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What kind of microscope are you using?  Usually Nikon objectives are not compatible whit other brands of microscope.

Monique Vasseur
Microscopie et imagerie
Département de biochimie
Université de Montréal
C.P. 6128, succursale Centre-ville
Montréal QC    H3C 3J7   Canada
tél. (514) 343-6111 poste 5148
-----Message d'origine-----
De : Confocal Microscopy List [mailto:[log in to unmask]] De la part de Pedro J Camello
Envoyé : 3 février 2008 10:15
À : [log in to unmask]
Objet : Help with Z slices

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I´m trying to set a Biorad 1024 (Nikon 60x, oil) to get optical slices of
labelled cells. Using a sample of 200 nm fluorescent beads I got Z
resolution of 327 nm (average of 6 beads, range 110 to 520 nm) with the
minimum iris (0.7).

My first question is: if I want to make 1 µ optical slices, should I get
the resolution for higher iris aperture untill I get 1 µ axial resolution?
(I´ve recorded vertical sections for the same beads from 0.7 to 8 iris
apertute)

The other question is about the axial (vertical) images generated by the
confocal. I expected to get an elliptical image for each bead, stretching
from top to bottom of the image. However, the lowest part of each bead is
shifted to the right, so that the fluorescent ellipses are slightly
tilted. It is supposed that the microscope is correctly aligned (the laser
has been recently replaced and the engineer from Zeiss checked
everything). If alignment is fine, could be that internal settings
regarding magnification and objetive are wrong? I made the measurements
using square pixels, but this feature depends on numbers entered in the
settings by the engineer.

I would appreciate some help for this artifact before starting real
experiments.

Thanks in advance.

Pedro


-- 
Dr Pedro J Camello
Dpt Physiology
Faculty of Veterinary Sciences
University of Extremadura
10071 Caceres
Spain
Ph: 927257100 Extension 1321
Fax:927257110