Udo,

As stated by one and all the, the resolution of an objective is determined
by its N.A. (a la Rayleigh),  and therefore, its useful magnification.
Sampling theory says that for non-periodic objects you need to acquire data
at 2.3 times the highest frequency you wish to measure. That means that if
you want to get 0.2 um resolution data from your 1.4 N.A. lens you have to
over sample your image with step sizes of about 0.087 um. This will be true
for your 63x or 100x. On that basis, if you use a fixed 512x512 frame with
0.087 um step size per pixel, you end up with same field of view regardless
of the obj. magnification. The zoom setting for the two lenses will be
different, but that is all. One can oversample with even smaller step
sizes, but this will not improve resolution and will cause more bleaching.

That said, not all 1.4 NA lenses are created equal. I personally don't
think there is any substitute for testing different lenses and picking the
best one. As a practical matter, in some instances, it may be desirable to
use the higher mag. objective when previewing by eye. That, I think is a
matter of personal preference and visual acuity. One of my technician needs
twice the magnification to see the same detail that I do!

One final word, the objective magnification with a CCD wide field
microscope camera is important. Depending on the camera pixel size a, 60x
objective may give too small an image so that the sampling requirement
isn't met for full resolution, whereas a 100x will.



Mario M. Moronne, Ph.D.
Dept. of Subcellular Structure M/S 6-2100
Lawrence Berkeley National Laboratory
1 Cyclotron Rd.
Berkeley, CA
94720

ph (510) 486-4236
FAX (510) 486-5664
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