Johannes,

That was not too much physics. Don't hold back; we just like things concise.


>unfortunately, there is going to be some physics to answer this question by
>Dr. Zucker. I beg your pardon.


>Just as a comment, it's not the main question which is going to be answered
>below:
>In fact, you do NOT increase resolution by means of deconvolution.
>Resolution is not a matter of software, it is something which is inherent
>to your optical system. What you do by deconvolution is to reduce the
>contribution by out-of-focus signals to the image of a certain focussing.
>This is why a properly adjusted confocal microscope with sufficiently small
>pinhole does have a better resolution than a widefield deconvolution
>system. Deconvolution does not ADD information to the raw data images, it
>substracts un-desired information.

My comment. For the most part this is true; however, as a practical
matter, out of focus light can seriously obscure detail. So, although
the spatial resolution one gets might not be theoretically better,
what one is able to actually visualize can be significantly improved
using deconvolution and the statistics can be far superior compared
to confocal because of the poor throughput of most confocals,
especially when you go to a very small pinhole and one is using red
emitting fluors. This is further exacerbated by bleaching problems
when using repeated scanning to reduce noise.


>
>>In order to do this effectively you
>>are supposed to use 0.17 micron beads ( balls) or 0.5 micron beads( balls)
>>with high power objectives to get a reference particle for the software.
>
>The size of the beads should be sub-resolution.

The size of the bead does not have to be sub-resolution to determine
the PSF. Even very large beads (10 um) have been used for this
purpose. Basically, you look at the spacing of the Airy disk minima
and maxima. The Bitplane software, which I have not used, may have
its own constraints.

>
>>While doing this test to acquire the necessary reference images from  the
>>0.17 u beads, they appeared as doublets( two beads) and the 0.5 micron
>>beads appeared egg shaped.
>
>This has to do with the fact, that you had your Nomarski prism installed,
>as you describe later on in your letter.

Most certainly this is the case. However, one can get particle
doubling if the mounting media is inappropriate. I once use an
acrylic polymer for mounting my 100 nm beads and got doubling. I
think this happened because the polymer formed an oriented crystal
like media that produced two refracted paths sort of like a calcite
crystal. It was annoying and fun to see at the same time. This was
with Wide Field imaging, by the way, not polarized laser illumination.

>
>>  What will this type of distortion observed on
>>beads do to the fluorescent images of cells and particles?
>
>It appears there just as it does for the spheres.

This assumes that the bead suspension media didn't compound the
problem. Cells might be different. But certainly, take out or the
prisms from the light path.

Thanks Johannes and Regards All,

Mario
--
_____________________________________________________________________
Mario M. Moronne, Ph.D.
NanoMed Technologies
ph (510) 528-2400
FAX (510) 528-8076
Berkeley, CA
94706

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