The condenser does not 'serve as an objective' - the objective is still
the image-forming lens.
Using a higher NA condenser will give you a brighter image, and higher
resolution, since this is classic Abbe imaging - just the ray paths are
reversed. For the same reason setting up correct Koehler imaging is
crucial.
The main reasons people get poor DIC imaging from a confocal are:
1. not filling the BFP of the objective (this will have a much greater
effect on the DIC image than the confocal one)
2. using a low NA condenser
3. not adjusting the condenser correctly
Guy
Optical Imaging Techniques in Cell Biology
by Guy Cox CRC Press / Taylor & Francis
http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
______________________________________________
Phone +61 2 9351 3176 Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
http://www.guycox.net
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]]
On Behalf Of MODEL, MICHAEL
Sent: Wednesday, 14 October 2009 11:08 PM
To: [log in to unmask]
Subject: Re: PSF with DIC
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
[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.
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