I'd like to weigh in on this one (from cytometry perspective and not on the sales or instrument specific issues). Several years ago I ran some cells through Leon Wheeless' slit scanning instrument that were stained with nucleolar antigens (p120 from Wade Bolton at Coulter) and DNA. Leon's group had for some time been deriving nuclear to cytoplasm ratios with acridine orange stained cells, and they have published extensively on this. In my experiment, the multiple nucleoli could easily be picked out of the traces, suggesting that significant sub-cellular localization was possible using antigen markers. Early work by Leon's group described an algorithmn for subtracting nuclear fluorescence from the entire trace (two dimensional analysis) that turns out to be mathmatically exact in three dimensions (unpublished), thus it appears that for some antigens (e.g., ~100% nuclear distribution), we could subtract ~exact non-specific staining on a cell by cell basis. Finally, sub-cellular translocalization is a major theme in cell signaling and cell cycle regulation and if possible should be a parameter (e.g., nuclear cyclin B1 should distinctly mark mitotic cells). Therefore, I have thought for some time that the best cytometer design is to use narrow beams, capture digital traces, and process/analzye the traces with cell biology in mind. However, I have a very defined end point (analysis of signaling molecules), and at the present time, other than the benefits described by Mario (dropping log amplifiers and easier compensation on multiparameter aquisition), with instruments that have large beams relative to cell size (DiVa) and without manufacturer software to capture files with traces (DiVa?? & Xcel), current instruments seem are a long way from what I want. jwj -- James W. Jacobberger, PhD Professor of Oncology Associate Professor of Genetics Case Western Reserve University Cancer Center & Dept. Genetics 10900 Euclid Ave.(BUT FOR COURIER SERVICES USE: 2109 Adelbert Rd. Cleveland, OH 44106-4944 ph: 216-368-4645 web site: http://josephine.cwru.edu Wednesday, January 23, 2002, 8:57:59 AM, you wrote: MR> ... or, is the grass greener on the other side of the picket fence? MR> I have learned that some instrument sales representatives are making MR> claims and misrepresentations about whether or not we are satisfied MR> with our high end sorter (BD DiVa), of course without my permission. MR> These claims are uninformed, and should be taken with less than a MR> grain of salt. While I am sure that most readers of this list are MR> savvy enough not to believe most of what sales reps say, there are MR> also plenty that don't have the necessary expertise to judge the MR> veracity of such statements. MR> First of all, a disclosure: in the last 14 years, I have neither MR> consulted for any instrument manufacturer, received any financial MR> remuneration from any, or hold financial interest in any. In fact, MR> as a government employee, we can't even receive discounts below MR> standard GSA pricing on anything we buy from them, no matter how many MR> sorters we buy. Finally, my laboratory is not under nondisclosure MR> agreement with BD; however, federal law prevents us from disclosing MR> private corporate information. MR> That said, we have had a BD Digital Vantage (DiVa) for nearly one MR> year. We have extensive experience with it now, and have tested it MR> for sensitivity, sorting capabilities, stability, etc. As we MR> published in a poster at CAC (the contents of which are therefore in MR> the public domain), we find the sensitivity of the digital MR> electronics on this DiVa to be at least as good as the analog MR> electronics on the same instrument for individual channel MR> measurements. However, it is with multi-color applications that the MR> advantage of the digital electronics becomes apparent: the lack of MR> log amplifiers combined with high resolution A/D conversion leads to MR> more accurate compensated data (for explanations, see my recent paper MR> in November Cytometry). In particular, compensating digital data MR> across lasers does not require the careful pulse matching (and delay MR> timing) that is so problematic on analog systems--i.e., we don't need MR> to look at pulse traces anymore. (A real bonus, given that we have a MR> fully-utilized 14-parameter system!) MR> As predicted, we found the sorting performance to be better than MR> analog, in terms of speed, given the essentially zero dead time. MR> Purity in 1-way, 2-way, or 4-way sorting was identical to analog in MR> 1- or 2-way sorting. We have successfully single-cell cloned, purity MR> sorted, yield sorted, etc. We have long since abandoned the analog MR> electronics in favor of the digital electronics. MR> Nonetheless, I think the biggest advantage of digital electronics is MR> still in the future. I firmly believe that once manufacturers MR> develop some sophisticated signal processing of the digital waveforms MR> (probably with relatively straightforward firmware upgrades to MR> current digital systems), we will achieve considerably better MR> sensitivity. And maybe even learn something about the subcellular MR> distribution of the fluorescence signals! MR> If you are considering a high end sorter, please disregard any claims MR> made by sales reps of any company about our level of satisfaction or MR> dissatisfaction with our instrument (and I've heard about both types MR> of claims, obviously from reps of different companies). If you are MR> in the market and care to discuss our experience with the DiVa, then MR> feel free to call me and we can talk candidly. MR> mr MR> (PS, one more disclosure: the content of this email represent my MR> views, and do not necessarily state or reflect those of the US MR> Government.)
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