Hi all,
Yep the confocal is pretty poor at imaging with transmission DIC, not helped
by the laser point scanning system that often leaves lines all across the
image [the engineers always blame mains interference, but I expect it's part
and parcel of the line scanning galvo mirror] - I suppose at least you get
the transmission image for free, light wise, when you scan the FITC channel.
But as you say feeding the light back in reverse through the condenser
doesn't help either, so the image is never as good as you see down the
eye-pieces or via a dedicated CCD camera on one of the microscopes ports
with standard halogen bulb illumination [in fact confocal transmission
images are really rather poor a lot of the time - or rather 'not of
publication quality' anyway - and many users give up on it and stick to
fluorescence only]. A bit of optical zoom often helps though I suppose - but
the image quality via a standard bright-field/phase contrast microscope with
a CCD camera is in another league.
Keith
---------------------------------------------------------------------------
Dr Keith J. Morris,
Molecular Cytogenetics and Microscopy Core,
Laboratory 00/069 and 00/070,
The Wellcome Trust Centre for Human Genetics,
Roosevelt Drive,
Oxford OX3 7BN,
United Kingdom.
Telephone: +44 (0)1865 287568
Email: [log in to unmask]
Web-pages: http://www.well.ox.ac.uk/molecular-cytogenetics-and-microscopy
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On
Behalf Of Ian Dobbie
Sent: 13 October 2009 02:08
To: [log in to unmask]
Subject: Re: PSF with DIC
John Runions <[log in to unmask]> writes:
> 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.
In general you do need these 4 components but laser scanning confocals
are a bit of a special case as the lasers are already polarised so you
can get away without one of the polarisers. The LSM's do DIC a bit
differently than most microscope as they use the polarised laser as
the input beam, split it in the prism before the objective, pass the
light through the sample, recombine the beams in the condenser and
then have a detector after the condenser. So they basically use the
microscope backwards, illuminating through the objective and detecting
through the condenser.
> 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.
The little slider under the objective on the Zeiss scopes is the DIC
prism that is splitting the two polarisations. You need to remove
this to optimise your images. It should have come with a little black
plastic case to put it in when its not on the scope.
Ian
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