> Our group is interested in co-localisation of fluorophores within >intercellular compartments. The question which has entered our >thoughts is with what confidence may one say that both fluorophores are truly >co-localised? That is to say , the two fluorophores are within X nm >of each other, given that the axial (x,y) resolution @ 488nm is say 200nm and >the z resolution is say 300 nm ( limited by mechanical considerations ? ). >this would then influence whether or not one may say two antigens were in the >same compartment, given the size of secretory or endosomal compartments. > > Patrick Corley I think that this resolution is a too optimistic guess, especially for the z-axis. If you really need such a resolution there you should test it for your own machine, e.g. with fluorescent beads (for both channels) >Try to avoid sequential scanning! >It induces positional shift between the red and the green >image. If you scan both colours simultaneously, you will >have also some shift (50-200 nm) but you can correct for >that because it is for all recordings the same. Re-scanning, >however, induces unpredictable shift, so, correction is not >possible. > >Erik Manders IMHO it is not neccessarily so: with the Leica TCS-System and the appropriate macros you can scan the different colors of every confocal section sequentially *before* changing the z-plane to the next confocal section, therefore a z-shift due to a (incorrect) re-positioning of the z-stage can not occure. A (still possible (see Mail of S. N. Pagakis)) z-shift should therefore be the same as with simultaneous scanning. Greetings Steffen Dietzel --------------------------------------------------------------------- If you want to protest against the French nuclear tests look at one of the following World-Wide-Web Sites and the links given there: http://www.icepp.s.u-tokyo.ac.jp/~keshi/ http://www.informatik.rwth-aachen.de/AEGEE/oneEurope/protest.html --------------------------------------------------------------------- Steffen Dietzel Institute of Human Genetics, University of Heidelberg Im Neuenheimer Feld 328, D-69121 Heidelberg, Germany phone:+49/6221/56-3998 or -3119 fax: -5332 personal e-mail: [log in to unmask] listservmail: [log in to unmask] ----------------------------------------------------------------------