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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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]
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