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----- Original Message -----
From: John Runions <[hidden email]>
To: [hidden email] <[hidden email]>
Sent: Fri Oct 09 01:13:58 2009
Subject: Re: PSF with DIC
Hi All,
After reading Ian and Robert's comments, I appreciate that there might be degradation of the PSF if DIC optics are in the confocal image forming pathway. I am just a bit confused about which optical parts should be removed. Different manufacturers have different names for equivalent bits. I usually think of there being four components in the image forming pathway for DIC - two polarisers, and two DIC prisms. These have various names depending on who you talk to, e.g. analyser, Wollaston prism etc.
My question is Zeiss specific. In their microscopes, there is a piece of glass that I call the objective prism in the back focal plane of the objective. Will it affect the PSF of confocal images. It is a fiddly and expensive bit to remove and I worry about doing so if there is not going to be image degradation.
Thanks for your help, John.
Ian Dobbie wrote:
[hidden email] writes:
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 μ m, PSF beads) and a distortion of
larger particles. PSF beads show two spots and 0.5 μm 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.
On my first reading of this I thought by interference contrast filter
Robert was referring to the polariser. On a second reading I realise
that it refers to the DIC prism. I wrote this extended reply before
realising that we are saying the same thing but I am posting this
anyway as a second description might help people understand what is
going on and why this happens.
DIC works by sheering the two polarizations relative to each
other with the condenser prism. The beams then pass through slightly
different sections of the sample, and are recombined with the second
(objective) prism. This produces an image of relative phase shift
between the two beams.
In epi-fluorescence the excitation beam passes through the DIC
(objective) prism and is split into two beams, offset relative to one
another. The fluorescence from these two regions is them shifted back
as the emission passes back through the (objective) DIC prism. This
produces a double image shifted by the sheer in the DIC prism. The
sheer tends to be a fraction of the resolution, say 1/3rd but varies
with lens, manufacture etc... In conventional wide field this is
generally not noticeable. On a properly set up confocal this leads to
a pronounced broadening of the PSF in the sheer direction, at 45
degrees to the x and y sample axis.
As Robert says, the take home message is it is best to remove any DIC
optics before taking confocal images.
Ian
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John Runions, Ph.D.
School of Life Sciences
Oxford Brookes University
Oxford, UK
OX3 0BP
email: [hidden email]
phone: +44 (0) 1865 483 964
Runions’ lab web site <http://www.brookes.ac.uk/lifesci/runions/HTMLpages/index.html%21>
Visit The Illuminated Plant Cell <http://www.illuminatedcell.com/ER.html> dot com
Oxford Brookes Master's in Bioimaging with Molecular Technology <http://www.brookes.ac.uk/studying/courses/postgraduate/2007/bmt>
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