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Well, Jim's given his approach.  Personally I wouldn't
even try to adjust the correction collar in confocal
opration - I'd always set it in widefield before starting
confocal acquisition.  There is no reason for it to be
different in confocal mode.  Furthermore, there's no
reason for it to be different in fluorescence than in
brightfield.  Therefore if your sample would bleach while
you adjusted in fluorescence, go to regular transmitted
light.  There will almost always be some sort of dark
speck you can use - maybe a liposome, maybe (dare I say
it?) a speck of contaminant.  In fluorescence we are often
looking at sub-resolution structures so the sample itself
will usually offer suitable features.  Yes, it is a little
bit tedious because the focus changes, but like everything
else, the more often you do it the easier it gets.

                                                  Guy


>>Guy- What procedure do you use to set the correction collar?  I have
>>always found this challenging due to the need to refocus as you
>>rotate.  I am usually trying to use it on our confocal system and I
>>have not sure whether DIC, widefield or confocal is the best way to
>>determine the right setting.    Dave
>>
>>Dr. David Knecht
>>Department of Molecular and Cell Biology
>>U-3125
>>91 N. Eagleville Rd.
>>University of Connecticut
>>Storrs, CT 06269
>>860-486-2200
>>860-486-4331 (fax)
>
>   I know you asked Guy, but a it is night there and we are just
> getting started here in Vancouver, let me just say something about
> adjusting for SA in confocal.  You really can only recognize that SA
> is present if you focus above-and below and see a different result in
> each direction. This poses a problem in confocal because when you
> focus above or below an object, it goes out of the optical section
> and disappears. So the first rule is that you must open the  pinhole
> up all the way.
>
> The second problem is that, if you have a thick specimen, then of
> course the image always looks different when you focus up than when
> you focus down, because you are imaging different slices of the
> specimen. So you need a planar specimen. They are hard to come by and
> most people I know, use sub-resolution fluorescent beads sprinkled
> throughout the specimen.  If you have SA, the image of the bead will
> look like a "bullseye" in one direction (we call this "rings") and a
> fuzzy blob in the other direction. When it is correctly adjusted, it
> is a "fuzzy-blob" on both sides.
>
> You can't just look at one plane, you must scan as fast as you can
> with a small box size and constantly focus up and down at the same
> time that you slowly turn the collar until you have a fuzzy blob
> above and below.
>
> Good luck.
>
> Jim P.
> --
>                     ****************************************
> Prof. James B. Pawley,                                     Phone:
> 604-822-7801
> 3D Microscopy of Living Cells: Summer Course   CELL: 778-869-2348
>
> "If it isn't diffraction, it is statistics":Microscopist's complaint,
> Anon.
>


--
Associate Professor Guy Cox
Electron Microscope Unit,
University of Sydney,
NSW 2006, Australia

Phone:+61 2 9351 3176    Fax:+61 2 9351 7682
http://www.guycox.net