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Dear Pedro,

I have seen a tilted PSF on our Biorad MRC1024 as well in the past. It
turned out to be a misalignment of the laser -  or at least, it could be
corrected by adjusting the entrance angle of the laser beam into the
scanhead.

Best regards,

Kevin


Kevin Braeckmans, Ph.D.
Lab. General Biochemistry and Physical Pharmacy
Ghent University
Harelbekestraat 72
9000 Ghent
Belgium
Tel: +32 (0)9 264.80.78
Fax: +32 (0)9 264.81.89

> -----Oorspronkelijk bericht-----
> Van: Confocal Microscopy List [mailto:[log in to unmask]]
> Namens Pedro J Camello
> Verzonden: zondag 3 februari 2008 16:15
> Aan: [log in to unmask]
> Onderwerp: Help with Z slices
> 
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> 
> 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