Re: PSF with DIC

Posted by McDonald, David L on
URL: http://confocal-microscopy-list.275.s1.nabble.com/PSF-with-DIC-tp3781952p3795775.html

This discussion prompted me to do a few simple
image quality tests on our Deltavision system,
Olympus IX70 scope.  One thing I found was if you
collect fluorescence and DIC separately, so no
DIC components are in the light path during
fluorescence acquisition, the DIC image is
noticeably shifted laterally and doesn't line up
with the fluorescent image.  Of course you can
collect all the channels together with the DIC
stuff in the light path and there is no shift
(didn't notice any significant image degradation,
will try some more involved tests) but
fluorescent intensities are reduced by about 70%,
mainly due to the analyzer on this system.

Dave

Dave McDonald
Scientific Imaging Lab
Fred Hutchinson Cancer Research Center
1100 Fairview Avenue North, DE-512
Seattle, WA 98109
206-667-4205
http://www.fhcrc.org


At 03:18 AM 10/9/2009, you wrote:

>When using DIC with a confocal setup, there is only one part which
>interferes with the laser/emission: the objective Nomarski prism. The two
>other parts (condenser Nomarski prism, analyzer) sit in the condenser
>which is not part of the confocal beam path. A polarizer is not necessary,
>since the laser is already polarized.
>
>In Zeiss setups, you put the objective prism manually in a slit below the
>objective. And yes, in my experience it does degrade image quality. This
>is especially true if you are working at the resolution limit. For best
>image quality, you have to remove the prism. That's actually one thing I
>like at the Leica stands (i.e. DMI6000): the prism is motorized and can be
>moved out automatically between two images, which means you can set up a
>time-lapse with DIC and fluorescence images w/o the latter being worse
>than necessary.
>
>Michael
>
>
> >            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
> >  --
> >       Runions signature        (Sent from my cra%#y non-Blackberry
> > electronic device that still has wires) Â
> > *********************************
> >  John Runions, Ph.D.
> >  School of Life Sciences
> >  Oxford Brookes University
> >  Oxford, UK
> >  OX3 0BP
> >
> >  email:Â  [hidden email]
> >  phone: [phone] Runions’ lab web site   Visit The
> > Illuminated Plant Cell dot com
> >  Oxford Brookes Master's in Bioimaging with Molecular Technology