On Feb 13,  8:02am, James Pawley wrote:
> Subject: Re: Enhanced PMTs: or more likely z-resolution
>
> 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".
>
You are correct -- I did not see zeros, since I did not see the signal pick up
on the other side of the major peak.  Also, the z increment reported by the
software was almost a factor of two off. Finally, the minimum step on the
axiovert with the Biorad stepper is 180nm, so I cannot really measure the axial
light distribution very accurately.
What I can say is that when imaging the nanobeads, I can go from almost no
signal through the peak and back down in about 6-8 sections (1080nm-1440nm).
However, this is probably  caused by having the tail of the distribution below
the noise. So I cannot get the exact PSF this way, but I think the response is
not bad. If I had a way to image two beads at a known distance from each other,
that would be very interesting, since that really defines resolution.

Opening the iris from 0.7mm to 2mm does not change things much (with a 63x
objective), so I am not at the diffraction limit axially.  0.7mm is chosen as
the "optimal pinhole size" based on 50-60% of the airy disk diameter (1.3mm
with 60x objective).

Thank you for pointing out the problems in my measurement. I will go back and
try to do it more carefully.  One of these days I will get it right...

--aryeh


--
Aryeh Weiss                          | email: [log in to unmask]
Department of Electronics            | URL:   http://optics.jct.ac.il/~aryeh
Jerusalem College of Technology      | phone: 972-2-6751146
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Jerusalem, Israel                    | ham radio: 4X1PB/KA1PB