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david,

on confocal I use xz scans (which only works really well if you have a fast
z-focus). if its only to correct for the variations in glass thickness, I use
the reflection of the upper glass edge and optimise for sharpness and
intensity
of the reflection (pinhole max. open). Using an intensity color coding LUT
helps.
Of course, imaging deep inside you want to also correct for aberrations
introduced by the sample. If you have a membrane label it works nicely as
descibed above. For beads or sub-resolution structures inside the sample, make
sure to hit the center of the bead/structure in y when scanning xz. In this
case I optimise for symmetrical shape of the psf.

cheers, jens

Quoting James Pawley <[log in to unmask]>:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>> 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.
>



--
Dr. Jens Rietdorf
EMBL, Meyerhofstr.1,
D-69117 Heidelberg,
Cell Biology/Biophysics Program,
Advanced Light Microscopy Facility
Phone ++49(0)6221 387-467 FAX-306
http://www.embl.de/almf