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
-----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
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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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