From: Bob Leif To: cyto-inbox Since the Scanning Flow Cytometer provides a real angular distribution of light scattering, it is greatly to be preferred to that of a commercial flow cytometer. However, other technology exists to perform definitive measurements. Unfortunately at present it is not commercially available. The combination of the Coulter effects(1), electronic cell volume and opacity with a low angle shape measurement(2) will provide definitive information. If the isovolumic condition is to be tested, precise buoyant density separations(3) will provide very useful information. I would be exceedingly surprised if any present procedure were truly volumetric. What actually happens is the results of the physical measurements are employed as feedback to adjust the chemical parameters. This means that different physical measurement approaches could result in different chemical formulations. 1) R. C. Leif, S. Schwartz, C. M. Rodriguez, L. Pell-Fernandez, M. Groves, S. B. Leif, M. Cayer, H. Crews; “Two Dimensional Impedance Studies of BSA Buoyant Density Separated Human Erythrocytes”. Cytometry 6 pp. 13-21 (1985). 2) 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). 3) R. C. Leif; “The Buoyant Density Separation of Cells”. Edited by G. L. Wied and G. F. Bahr, Automated Cell Identification and Sorting Academic Press, New York, pp. 21-96 (1970). -----Original Message----- From: Pit Tarasov [mailto:scibox@online.nsk.su] Sent: Wednesday, August 02, 2000 2:09 AM To: cyto-inbox Subject: Re[2]: RBC surface area, volume, and topology Dear Howard Shapiro, I noticed in the Mail List's archive your letter about RBC analysis and have also some questions about isovolumetric sphering ( in addition to Julie Pribyl's ) if you do not mind: Julie Pribyl wrote: >> Our lab is currently researching how high cholesterol levels affect the >> diffusion of oxygen >> through red blood cells. My job for the summer will be trying to find a >> way to calculate an average surface area, average volume, and topology of >> the red blood cells. Will FSC and SSC data provide enough information? Howard Shapiro wrote: ... > It is essentially impossible to get the information from FSC and SSC. > However, the red cells must be subjected to a chemical > treatment which transforms them from their native biconcave disc > shape to spheres without changing volume (the process is called > ISOVOLUMETRIC SPHERING) in order to prevent the normal asymmetry > of the cells from producing spurious scatter measurement > values. .... > Thus, while the volume can be measured, the topology is lost. > Even then, the volume measurement is only possible because rigorous > standardization and calibration procedures have been developed, and > we don't have anything like that for FSC and SSC (although some work > has been done on FSC and bacterial volumes). It's easy to collect a > lot of FSC and SSC values from cells under different conditions, and > hard to derive reliable quantitative information from them > This is a really, really hard problem...... We have an opportunity (Everyone is invited to collaborate) to perform direct real time measurements on the Scanning Flow Cytometer refractive index and size of individual RBC's (in sphered state, and sizes of any other spheric particles in "biological region") with resolution close to electron microscope. ??????? Could someone tell (I've heard about it before and very interested, as a part of my job) what is "ISOVOLUMETRIC SPHERING"? Any literature references, protocols, explanations will be appreciated with gratitude. ??????? Being predictably SPHERED (not necessarily isovolumetrically) RBCs are easy to study on SFC. Pit Tarasov for e-mail: scibox@online.nsk.su for air mail: Pit Tarasov Institute of Chemical Kinetics & Combustion Institutskaya 3 Novosibirsk 630090, Russian Federation. P.S.: I beg everyone's pardon for a bit boring consequent introduction to what is a Scanning Flow Cytometry (i.e. what we can do): ************************************************************ Though the method utilising two angular intervals (2ALS)was introduced by D. H. Tycko, et al. in their paper "Flow-cytometric light scattering measurement of red blood cell volume and hemoglobin concentration," Applied Optics 24, 1355-1365 (1985) and can produce absolute measurements of particle volume and refractive index that relates to the hemoglobin concentration if a suitable CALIBRATION sample is available. The Flying Light-Scattering Indicatrix (FLSI) method based on the Scanning Flow Cytometer (SFC) was introduced in series of papers in our laboratory: A. V. Chernyshev et al, "Measurement of scattering properties of individual particles with a scanning flow cytometer," Applied Optics 34, 6301 - 6305 (1995). The SFC allows the measurement of the entire angular light-scattering pattern, (indicatrix), of individual particles. And on the particles with CHARACTERISTIC RADIUS of CURVATURE > the wavelength of laser beam (spheres, for example - beads, sphered RBCs) indicatrix provides the resolution close to EM - analogous to Kotelniokhov-Shenon theorem in signal sampling: In order to provide a real-time analysis of the particles, the FLSI (it gives good size, RI resolution for particles with the shape close to SPHERICAL) method was theoretically supported with a parametric solution of the inverse light-scattering problem. We have applied this method (with corrections to imaginary part of Hb refractive index - absorbtion) to determine the size & Hb content of individual RBC's simply sphered in solute of reduced tonicity. K. A Sem'yanov et al, "Non-Calibration Method To Determine The Size And Hemoglobin Concentration Of Individual Red Blood Cells From Light Scattering" Applied Optics (~september 2000, in press) We have also reported native RBC's measurements and comparison with double-wave WKB modelling (rough approximation in wave optics, at present we implement the T-matrix method). The results were in a good agreement and it has been shown that the main polymorphism in the measured indicatrices caused by different orientation of non-spherical cells in the testing zone of the SFC (shear forses rotate particles in a closed flow and bring them into testing zone in the various orientations). Alexander N. Shvalov, Juhani T. Soini, Andrei V. Chernyshev, Pit A. Tarasov, Erkki Soini, Valeri P. Maltsev, "Light-scattering properties of individual erythrocytes," Applied Optics 38, 230-235 (1999). ************************************************************
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