Aryeh wrote:

>As an aside -- concerning optical sectioning. I just tested the axial
>resolution of my MRC-1024/Axiovert combo using the Bangs labs 63nm
>beads. I found that at best I was getting about 700nm full width
>between the first zeros at 0.7mm iris (63x/1.4 objective). However, my
>system is not vibration isolated, and the bead was in water (although I
>gave it time to settle). I also tested at 2mm and 4mm iris
>settings. At 2mm iris diameter I still had about 1 micron axial
>resolution. At 4mm it was about 1.3 micron between the zeros.
>The initial data I took were noisy, so I plan to redo it to get more
>accurate results -- but the initial numbers are in the right range and
>confirm what I see when imaging specimens.
>The moral is, unless you need the ultimate in resolution, you can do
>very well by opening up the iris (or pinhole) and sacrificing some
>axial resolution. You gain a lot in sensitivity, and dont lose that
>much in resolution.

Actually the true moral of this tale is that if your sample is in water
you are NOT using an NA 1.4 lens!!!! The NA you are actually using will
be ~1.22 but the image will be so seriously distorted by spherical
aberration that this figure doesn't mean much.  In this case opening
up the pinhole won't make matters that much worse ......

If you really want to image samples in water you need a water immersion
lens!

                                Guy Cox


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