>In my experience, when everything works properly, laser scanning DIC
>or bright field can be about as good as with normal illumination.
>Not sure why, since the condenser (which serves as an objective in
>this case) has a much lower NA, 0.55 on our scope. Maybe the absence
>of chromatic aberrations with monochromatic illumination partially
>compensates for NA?
>
>Mike
You might be right Michael. Zeiss used to specify that their DIC
worked best at only one wavelength: the green line of the mercury arc
and they used to sell the "interference green" filter to select it.
Jim P.
>-----Original Message-----
>From: Confocal Microscopy List
>[mailto:[log in to unmask]] On Behalf Of James Pawley
>Sent: Wednesday, October 14, 2009 8:50 AM
>To: [log in to unmask]
>Subject: Re: PSF with DIC
>
>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
>[log in to unmask]
>3D Microscopy of Living Cells Course, June 13-24, 2009, UBC, Vancouver Canada
>Info: http://www.3dcourse.ubc.ca/ Applications due by March 15, 2009
> "If it ain't diffraction, it must be statistics." Anon.
--
**********************************************
Prof. James B. Pawley, Ph. 608-263-3147
Room 223, Zoology Research Building,
FAX 608-265-5315
1117 Johnson Ave., Madison, WI, 53706
[log in to unmask]
3D Microscopy of Living Cells Course, June 13-25, 2009, UBC, Vancouver Canada
Info: http://www.3dcourse.ubc.ca/ Applications due by March 15, 2009
"If it ain't diffraction, it must be statistics." Anon.
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