing is quite usual. As you probably know, the sorter sorts drops (not cells) and in the case of the Aria (as in most conventional sorters) it does so by charging the stream at the position where the cell of interest is, before the actual drop is formed. Therefore, when this drop is formed and passes through the electric potential created by the electric plates, it will be deflected into the respective tube. However, what you need to understand is that whenever a certain region of the stream is charged, it will attract opposite charges from the neighboring region and reppel like charges. Thus, the neighboring virtual drops will be slightly charged also, both positively and negatively (since when you test-sort the machine is applying pos and neg charges to the stream). If you don't correct for this, you will see two (or more!) streams appearing on both sides of the middle stream. These represent all the different charge intensities that are being created. You can correct this in at least two ways: 1. You can neutralize the neighboring charges, by applying equally opposite charges to those regions and eliminate this undesirable deflection. In the bottom part of your stream view window, you can adjust the compensating charge of the 2nd, 3rd and 4th neighboring drops. The value should be highest for the 2nd drop and lowest for 4th drop (should decrease exponentially!?) since the charge effect is less prounouced as you move away from your drop. Try moving the 2nd drop first until you find the optimal correction. Then change the 3rd and 4th sequentially. 2. Or, you can simply find a new position of your stream (by altering the amplitude) where this phenomenon is reduced. Sometimes changing the position will be enough, but most of the times you'll have to do both: change to a better position, and then correct for the undesirable deflection. Once you've established a good position for your stream in a given pressure setting, it should remain stable from day to day for quite a while (I have the same settings for months, and I only tweak the 2nd and 3rd drop slightly because I'm a bit picky! ;) Hope this helps. Good luck, Rui. _____________________________ Rui Gardner Flow Cytometry Lab Manager Instituto Gulbenkian de Ciência Apart. 14, PT-2781-901 Oeiras PORTUGAL Email: ruig@igc.gulbenkian.pt Phone: +351 21 440 7951 Fax: +351 21 440 7970 On 12/5/07 10:11 PM, "Vinko Tosevski" <vinko.tosevski@medri.hr> wrote: > 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 > > > This attachment - 'DSC00502.JPG' - 919.53 KBytes - can be viewed at > http://www.cyto.purdue.edu/MD-parts/54e17b60744833131d3be63bff34cfc5f7c109b4.J > PG >Received on Fri Dec 7 14:58:00 2007
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