The other consideration is that in confocal, the illumination comes down
through the objective, then specimen, then condenser, then to the
transmitted light detector, at least it does on our Leica SP2 - so the
objective is acting as condenser and vice versa. And because the laser
illumination is polarised, we only use the condenser polariser, of course,
which has to be oriented against the polarisation of the laser, which is not
necessarily at the same angle as in the analyser above the objectives.
Also, at least on our instrument, you only have true Kohler in one position
of the stage plus condenser - as soon as you do a z-stack, you lose true
Kohler except in one slice of the stack.
cheers, Rosemary
Rosemary White
CSIRO Plant Industry
GPO Box 1600
Canberra, ACT 2601
Australia
ph 61 2 6246 5475
fx 61 2 6246 5334
On 15/10/09 2:17 AM, "Guy Cox" <[log in to unmask]> wrote:
> Perfectly true. But since we are forming images point by point it is
> the spot formed by the objective that primarily determines the
> resolution, and forms the image. But the condenser needs to be able to
> collect the wide-angle diffracted rays to give the full resolution.
> (Just as in widefield imaging if we reduce the condenser NA we move
> towards only half the maximal resolution given by the objective).
>
> 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: Thursday, 15 October 2009 1:06 AM
> To: [log in to unmask]
> Subject: Re: PSF with DIC
>
> Could you please explain? I thought that the objective, by definition,
> is the lens that collects light from the sample and condenser is what
> illuminates it...
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[log in to unmask]]
> On Behalf Of Guy Cox
> Sent: Wednesday, October 14, 2009 10:59 AM
> To: [log in to unmask]
> Subject: Re: PSF with DIC
>
> 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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