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I can speak to number 3 from experience.  The oil can make a huge
difference.  We recently upgraded our Leica with the AOBS, and when we
reinstalled it a few weeks ago, the axial resolution was coming out at
around 700nm; Leica specs it at <350nm.  The service guy and I beat our
heads for two days over this, both of us fearing that we would have to
ship the whole thing back to Germany.  Finally we borrowed some Zeiss
oil from a friendly lab, and voila...325nm z resolution.  The RI of the
oils were supposedly the same, both non-fluorescent, etc. etc.  But they
were different in some way.  We had been using sub-par oil for almost 4
months, with the concommitant decrease in resolution.  Images looked
fine, and there was no way to tell without actually testing the
systems.  Bob is definitely right on this count:  we should test our
systems regularly!!!  I know I will.

-Russell McConnell



Robert Zucker wrote:

>
>
>Axial update-summary Over 50 people who have asked for the PDF file and
>a few scientists who have confirmed our observations in their respective
>labs and offered additional insight. Below is the summary of some of the
>comments that I received.
>
>1. We use 488 excitation. Other wavelengths can also be used but the
>longer the wavelength the greater the axial resolution
>2. We zoom now at 10 x. Lower zooms will decrease the accuracy. Higher
>zooms may influence the tests due to scanning instability.
>3. Other factors to consider in the interpretation of the tests are
>immersion oil and temperature. These are considered minor but have not
>been tested. Does anyone have data?
>4. If the setting of the offset value  (background) is too high it will
>introduce between 7-9 nm decreases in the axial resolution.
>5. The Axial Resolution pattern is very important and the FWHM should be
>replaced by a value 2/3 down from the peak maximum and not at the ½
>point.
>6. Both Alignment and dirty objectives can influence the axial
>resolution test.
>7) The part can be both from Biorad # TL2MRC339610 or  Spherotech.
>(Mirror slide)
>
>Conclusions—this test measures following:  1) lens manufacturing quality
>2) system alignment 3) dirty lenses.  Do it!!!
>
>Bob
>
>Robert M. Zucker, PhD
>U.S. Environmental Protection Agency
>Office of Research and Development
>National Health and Environmental Effects Research Laboratory
>Reproductive Toxicology Division, MD 72
>Research Triangle Park, North Carolina, 27711
>Tel: 919-541-1585; fax 919-541-4017
>e-mail: [log in to unmask]
>

--
Russell McConnell
Manager, Tufts-NEMC Imaging Facility
Department of Neuroscience
http://www.neurosci.tufts.edu/Imaging