>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.....


>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 ......
>                                Guy Cox


In addition to Guy's point, Aryeh mentions that his measure of resolution
is " full width between the first zeros" . This would seem to indicate the
DIAMETER of the first dark ring rather than its RADIUS which is the basis
of the Rayleigh Criterion of resolution.  (i.e. the "Rayleigh Resolution:
in Z seems to be about 350nm).

This is very good and one must wonder how it is so good.  As the beads were
right next to the coverslip, spherical aberration should not be too much of
a problem (though NA is indeed limited as Guy says).  If the bead bleaches,
this may make the signal decrease faster than it should as one focuses past
it? On the other hand, as this is a small object, the signal level may be
so low that the it is hard to measure the "zeros".

Jim Pawley

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