Et tu Howard? My guess is that the huge majority of flow done on the planet in which I live is performed on analytical instruments with fixed optics. Wonders of modern multiparameter flow aside, 2 color is still more prevalent than 3, 3 more prevalent than 4, and four is way more prevalent than 5 or more! In our core facility (which features 2 identical 4 color analyzers) I preach constantly for users to label their axes, but nothing like Howard and Paul have suggested. Antibody, fluorochrome, and detector are what you really want to use in the label, especially the first two, since this vitally important information should be included in the header of the listmode file where it will be linked to the primary data forever. How about the other suggested info? Center of emissions wavelength: Incomplete (it has been already acknowledged that band-width is important too) and entirely unnecessary on instruments in which the optics are standard and specified by the manufacturer. These are constants within experiments, between experiments, and between labs. In our lab, and in all others with unmodified Coulter XLs cytometers, FL1 can be used perfectly intelligibly in an axis label as shorthand for: The light from a 488 nm argon laser has been focused by 2 beam shaping lenses; light scattered at 90o within a quartz cuvette was passed through a collimating lens, a 488 dichroic longpass filter, through a 488 blocking filter filter, a 550 nm dichroic longpass filter and a 524 bandpass filter (505 - 545 nm) before being sensed by the photomultiplier tube arbitrarily designated FL1. The analog signal has been amplified, digitized, compensated against signals from other detectors, log-transformed and binned into 1024 channels which is represented on a four decade scale. If I have made modifications to the instrument, or if I am using an instrument which has easily replaceable optics (like our MoFlo), then it certainly behooves me to detail this information in the materials and methods, but PLEASE, not in the axis label (see Chart Junk, below). Compensation: I agree that the vast majority of published histograms show compensated data, and that the majority of flow sins are committed in the name of compensation. But include compensation in the axis label???? Cytomation has tried this in their Summit software and once you pass 2 colors it is an unintelligible mess (e.g. FL1 - X% FL2 - Y% FL3....). Perhaps (perhaps) the compensation matrix and other "cytosettings" should be routinely included in the materials and methods section (difficult to do if these settings change slightly each day as the result of QC procedures). I say perhaps because this information will only provide a rough guide for others wishing to reproduce the work, even if they have identical instrumentation. On the positive side, it will allow the manuscript reviewer to decide if anything particularly egregious has been done to instrument or the data. As an aside, I am a huge fan of saving high resolution uncompensated data in the listmode file and performing compensation in software. Summary: When you are sitting at the instrument acquiring your data: please label each parameter with the antibody (or dye), fluorochrome (if this applies), and (do I speak heresy?) the fluorescence channel (FL1, FL2, etc.). This information will be forever linked to your listmode datafile and you will be thankful when you pull the data up for reanalysis several months later. When your are trying to publish your data the goal is to create a graphic that allows the reader to understand at a glance what you measured and to let the data speak for themselves. Axis labels are but a small part of this process. Read Edward Tufte, "The Visual Display of Quantitative Information, Graphic Press, Cheshire, Ct, 1983, for an explanation of "Chart Junk" and why you don't want to clutter your graphic with redundant or superfluous information. But beware, you may still have to please Paul or Howard. Albert Donnenberg Pittsburgh, PA
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