From: Bob Leif To: cyto-inbox Howard wrote, " And, as far as sizing goes, while Coulter volume has its problems, it's far more accurate than scatter measurements." I agree that Coulter volume is far more accurate than scatter measurements. However, this statement should include the modifier than scatter measurements made with today's commercial flow cytometers. A major problem with low angle light scatter is that the optics have been optimized for the fluorescence measurements. The cone angle is too big. Al Brunsting made rather precise bulk static light scatter measurements, years ago at Los Alamos. Frank et al. (1) were able to measure the length to width ratio of erythrocytes. Although this work was based on my idea, the vast majority of it was done after I left Coulter. Coulter volume is both precise and has only two problems: 1) For most cells, the orifice should be 70 to 100 microns. 2) Even though the late Mack Fulwyler made it work for a sorter, there is a significant problem concerning the location of the second Coulter electrode in a sorter, particularly in a system that requires a high numerical aperture fluorescence optical detection system. The problem of the gas bubbles produced at the electrodes was solved years ago (2,3). The use of the combination of Coulter DC and AC impedance and median angle (45 degree) light scatter does produce good discrimination among leukocytes in the Beckman Coulter hematology analyzers. It also works with fluorescence (4). 1) Apparatus and Method for Determination of Individual Red Blood Cell Shape Coulter Corp., R. S. Frank, J. L. Wyatt, W. Gong, C. M. Rodriguez, and R. C. Leif. 5,798,827 (1998). 2) Use of Fluid Retarding Ion Conducting Material, Coulter Electronics, Inc., R. C. Leif, 4,258,316 (1981). 3) R. C. Leif, V. Guarino and N. Lefkove; "The AMAC IIA, A True Bridge Circuit Coulter-Type Electronic Cell Volume Transducer". J. Histochem. Cytochem. 27, pp. 225-233 (1979). 4) R. C. Leif, M. L. Cayer, W. Dailey, T. Stribling, and K. Gordon, "The use of a Spherical Multiparameter Transducer for Flow Cytometry". Cytometry 20, pp 185-190 (1995). -----Original Message----- From: Howard Shapiro [mailto:hms@shapirolab.com] Sent: Sunday, June 30, 2002 5:12 PM To: cyto-inbox Subject: Cell Sizes, FSC, and SSC Annette Byrne wrote: > Hello all > > Does anyone have an info on relative sizes of the following cell types: > Fibroblasts, endothelial cells, smooth muscle cells and macrophages. I am > trying to make a gross distintion of a mixed cell population containing the > cells above based on their FSC/SSC. > > If anyone could order them based on largest to smallest cell type I would > be eternally grateful ! > FSC does not measure cell size!!!!!!!! In some instruments, 5.5 um plastic beads have bigger FSC signals than 5.0 um plastic beads (and in other instruments, they don't). These beads are relatively uniform and smooth-surfaced and spherical, and all have pretty much the same refractive index; FSC (and SSC) signals are influenced by both size and refractive index. The refractive indices of cells, by contrast, vary with the cells' content of water and of proteins and other macromolecules. If you can't reliably use FSC to tell that a large lymphocyte is bigger than a small one, you certainly can't use FSC to compare relative sizes of different cell types. If you happened to have access to pure populations of the cell types you were interested in, you could produce FSC vs. SSC plots for each, which might help in resolving a mixture, provided the cells in the mixture had not been exposed to conditions that could change their refractive indices or sizes. The best way to identify mixed cell types on FSC vs. SSC plots is to gate individual cell types based on the presence of a specific marker, but you have to be sure of the marker specificity - see my reply to a posting from Ray Hester on reticulocytes. And, as far as sizing goes, while Coulter volume has its problems, it's far more accurate than scatter measurements. -Howard
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