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Re Jim Pawley's comments:
First off 1 Airy means that the diameter of the pinhole equals
the diameter of the Airy disk (and of course the radius of the
pinhole equals the radius of the Airy disk). Under these conditions
the Rayleigh 2-point resolution will be equal to the radius of
the Airy disk.
It is true that in confocal microscopy one is exciting the sample
above and below the plane of focus so some fluorescence is wasted.
In principle one could recover some of this by deconvolution and
a partly open pinhole. Like any other deconvolution method this
would work best with a sparse dataset. However there is a very
important practical reason why this is actually a VERY bad idea.
That is that practical confocal microscopes do not expand the
beam sufficiently to fill the back focal plane of the objective
with a uniform field (even microscopes with beam-expanding optics.)
So if you open the pinhole beyond 1 Airy, instead of getting wide-
field resolution, as theory would suggest, your resolution gets
dranatically worse. The theory and practice are covered in a
recent paper:
Guy Cox & Colin Sheppard, 2004. Practical limits of resolution in confocal
and
non-linear microscopy. Microscopy Research & Technique, 63, 18-22
The practical answer is to use multiphoton excitation, so you
don't excite outside the focal volume and no photons are wasted.
Where deconvolution is most useful in confocal microscopy is in
improving the Z-resolution (or at least the perceived Z-resolution).
Not only is the resolution worse in Z, the spread function is
a rather problematic shape which doesn't go to zero (ie some
small amount of out of focus light always gets through the
pinhole). This also seems to be the most useful factor for
Perveen's work. See:
G.C. Cox and C. Sheppard, 1993. Effects of image deconvolution on optical
sectioning in conventional and confocal microscopes. Bio-Imaging 1, 82-95.
G.C. Cox and Colin Sheppard, 1999 Appropriate Image Processing for Confocal
Microscopy. In: P.C. Cheng, P P Hwang, J L. Wu, G Wang & H Kim (eds)
Focus on Multidimensional Microscopy. World Scientific Publishing,
Singapore,
New Jersey, London & Hong Kong. Volume 2, pp 42-54 ISBN 981-02-3992-0
In this case it is definitely best to work with a 1-Airy pinhole.
Guy Cox
Assoc. Prof. Guy Cox, ooOOOOOOoo
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