Steffen,
 
I think that all will agree that accuracy in the measurement of position
CAN be very high but that it will depend strongly on the stability of the
stage and the S/N of the signal.
 
However, before you spend too much time trying to maximize these, it is
good to remember that hybridizing specimens requires heating them to a
level at which much (almost all?) of the structural detail is lost (as
seen, for instance, in SEM where frizzy chromosomes becose like pieces of
Jello.) so any measure of location that you make in the LM will almost
certainly be a measurement on a specimen in which the positions of what you
are measuring differ significantly from what they were in the living state.
 
Don't mean to discourage you...?
 
Jim Pawley
 
>I have a question concerning the resolution of the confocal microscope:
>
>Normally the resolution of a confocal is given as the distance by which two
>points must be separated to resolve them in two points (about 300x600nm x,y
>and z.). But this is not the type of experiment I am interested in. I want
>to determine the gravity centers of point-like Fluorescence in situ
>hybridization signals (cosmid or YAC-probes) and then (by 3D image analysis)
>the shortest distance of these gravity centers to a nearby surface of a
>large structure recorded in another color (a chromosome). This larger
>structure has a strong gray-level increase over several pixels at the border
>and in the core the gray-levels move around a mean.
>
>My questions are:
>(1)  How accurate (in nm) can the gravity center of a point like signal can
>be determined (relative to other signals) and
>(2)  How accurate can the position of a "surface" be determined, lets say at
>a particular threshold?
>
>I am working on a Leica TCS System and use mainly the 63x, 1.32NA PL APO lens
>
>Any ideas about it?
>
>Greetings
>
>Steffen Dietzel
 
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