Martin Bos and Marieke van Marle, from The Netherlands state:
 
>We are studying dairy systems with fluorescence-CSLM, using rhodamine as
>dye, YHS filter block, but find that the intensity near the edges of the
>picture is considerably less than near the center. At high zoom factors
>this is not such a problem, but at lower zoom factors it gets worse. Does
>anyone know a simple explanation behind this? Could this be helped by a
>better alignment of the microscope? Though experienced with operating the
>MRC600, we are novices to aligning it. The operator at our department does
>not seem very concerned, so we would appreciate some outside help.
 
     I have also found this to be the case, particularly with lower
magnification objectives (our lower magnification objectives are also of
lower quality/price!). It is probably due to the complete field of view not
being exactly in focus, which results in a loss of signal towards the edge
of the field because of the confocal nature of the microscope. If you look
at an image under normal transmission illumination you may be able to see
that there is a loss of focus on the edges. One of the reasons good
objectives are so expensive is that it is difficult to make a completely
flat field of view. Of course your confocal should be properly aligned
(contact BioRad if you think this could be a problem), but even a perfectly
aligned system will often show less fluorescence near the edges. Often a
small level of zoom (say about 2) gets rid of most of the problem.
 
Good luck, I hope this is of some help.
Bye, Alan Hibbs.
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Dr. Alan R. Hibbs                                phone: 07 362 0416
Queensland Institute of Medical Research           FAX: 07 362 0104
PO Royal Brisbane Hospital                       email: [log in to unmask]
Brisbane, Qld 4029
Australia
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