Mike
We have seen this effect on Leica, Nikon, and Zeiss confocal
microscopes. There is an incompatibly between the optics of a
confocal microscope and the optics to generate DIC images
(described below). A short description of the observation has
been published in a review article on Evaluation of Confocal
Performance(1). For optimum resolution it is essential to remove
the interference DIC filters from the light path. I have included
the description below from the review article. There is also an
image of PSF beads and 0.5 beads in the article.
Please contact me for additional information.
Best wishes
Bob
1. Zucker, R.M. Evaluation of Confocal Microscopy System Performance.
Cell Imaging Techniques. Douglas Taajets editor Humana Press
Chapter 5 77-135 2005
4.23. Interference Contrast and Confocal
Interference contrast is a very useful parameter in microscopy and it
can be
combined with fluorescence. However, because the microscope system was
designed for light to traverse through two interference filters, when
this optical
system is applied to a confocal microscope there is distortion in the
fluorescence
signals. The fluorescent light traverses the interference contrast
filter and
excites the sample, and then the emitted fluorescence travels back down
through the same interference contrast filter and back through the scan
head.
The resulting image shows a duplication of very small particles (0.17 ì
m, PSF
beads) and a distortion of larger particles. PSF beads show two spots
and 0.5 ìm
beads show an egg shaped image instead of a round image. The same
distortion
that is observed on beads will occur on biological structures in cells (
see Fig.
15). For optimum resolution of data that will be deconvoluted later, it
is recommended
to remove the interference filters when acquiring an image.
Robert M. Zucker, PhD
U.S. Environmental Protection Agency
Office of Research and Development
National Health and Environmental Effects Research Laboratory.
Toxicology Assessment Division
Telephone: 919-541-1585 Fax: 919-541-4017
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Mail address: USEPA,ORD,NHEERL,TAD
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2525 E.NC Highway 54
Durham, NC, 27713
From: "MODEL, MICHAEL" <[log in to unmask]>
To: [log in to unmask]
Date: 10/07/2009 09:43 AM
Subject: PSF with DIC
Sent by: Confocal Microscopy List <[log in to unmask]>
While I'm here...has anyone properly investigated the effect of the DIC
objective prism in confocal fluorescence imaging? I had always assumed
(rightly or wrongly) that it's presence didn't influence the PSF, but
last week I was imaging some subresolution beads and found that,
particularly on our IX81-based FV1000 confocals, the DIC objective prism
had quite a pronounced effect on the psf. Specifically the psf was
distorted along a diagonal axis and at the point of focus, the bead
appeared significantly larger with the prism in place. The implication
of this is that for confocal fluorescence imaging, the resolution of the
microscope is reduced when the DIC objective prism is in place. I've
also looked on our Zeiss Axiovert 200 and Nikon TE-2000 based systems
which employ a slightly different method of DIC and there the effect is
much less pronounced although noticeable.
Simon
Simon - thanks for your input on the noise issue - and as for the DIC
prism, I haven't looked at the PSF but have noticed slight deterioration
in resolution of fluorescent details, and try to remember to take it out
when not needed. (Besides, the handle gets on the way of our Prior stage
and has been broken several times).
Mike
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