I generally refrain from commenting on this list though I follow it daily with interest and occasional incredulity (the last time I raised an issue was to attempt to get people to stop using the term "FACS analysis" when they clearly had no idea what they were talking about and what the term meant). Being one of those present at the birth of flow cytometry we old guys tend to bemoan the apparent lack of knowledge of many (but certainly not all) currently in the field. After all, back then virtually all those capable of getting reproducible data out of a flow cytometer could take it apart and put it back together (with no extra parts left over), were knowledgeable or expert in fluidics, optics and electronic (at least those of a flow cytometer) and were serious cytochemists (we actually made our own buffers starting with H2O and created most of the staining techniques that are now purchased in kits). This was a fairly high hurdle to surmount and meant that a significant amount of knowledge needed to be accumulated before you could play in the field. With the rise of core facilities and turnkey systems the bar has been virtually thrown on the ground. Thus, at least one of the issues has become how to deal with the sometimes simplistic questions that arise and how harsh to be towards those that misuse terminology either through laziness or ignorance. The Purdue website is a democracy and it should be open to all and to the extent that the majority tolerates a wide range in expertise of users of the site then so be it. Perhaps the narrower question is more concerned with the question of data presentation discussed earlier. This whole field is a lot more complicated that many in it actually realize. Developing a good foundation of knowledge for users is important because cytometry is a tool used to develop data that may be used to answer important questions in science. If it is misused as it sometimes is, then misinformation gets attached to important scientific questions. It is important to users to set as high a standard as is practical for the field to avoid misuse and misinterpretation of data to the extent possible. In addition, it is also important to say what you mean and understand what you say. Thus, get rid of as much meaningless "jargon" as is possible. Howard has laid out some of the rules for how one should label axes though that is just one area. Think about what you are doing - I have yet to see a flow cytometer that measures "FL1s" - imagine one cell having twice as many FL1s as another? Also, help to educate those you meet or who use your facility, students you mentor or investigators you train. Don't believe everything you read. Because a kit has been designed to measure something (i.e., apoptosis) understand how it works and why (assuming it does, in fact, work). Also think about whether it will work in your "system" - are you misusing the kit because it was not designed to be used in the way you are using it? Finally, to get back to the original issue, newcomers to the field, in my estimation, should be encouraged to do some homework before asking questions. If you have access to this site you have access to a search engine or more specifically databases such as PubMed, Medlines, etc. There are literally dozens of books or compendia of reviews of varying degrees of sophistication which cover virtually all aspects of the field. There is the journal Cytometry. Do a search to see what is in the literature. Realize that your questions may have already been asked and answered a dozen times and can be found in the archives. Some may feel that this website should be used as a search engine - ask whatever question you want in whatever form you choose and expect to get educated by more experienced users. This does tend to happen because many experienced users are willing to take the time to educate people new to the field in that way. Others think that questions like "I want to measure DNA in my cells, what dye should I use?" represents an embarrassing lack of scientific discipline. I for one would much rather see the question asked as "I looked in Howard's or Alice's, etc. book, checked out Methods in Cell Biology and Current Protocols in Cytometry and I still don't understand it or I still can't get it to work". However, that's just my opinon. Frank -----Original Message----- From: Howard Shapiro [SMTP:hms@shapirolab.com] Sent: Wednesday, December 19, 2001 6:27 AM To: Cytometry Mailing List Subject: Re: Paul's Tutorial on FL1, FL2, etc. Paul Robinson wrote- "If you want to discuss fluorescence signals, it is appropriate to use a terminology that is interpretable in a scientific manner. Here are my suggestions, and these are the ones that we require for those who contribute to Current Protocols in Cytometry, the manual that I hope all of you have in your labs!!! To express a fluorescence signal on any machine we request the fluorochrome (example follows), and the center of the wavelength band. e.g. FITC-525 nm, PE-575 nm. If there is an antibody attached, we would suggest it be written CD4-FITC-525 nm. Of course, there are many times when we might simply measure a frequency band, so FITC may not be appropriate. In such case it might be "green fluorescence-515 nm" or similar. If you are using a system with fixed filters that might be "green fluorescence- 525 nm". On flexible systems where all sorts of filter changes can be made, then you obviously must express the specifications of your detection in detail. If you don't know what the exact specifications are for each PMT, you shoudl find out, print them out and paste them on the front of the machine. This is not perfect, as it does not necessarily indicate the width of the band, etc. However, all of these can be stated in figure legends. If you want to indicate where on your cytometer this is measured from, you can add the PMT designation as well. e.g. CD4-FITC-523 nm-FL1 No system is perfect, but the system I have suggested is totally reproducible by any one with any machine. There are many minor modifications to the above that are perfectly acceptable. I take this issue quite seriously, and if you are unlucky enough to have your manuscripts sent to me for review, I won't accept FL1, FL2, etc. I just return the M/S and suggest that the author write in an acceptable scientific format. That might sound tough, but in fact there are few if any journals that will accept figure references that lack units. That is in fact what we constantly see in flow cytometry. We need to raise our standards to what the rest of the community requires." I would take what Paul wrote a little bit further. What the *instrument* initially measures are *optical* properties of cells or other particles: For "Small angle light scattering", aka forward scatter, FALS, etc., the measurement depends on the wavelength(s) used, the smallest and largest included angles, and the shape and orientation of the blocker bar, if used. For "Orthogonal light scattering", aka RALS, side scatter, 90 degree scatter, etc., the measurement also depends on the wavelength(s) used; it is less dependent on angular range than the small angle scattering measurement, but more dependent on polarization (in fact, one can measure depolarized orthogonal scatter as a separate parameter; it is useful, for example, for identifying eosinophils). Fluorescence measurements are also polarization dependent, and measurements of fluorescence in planes of polarization perpendicular and parallel to the excitation beam provide information about the mobility of fluorescent materials in or on cells. However, we usually tend to neglect polarization in specifying how scatter and measurements were made, sometimes (see Asbury et al in the June 2000 Cytometry) at our peril. Obviously, one needs to specify the excitation wavelength (or range in instruments not using strongly monochromatic sources, e.g., lasers), and the emission passband. This can be done most precisely when band pass filters, characterizable by center wavelength and bandwidth, are used; if you use a long pass filter, the detector bandwidth may be more important than filter characteristics in limiting response at the long end, as most people who work with Cy7 tandems have noted by now. But that describes the raw measurements we make, *not* how we should label the axes of our data displays. On the scatter side, I don't have a problem with FALS, RALS, or forward and side scatter, but the FALS signal should *not* be labeled "cell size" unless the experimenter can produce a calibration curve (for example, the B-D FACScan, according to Current Protocols, produces a bigger signal from a 5 um bead than from a 5.5 um bead). And the fluorescence channels aren't what the raw measurements are, in most cases, because we *compensate* them. The machine, using 488 nm excitation, may, for example, measure 525/15 nm fluorescence, 575/20 nm fluorescence, and 670/20 nm fluorescence (n1/n2 here give center frequency and bandwidth). We use compensation to get the intensity of fluorescence from, for example, fluorescein (not "FITC", or fluorescein isothiocyanate; that is what the antibody was conjugated with and there ought not to be any in the sample), phycoerythrin (PE), and PE-Cy5 tandem conjugate labels. It is acceptable to specify either the fluorescent label, or, more scientifically interesting in terms of the biological characteristics of the cells, the antibody or other reagent to which it was bound in the label(legend) of a display. It is inappropriate to label an axis with the optical characteristics of the channel if the data are compensated. Of course, everybody who reads this list does the flow and the compensation correctly, so we can be believed if we neglect to tell people which machine we used, what the wavelengths were, and which antibody had which fluorescent label, and just put the antibody names on the axes. Well, maybe not, but, in an ideal world, that's what we'd want. And for non-antibody measurements, we ought to label axes as "DNA content", "RNA content", etc. Membrane potential, pH, calcium, etc. are a bit dicy if they are not calibrated, but, in an ideal world, they are, *or* there is some disclaimer about the imprecise nature of the measurement somewhere in the paper. More to come in the 4th Edition. -Howard
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