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This is an interesting idea. Are you saying on your confocal that the
light that illuminates the rear aperture has a narrower radius than the
field stop -and that you can see the entire beam profile? I'd say on
our system that the beam FWHM is >> the width of the rear aperture (we
can't see the whole beam because it's clipped by stops elsewhere and it
ends up as a more or less uniform circle of light about 1.5cm across).
My argument for opening the pinhole a bit when using deconvolution would
be that as the pinhole is opened sub resolution beads get brighter. Thus
photons from the focal plane are being lost. Recovery of those photons
in a photon noise limited system should improve final image quality -if
resolution is being limited by shot noise..
Cheers
Guy Cox wrote:
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>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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