Hello Robert, Check out the new journal article in this month's Cytometry: "Polarization of Scatter and Fluorescence Signals in Flow Cytometry" Volume 40 Number 2, June 1, 2000. This might address your problem. I have found that in air-in-stream sorters/analyzers, slight adjustments to the Z-axis, scatter bars, and slight adjustments to the laser beam path, laser power, and the FL focus, CAN help you get signals that look like they do on another instrument. But this is an art. However, it can be qualified using bead comparisons, or CRBC comparisons between the two instruments, comparing channel numbers and scatter patterns using the same filters. I had a similar problem with a FACSCAN vs. FACSTARplus. My supervisor was not satisfied until the scatter and FL signals were nearly identical. Well, this is nearly impossible, without having both instruments in your hands, and working with ALL the variables in setting up a good interogation point on a stream-in-air system to mimic what is seen in a cuvette flow chamber. I was able to closely match the same signals, and use settings appropriate for each instrument to get similar results. I used beads of many types, and CRBC's and tried to "mimic" the signal produced by the FACSCAN. This worked well, but it was not exact. I am sure there are better, published, ways to do this. It is a given that without putting the screws to both instruments, the plots may not look the same, but the analysis-end result should be similar if the experiment is designed well (multiple controls, right filters, etc.), and you accept the slight differences between the two instruments. Howard Shapiro has an interesting editorial about the article I mentioned above in the same journal. He states-- in general, polaraization of the laser beam and how it interacts with the particles, filters, lenses, and mirrors affects end results. And not all instruments polarize alike! Yikes. The above article lends credence to my practice of telling investigators not to expect the results to be exactly the same on different instruments (but this is not an excuse for a misaligned, or otherwise poorly working instrument). And I suggest using one instrument throughout an experiment's long term execution. Also, when going from an analyzer to a sorter---for the purpose of sorting, ( after much analysis of what to sort on the analyzer), a problem arises when investigators are not seeing plots exactly like they have in their hands. And guess who gets the blame? Granted, making sure you have the correct filters (or improved filters) is always a good idea. Jens Fleischer <jfleischer@knuut.de> on 05/31/2000 06:48:22 PM Please respond to jfleischer@knuut.de To: cyto-inbox cc: (bcc: Christopher S Boyce/BII) Subject: Re: Signal detection and sensitivity Robert Adair McGilp wrote: > I have an investigator who has been using a FACSort to analyse his FITC/ > PE/Cychrome labelled samples and gets good resolution of the populations > of interest (dual positives). He feels that we have been unable to > reproduce the results on our Vantage SE. We have moved the z axis so that > the nozzle is a good inch above the laser intercept and this gives a good > approximation of the results he has seen on the analyser. Be sure to insert the appropriate filter in front of the FL3 detector. Pe-Cy5 has its emission maximum at 675 nm. BD has a 675/40 bandpass filter and a 650 longpass. Both should give a good resolution. In the standard configuration you often find a 630/22 or 610/20 designed for propidium iodide in front of FL3, which will not give good intensities for PE-Cy5. Jens
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