Jim (Jake) Jacobberger wrote- >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turns out to be mathmatically exact in three dimensions (unpublished), 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. The digital pulse processing in the DiVa and Luminex instruments (and in Rick Thomas's NPE system) was anticipated in Leon Wheeless's slit-scanning flow cytometers decades ago, but the newer instruments (or at least the DiVa and Luminex do not get a large enough number of samples per pulse to permit much information to be derived from pulse shape. The basic problem is that both the DiVa and Luminex need to achieve a dynamic range in the neighborhood of four decades, which requires at least a 14-bit analog-to-digital converter (ADC). The fastest sample rate available from affordable 14-bit converters was, until recently, 10 MHz; the DiVa uses a 10 MHz converter, which means it can get 32 samples from a 3.2 usec pulse, 16 from a 1.6 usec pulse, 8 from an 800 nsec pulse, etc. You'd probably want at least 32 samples to do good shape analysis. The options, not mutually exclusive, are to flow at a lower velocity, giving longer pulses, and to give up dynamic range in favor of more rapid sampling - modern digital storage oscilloscopes use 8- and 10-bit ADC's that sample at gigahertz rates. David Galbraith and Jeff Rodriguez and their research groups at the U. of Arizona collaborated on some work on pulse shape analysis a few years back using fast ADC's; see Zilmer NA, Godavarti M, Rodriguez JJ, Yopp TA, Lambert GM, Galbraith DW: Flow cytometric analysis using digital signal processing. Cytometry 20:102, 1995. For what it's worth, Analog Devices now has a 14-bit, 105 MHz ADC. This could do pretty good pulse shape collection at conventional flow rates, provided you could find a DSP chip to keep up with its data acquisition rate. The system design gets very complex; if a "killer application" - perhaps Pap smears? - could be found, I'm sure somebody would find the time and money to build the machine. -Howard
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