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
This archive was generated by hypermail 2b29 : Sun Jan 05 2003 - 19:01:44 EST