Hi all.
I agree with Keith, but would just like to add a bit more about why
the lines are often more evident in the DIC image.
Basically, DIC is a fairly low contrast technique but (compared to
fluorescence), the signal level (in photons/pixel) is usually very
high and the background isn't black (one can't see variations in an
absence of signal).
Consequently, laser instability (perhaps 1-2%) that is not
perceptible in fluorescence where the signal level is almost always
less than 100 photons/pixel. Under these conditions, Poisson Noise
(>10%) swamps out the laser (or fiber coupling) instability. This
instability becomes visible in DIC because the signal level may be
10-100x higher and the background isn't a zero signal area.
The point is that the laser/fiber-coupling instability is probably
always there and in some cases, it might be worth reducing it.
Cheers,
Jim Pawley
>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
--
**********************************************
Prof. James B. Pawley, Ph. 608-263-3147
Room 223, Zoology Research Building,
FAX 608-265-5315
1117 Johnson Ave., Madison, WI, 53706
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3D Microscopy of Living Cells Course, June 13-24, 2009, UBC, Vancouver Canada
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"If it ain't diffraction, it must be statistics." Anon.
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