>        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
 
 
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Steffen Dietzel
Institute of Human Genetics, University of Heidelberg
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