Snezna Rogelj wrote- >Hello, this is probably one of those "stupid" questions that anyone in >posession of a flow cytometer should be ashamed to ask: > >We would like to determine the size distribution of polymer/lipid micelles >which we can fluorescently stain with nile red. >We have fluorescent beads >from Molecular Probes that are about 0.02um and about 1um in diameter to use >as size standards. If you really mean 0.02 um, you won't be able to detect those beads in either forward or side scatter; you would probably have a fighting chance at detecting 0.2 um beads in side scatter in an instrument in which observation is done in a cuvette, but forward scatter might be a problem. > We suspect the micelles to be somewhere around 0.5 um in >diamater. How do we go about translating the FSC readings to sub-microns? There isn't any way; the refractive index of the beads is different from the refractive index of the polymer/lipid micelles, so, even if you could make some kind of calibration curve for the beads, it would be no help for the micelles. > Is >it a linear parameter? Not even remotely. Forward scatter isn't very good for sizing in most cases; this is almost certainly one of them. For particles well below 0.25 um in size, and down to perhaps 0.06 um, side scatter can be used for sizing, as the scattering follows Rayleigh's formula (inverse sixth-power dependence); your micelles are probably too big. We really need a good size measurement for flow cytometry, but there isn't one, at least on current commercial instruments. -Howard -Howard
This archive was generated by hypermail 2b29 : Sun Jan 05 2003 - 19:01:23 EST