PSF with DIC

[Donnelly, Tom]

The analyzer on the DeltaVision is in the ex filterwheel and is rotated out of the light path during fluoresence imaging.

If there is an analyzer in the path, consider contacting service or applications.

Tom
--------------------------
Sent using BlackBerry

This message was sent via Blackberry.

----- Original Message -----
From: Dave McDonald <[hidden email]>
To: [hidden email] <[hidden email]>
Sent: Fri Oct 09 09:25:42 2009
Subject: Re: PSF with DIC

This discussion prompted me to do a few simple
image quality tests on our Deltavision system,
Olympus IX70 scope.  One thing I found was if you
collect fluorescence and DIC separately, so no
DIC components are in the light path during
fluorescence acquisition, the DIC image is
noticeably shifted laterally and doesn't line up
with the fluorescent image.  Of course you can
collect all the channels together with the DIC
stuff in the light path and there is no shift
(didn't notice any significant image degradation,
will try some more involved tests) but
fluorescent intensities are reduced by about 70%,
mainly due to the analyzer on this system.

Dave

Dave McDonald
Scientific Imaging Lab
Fred Hutchinson Cancer Research Center
1100 Fairview Avenue North, DE-512
Seattle, WA 98109
206-667-4205
http://www.fhcrc.org


At 03:18 AM 10/9/2009, you wrote:
>When using DIC with a confocal setup, there is only one part which
>interferes with the laser/emission: the objective Nomarski prism. The two
>other parts (condenser Nomarski prism, analyzer) sit in the condenser
>which is not part of the confocal beam path. A polarizer is not necessary,
>since the laser is already polarized.
>
>In Zeiss setups, you put the objective prism manually in a slit below the
>objective. And yes, in my experience it does degrade image quality. This
>is especially true if you are working at the resolution limit. For best
>image quality, you have to remove the prism. That's actually one thing I
>like at the Leica stands (i.e. DMI6000): the prism is motorized and can be
>moved out automatically between two images, which means you can set up a
>time-lapse with DIC and fluorescence images w/o the latter being worse
>than necessary.
>
>Michael
>
>
> >            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.
> >
> >  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.
> >
> >  Thanks for your help, John.
> >
> >
> >  Ian Dobbie wrote:    [hidden email] writes:
> > 4.23. Interference Contrast and Confocal
> > Interference contrast is a very useful parameter in microscopy and it can
> > be combined with fluorescence. However, because the microscope system was
> > designed for light to traverse through two interference filters, when
> > this optical system is applied to a confocal microscope there is
> > distortion in the fluorescence signals. The fluorescent light traverses
> > the interference contrast filter and excites the sample, and then the
> > emitted fluorescence travels back down through the same interference
> > contrast filter and back through the scan head.  The resulting image
> > shows a duplication of very small particles (0.17 μ m, PSF beads) and a
> > distortion of larger particles. PSF beads show two spots and 0.5 μm
> > beads show an egg shaped image instead of a round image. The same
> > distortion that is observed on beads will occur on biological structures
> > in cells ( see Fig.  15). For optimum resolution of data that will be
> > deconvoluted later, it is recommended to remove the interference filters
> > when acquiring an image.            On my first reading of this I thought
> > by interference contrast filter Robert was referring to the polariser. On
> > a second reading I realise that it refers to the DIC prism. I wrote this
> > extended reply before realising that we are saying the same thing but I
> > am posting this anyway as a second description might help people
> > understand what is going on and why this happens.   DIC works by sheering
> > the two polarizations relative to each other with the condenser prism.
> > The beams then pass through slightly different sections of the sample,
> > and are recombined with the second (objective) prism. This produces an
> > image of relative phase shift between the two beams.  In epi-fluorescence
> > the excitation beam passes through the DIC (objective) prism and is split
> > into two beams, offset relative to one another. The fluorescence from
> > these two regions is them shifted back as the emission passes back
> > through the (objective) DIC prism. This produces a double image shifted
> > by the sheer in the DIC prism. The sheer tends to be a fraction of the
> > resolution, say 1/3rd but varies with lens, manufacture etc... In
> > conventional wide field this is generally not noticeable. On a properly
> > set up confocal this leads to a pronounced broadening of the PSF in the
> > sheer direction, at 45 degrees to the x and y sample axis.  As Robert
> > says, the take home message is it is best to remove any DIC optics before
> > taking confocal images.  Ian
> >  --
> >       Runions signature        (Sent from my cra%#y non-Blackberry
> > electronic device that still has wires) Â
> > *********************************
> >  John Runions, Ph.D.
> >  School of Life Sciences
> >  Oxford Brookes University
> >  Oxford, UK
> >  OX3 0BP
> >
> >  email:  [hidden email]
> >  phone: [phone] Runions’ lab web site   Visit The
> > Illuminated Plant Cell dot com
> >  Oxford Brookes Master's in Bioimaging with Molecular Technology

This email message, together with any attachments, is for the sole use of the intended recipient(s) and is the confidential information of Applied Precision Inc. If you are not the intended recipient, your review, use, disclosure, copying or dissemination of this email message or its attachments, or the information contained therein, is strictly prohibited. If you are not the intended recipient or if you think this email was sent to you in error, please notify the sender by reply email and delete this message and its attachments, as well as all copies, from your system.

[Robert Carter-2]

I use our lab's Deltavision (IX-70 base) almost exclusively for the live-cell
imaging.  I've already completed a good bit of hi-res imaging and fixed, stained
cells before proceeding to live-cell, time-lapse experiments.

The PSF is affected and resolution deteriorates when the DIC objective prism
is left in the light path during epi-fluorescence imaging.  That said, I leave the
objective prism removed for fixed-cell imaging because I only need the epi-
fluorescence. It's in place for live-cell work though because DIC is highly-
useful.

For live-cell, compromises always have to be made to cut down on the
phototoxicity.  DIC is no exception.  I am imaging HPV plasmids bound
inducibly by a GFP fusion peptide.  Some rules I learned from the instructors
and company reps at the 2002 Cold Spring Harbor course have served me very
well and yielded great success.  
  1)Use pixel binning - live-cell is about observing the dynamics of the  
     biological process.  2x2 binning allows is more than sufficient to
     corroborate fixed cell images and allow for accurate reporting of dynamic
     processes.
  2)During live-cell imaging, I collect the bright-field and fluorescent image
    channels through the GFP emission filter.  The bright-field image quality is
    just fine even though I'm not using the Analyser.  I might collect eight or
    ten z-plane images per cell per time point.  I am eliminating fifteen to
    nineteen emission filter changes in this instance.  This saves a lot of time
    and keeps the cells happier for longer.
  3)Use higher-intensity illumination together with shorter exposure times.  The
    imaging for each time point is completed sooner, cells have
    more "rest"/"recovery" time between time points, and they stay healthy for
    much longer.  This is crucial, especially more mitotic cells.  I can routinely
    image twelve to fifteen mitotic cells prior to the next time point's imaging
    pass.

I hate losing three-fourths of the signal so I take the objective prism out and
safely stow it until I need DIC.  I had my bosses put a keycode access lock on
our scope rooms.  Each user has his own code, and each time the user enters
the room, a person-specific log entry is generated.  It's amazing how much
less damage and destruction occurred once we implemented this policy.  Just
ensure that users are comfortable taking the multi-thousand dollar objective
prism out when DIC isn't required.

This is my first posting although I am a long-time subscriber to the confocal
list.

Hope this helps and isn't too redundant.

Best luck,

Rob Carter
Lab of Tom Broker and Louise Chow at UAB
505 MCLM Bldg
Birmingham, AL 35294
205-975-8304

[Rietdorf, Jens]

Dear,

though the prism does distort the image, I do not see how it would
reduce the NA, so basically the resolving power should be unaffected and
it would 'just' need a clever piece of de-distortion software to
restore. Has anyone of you tried to use a measured PSF to de-convolve
such a distorted image?

Thanks, jens

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Robert Carter
Sent: Saturday, October 10, 2009 12:05 PM
To: [hidden email]
Subject: Re: PSF with DIC

I use our lab's Deltavision (IX-70 base) almost exclusively for the
live-cell
imaging.  I've already completed a good bit of hi-res imaging and fixed,
stained
cells before proceeding to live-cell, time-lapse experiments.

The PSF is affected and resolution deteriorates when the DIC objective
prism
is left in the light path during epi-fluorescence imaging.  That said, I
leave the
objective prism removed for fixed-cell imaging because I only need the
epi-
fluorescence. It's in place for live-cell work though because DIC is
highly-
useful.

For live-cell, compromises always have to be made to cut down on the
phototoxicity.  DIC is no exception.  I am imaging HPV plasmids bound
inducibly by a GFP fusion peptide.  Some rules I learned from the
instructors
and company reps at the 2002 Cold Spring Harbor course have served me
very
well and yielded great success.  
  1)Use pixel binning - live-cell is about observing the dynamics of the

     biological process.  2x2 binning allows is more than sufficient to
     corroborate fixed cell images and allow for accurate reporting of
dynamic
     processes.
  2)During live-cell imaging, I collect the bright-field and fluorescent
image
    channels through the GFP emission filter.  The bright-field image
quality is
    just fine even though I'm not using the Analyser.  I might collect
eight or
    ten z-plane images per cell per time point.  I am eliminating
fifteen to
    nineteen emission filter changes in this instance.  This saves a lot
of time
    and keeps the cells happier for longer.
  3)Use higher-intensity illumination together with shorter exposure
times.  The
    imaging for each time point is completed sooner, cells have
    more "rest"/"recovery" time between time points, and they stay
healthy for
    much longer.  This is crucial, especially more mitotic cells.  I can
routinely
    image twelve to fifteen mitotic cells prior to the next time point's
imaging
    pass.

I hate losing three-fourths of the signal so I take the objective prism
out and
safely stow it until I need DIC.  I had my bosses put a keycode access
lock on
our scope rooms.  Each user has his own code, and each time the user
enters
the room, a person-specific log entry is generated.  It's amazing how
much
less damage and destruction occurred once we implemented this policy.
Just
ensure that users are comfortable taking the multi-thousand dollar
objective
prism out when DIC isn't required.

This is my first posting although I am a long-time subscriber to the
confocal
list.

Hope this helps and isn't too redundant.

Best luck,

Rob Carter
Lab of Tom Broker and Louise Chow at UAB
505 MCLM Bldg
Birmingham, AL 35294
205-975-8304

[Guy Cox]

I think you are probably right but .... why would you do this rather
than just pull the slider out?

                                  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 [phone]     Fax [phone]
Mobile [phone]
______________________________________________
     http://www.guycox.net
-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Rietdorf, Jens
Sent: Sunday, 11 October 2009 6:00 AM
To: [hidden email]
Subject: Re: PSF with DIC

Dear,

though the prism does distort the image, I do not see how it would
reduce the NA, so basically the resolving power should be unaffected and
it would 'just' need a clever piece of de-distortion software to
restore. Has anyone of you tried to use a measured PSF to de-convolve
such a distorted image?

Thanks, jens

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Robert Carter
Sent: Saturday, October 10, 2009 12:05 PM
To: [hidden email]
Subject: Re: PSF with DIC

I use our lab's Deltavision (IX-70 base) almost exclusively for the
live-cell imaging.  I've already completed a good bit of hi-res imaging
and fixed, stained cells before proceeding to live-cell, time-lapse
experiments.

The PSF is affected and resolution deteriorates when the DIC objective
prism is left in the light path during epi-fluorescence imaging.  That
said, I leave the objective prism removed for fixed-cell imaging because
I only need the
epi-
fluorescence. It's in place for live-cell work though because DIC is
highly-
useful.

For live-cell, compromises always have to be made to cut down on the
phototoxicity.  DIC is no exception.  I am imaging HPV plasmids bound
inducibly by a GFP fusion peptide.  Some rules I learned from the
instructors and company reps at the 2002 Cold Spring Harbor course have
served me very well and yielded great success.  
  1)Use pixel binning - live-cell is about observing the dynamics of the

     biological process.  2x2 binning allows is more than sufficient to
     corroborate fixed cell images and allow for accurate reporting of
dynamic
     processes.
  2)During live-cell imaging, I collect the bright-field and fluorescent
image
    channels through the GFP emission filter.  The bright-field image
quality is
    just fine even though I'm not using the Analyser.  I might collect
eight or
    ten z-plane images per cell per time point.  I am eliminating
fifteen to
    nineteen emission filter changes in this instance.  This saves a lot
of time
    and keeps the cells happier for longer.
  3)Use higher-intensity illumination together with shorter exposure
times.  The
    imaging for each time point is completed sooner, cells have
    more "rest"/"recovery" time between time points, and they stay
healthy for
    much longer.  This is crucial, especially more mitotic cells.  I can
routinely
    image twelve to fifteen mitotic cells prior to the next time point's
imaging
    pass.

I hate losing three-fourths of the signal so I take the objective prism
out and safely stow it until I need DIC.  I had my bosses put a keycode
access lock on our scope rooms.  Each user has his own code, and each
time the user enters the room, a person-specific log entry is generated.
It's amazing how much less damage and destruction occurred once we
implemented this policy.
Just
ensure that users are comfortable taking the multi-thousand dollar
objective prism out when DIC isn't required.

This is my first posting although I am a long-time subscriber to the
confocal list.

Hope this helps and isn't too redundant.

Best luck,

Rob Carter
Lab of Tom Broker and Louise Chow at UAB
505 MCLM Bldg
Birmingham, AL 35294
205-975-8304

[James Pawley]

>Dear,
>
>though the prism does distort the image, I do not see how it would
>reduce the NA, so basically the resolving power should be unaffected and
>it would 'just' need a clever piece of de-distortion software to
>restore. Has anyone of you tried to use a measured PSF to de-convolve
>such a distorted image?
>
>Thanks, jens


Certainly, you could do this (keeping in mind that the DIC distortion
of the PSF changes somewhat over the field of view). In addition, you
could not use the trick of rotational averaging that allows one to
reduce the statistical noise in the measured PSF. But even a
distorted PSF is still a PSF and can be used to deconvolve.

But remember, a distorted PSF (which at the very least comes about
from having every point-object seem to be located in two
partially-overlapped positions) is bigger than an undistorted one.
And losing resolution in this way can never be good.

Jim Pawley

>-----Original Message-----
>From: Confocal Microscopy List [mailto:[hidden email]]
>On Behalf Of Robert Carter
>Sent: Saturday, October 10, 2009 12:05 PM
>To: [hidden email]
>Subject: Re: PSF with DIC
>
>I use our lab's Deltavision (IX-70 base) almost exclusively for the
>live-cell
>imaging.  I've already completed a good bit of hi-res imaging and fixed,
>stained
>cells before proceeding to live-cell, time-lapse experiments.
>
>The PSF is affected and resolution deteriorates when the DIC objective
>prism
>is left in the light path during epi-fluorescence imaging.  That said, I
>leave the
>objective prism removed for fixed-cell imaging because I only need the
>epi-
>fluorescence. It's in place for live-cell work though because DIC is
>highly-
>useful.
>
>For live-cell, compromises always have to be made to cut down on the
>phototoxicity.  DIC is no exception.  I am imaging HPV plasmids bound
>inducibly by a GFP fusion peptide.  Some rules I learned from the
>instructors
>and company reps at the 2002 Cold Spring Harbor course have served me
>very
>well and yielded great success.
>   1)Use pixel binning - live-cell is about observing the dynamics of the
>
>      biological process.  2x2 binning allows is more than sufficient to
>      corroborate fixed cell images and allow for accurate reporting of
>dynamic
>      processes.
>   2)During live-cell imaging, I collect the bright-field and fluorescent
>image
>     channels through the GFP emission filter.  The bright-field image
>quality is
>     just fine even though I'm not using the Analyser.  I might collect
>eight or
>     ten z-plane images per cell per time point.  I am eliminating
>fifteen to
>     nineteen emission filter changes in this instance.  This saves a lot
>of time
>     and keeps the cells happier for longer.
>   3)Use higher-intensity illumination together with shorter exposure
>times.  The
>     imaging for each time point is completed sooner, cells have
>     more "rest"/"recovery" time between time points, and they stay
>healthy for
>     much longer.  This is crucial, especially more mitotic cells.  I can
>routinely
>     image twelve to fifteen mitotic cells prior to the next time point's
>imaging
>     pass.
>
>I hate losing three-fourths of the signal so I take the objective prism
>out and
>safely stow it until I need DIC.  I had my bosses put a keycode access
>lock on
>our scope rooms.  Each user has his own code, and each time the user
>enters
>the room, a person-specific log entry is generated.  It's amazing how
>much
>less damage and destruction occurred once we implemented this policy.
>Just
>ensure that users are comfortable taking the multi-thousand dollar
>objective
>prism out when DIC isn't required.
>
>This is my first posting although I am a long-time subscriber to the
>confocal
>list.
>
>Hope this helps and isn't too redundant.
>
>Best luck,
>
>Rob Carter
>Lab of Tom Broker and Louise Chow at UAB
>505 MCLM Bldg
>Birmingham, AL 35294
>205-975-8304

--
James and Christine Pawley, 21 N. Prospect Ave. Madison, WI, 53726
Phone: [phone]

[Keith Morris]

Hi all,

 

The small glass DIC prism directly below the objective normally just pulls out to completely remove it [it does in all our inverted Zeiss/Nikon/Leica microscopes], but it always seemed to me to have minimal effect on imaging [and so we tend to leave it in]. There’s certainly no comment by Zeiss about removing it during confocal imaging [it is ‘clearly’ optically transparent]. I’ll have to try imaging with and without it next time to reassure myself that is the case – if others can see a difference with it out of the path, it’s no doubt something to look out for. Would the effect be more marked with a cheaper Plan Neofluar or an expensive Plan Apochromat I wonder. As John suggests I wouldn’t want our users removing this DIC prism on a regular basis or we might never see it again.

 

The DIC Polaroid [analyzer] filter further below* this in the laser path is a rather different matter as the laser light itself is also polarized, so it could therefore adversely affect the laser light [being similar to the effect of rotating twin Polaroid filters – i.e. a Polarizer].  So it is the bottom Polaroid filter that is only required when viewing down the microscope with the objectives [as the halogen light isn’t polarized], but this Polaroid filter isn’t needed with polarized laser light DIC transmission or fluorescence confocal imaging. Indeed our Zeiss 510 automatically withdraws this Polaroid filter from the light path when the laser is on - with our Leica SP2 you had to remember to manually withdraw this filter. You still leave the other Polaroid filter & prism in on the condenser if you want DIC transmission images, and naturally these has no effect at all on epi-fluorescence in any case]. Nothing to do with the ‘prism’ thread, but this Polaroid filter bit of the DIC optics is worth keeping out of the confocal laser path.

 

Regards

 

Keith

 

*on inverted microscopes

 

---------------------------------------------------------------------------
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:  [phone]
Email:  [hidden email]

Web-pages: http://www.well.ox.ac.uk/molecular-cytogenetics-and-microscopy

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of John Runions
Sent: 09 October 2009 09:14
To: [hidden email]
Subject: Re: PSF with DIC

 

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.

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.

Thanks for your help, John.


Ian Dobbie wrote:

[hidden email] writes:

4.23. Interference Contrast and Confocal

Interference contrast is a very useful parameter in microscopy and

it can be combined with fluorescence. However, because the

microscope system was designed for light to traverse through two

interference filters, when this optical system is applied to a

confocal microscope there is distortion in the fluorescence

signals. The fluorescent light traverses the interference contrast

filter and excites the sample, and then the emitted fluorescence

travels back down through the same interference contrast filter and

back through the scan head.  The resulting image shows a duplication

of very small particles (0.17 μ m, PSF beads) and a distortion of

larger particles. PSF beads show two spots and 0.5 μm beads show an

egg shaped image instead of a round image. The same distortion that

is observed on beads will occur on biological structures in cells (

see Fig.  15). For optimum resolution of data that will be

deconvoluted later, it is recommended to remove the interference

filters when acquiring an image.

On my first reading of this I thought by interference contrast filter

Robert was referring to the polariser. On a second reading I realise

that it refers to the DIC prism. I wrote this extended reply before

realising that we are saying the same thing but I am posting this

anyway as a second description might help people understand what is

going on and why this happens. 

DIC works by sheering the two polarizations relative to each

other with the condenser prism. The beams then pass through slightly

different sections of the sample, and are recombined with the second

(objective) prism. This produces an image of relative phase shift

between the two beams.

In epi-fluorescence the excitation beam passes through the DIC

(objective) prism and is split into two beams, offset relative to one

another. The fluorescence from these two regions is them shifted back

as the emission passes back through the (objective) DIC prism. This

produces a double image shifted by the sheer in the DIC prism. The

sheer tends to be a fraction of the resolution, say 1/3rd but varies

with lens, manufacture etc... In conventional wide field this is

generally not noticeable. On a properly set up confocal this leads to

a pronounced broadening of the PSF in the sheer direction, at 45

degrees to the x and y sample axis.

As Robert says, the take home message is it is best to remove any DIC

optics before taking confocal images.

Ian

 

--

(Sent from my cra%#y non-Blackberry electronic device that still has wires)

 

*********************************
John Runions, Ph.D.
School of Life Sciences
Oxford Brookes University
Oxford, UK
OX3 0BP

email:  [hidden email]
phone: [phone]

Runions’ lab web site

 

Visit The Illuminated Plant Cell dot com
Oxford Brookes Master's in Bioimaging with Molecular Technology

[mmodel]

Dear Joachim - I tried to reproduce your experiment. If I pull the eyepiece out so that the intermediate image is behind its focal plane I see a really sharp image of a slide projected onto a piece of paper as you would expect. If I keep the eyepiece in the normal position where the intermediate image should be closer than the focal plane I also see an image but it’s blurry. I think the object on a slide also serves as a partial shield for direct, undiffracted light, which is supposed to converge somewhere behind the eyepiece, and I think that’s what we see.

 

Mike

 

Michael Model, Ph.D.

Confocal Microscopy,

Dpt Biological Sciences,

1275 University Esplanade,

Kent State University, Kent, OH 44242

tel. [phone]

 

[Glen MacDonald-2]

Any adjustment of the analyzer will also shift the distortion in a  
manner depending on the implementation of the DIC in any particular  
microscope.  Each adjustment requiring then its own PSF.

Regards,
Glen

Glen MacDonald
Core for Communication Research
Virginia Merrill Bloedel Hearing Research Center
Box 357923
University of Washington
Seattle, WA 98195-7923  USA
[phone]
[hidden email]

******************************************************************************
The box said "Requires WindowsXP or better", so I bought a Macintosh.
******************************************************************************


On Oct 10, 2009, at 1:36 PM, James Pawley wrote:

>> Dear,
>>
>> though the prism does distort the image, I do not see how it would
>> reduce the NA, so basically the resolving power should be  
>> unaffected and
>> it would 'just' need a clever piece of de-distortion software to
>> restore. Has anyone of you tried to use a measured PSF to de-convolve
>> such a distorted image?
>>
>> Thanks, jens
>
>
> Certainly, you could do this (keeping in mind that the DIC  
> distortion of the PSF changes somewhat over the field of view). In  
> addition, you could not use the trick of rotational averaging that  
> allows one to reduce the statistical noise in the measured PSF. But  
> even a distorted PSF is still a PSF and can be used to deconvolve.
>
> But remember, a distorted PSF (which at the very least comes about  
> from having every point-object seem to be located in two partially-
> overlapped positions) is bigger than an undistorted one. And losing  
> resolution in this way can never be good.
>
> Jim Pawley
>
>
>> -----Original Message-----
>> From: Confocal Microscopy List  
>> [mailto:[hidden email]]
>> On Behalf Of Robert Carter
>> Sent: Saturday, October 10, 2009 12:05 PM
>> To: [hidden email]
>> Subject: Re: PSF with DIC
>>
>> I use our lab's Deltavision (IX-70 base) almost exclusively for the
>> live-cell
>> imaging.  I've already completed a good bit of hi-res imaging and  
>> fixed,
>> stained
>> cells before proceeding to live-cell, time-lapse experiments.
>>
>> The PSF is affected and resolution deteriorates when the DIC  
>> objective
>> prism
>> is left in the light path during epi-fluorescence imaging.  That  
>> said, I
>> leave the
>> objective prism removed for fixed-cell imaging because I only need  
>> the
>> epi-
>> fluorescence. It's in place for live-cell work though because DIC is
>> highly-
>> useful.
>>
>> For live-cell, compromises always have to be made to cut down on the
>> phototoxicity.  DIC is no exception.  I am imaging HPV plasmids bound
>> inducibly by a GFP fusion peptide.  Some rules I learned from the
>> instructors
>> and company reps at the 2002 Cold Spring Harbor course have served me
>> very
>> well and yielded great success.   1)Use pixel binning - live-cell  
>> is about observing the dynamics of the
>>
>>     biological process.  2x2 binning allows is more than sufficient  
>> to
>>     corroborate fixed cell images and allow for accurate reporting of
>> dynamic
>>     processes.
>>  2)During live-cell imaging, I collect the bright-field and  
>> fluorescent
>> image
>>    channels through the GFP emission filter.  The bright-field image
>> quality is
>>    just fine even though I'm not using the Analyser.  I might collect
>> eight or
>>    ten z-plane images per cell per time point.  I am eliminating
>> fifteen to
>>    nineteen emission filter changes in this instance.  This saves a  
>> lot
>> of time
>>    and keeps the cells happier for longer.
>>  3)Use higher-intensity illumination together with shorter exposure
>> times.  The
>>    imaging for each time point is completed sooner, cells have
>>    more "rest"/"recovery" time between time points, and they stay
>> healthy for
>>    much longer.  This is crucial, especially more mitotic cells.  I  
>> can
>> routinely
>>    image twelve to fifteen mitotic cells prior to the next time  
>> point's
>> imaging
>>    pass.
>>
>> I hate losing three-fourths of the signal so I take the objective  
>> prism
>> out and
>> safely stow it until I need DIC.  I had my bosses put a keycode  
>> access
>> lock on
>> our scope rooms.  Each user has his own code, and each time the user
>> enters
>> the room, a person-specific log entry is generated.  It's amazing how
>> much
>> less damage and destruction occurred once we implemented this policy.
>> Just
>> ensure that users are comfortable taking the multi-thousand dollar
>> objective
>> prism out when DIC isn't required.
>>
>> This is my first posting although I am a long-time subscriber to the
>> confocal
>> list.
>>
>> Hope this helps and isn't too redundant.
>>
>> Best luck,
>>
>> Rob Carter
>> Lab of Tom Broker and Louise Chow at UAB
>> 505 MCLM Bldg
>> Birmingham, AL 35294
>> 205-975-8304
>
>
> --
> James and Christine Pawley, 21 N. Prospect Ave. Madison, WI, 53726  
> Phone: [phone]

[Paul Maddox]

It should be noted that so called “short sheer” prisms are offered by several manufacturers (maybe all?) meant to be more compatible with fluorescence imaging.  These prisms sheer the two beamlets by a shorter distance than standard prisms resulting in less spread of the PSF.  Obviously the contrast generated in DIC will be less (although the resolution fractionally higher), but this can be usually be compensated for by post-processing.  If you are using DIC and fluorescence, they are worth a test drive.  

I also feel compelled to second comments made on this thread that, if at all possible, the analyzer should be moved (via a wheel or cube changer) for fluorescence imaging.

Paul

Paul S. Maddox, PhD
Assistant Professor
Institute for Research in Immunology and Cancer
Dept of Pathology and Cell Biol, U. de Montreal
P.O. Box 6128, Station Centre-Ville
Montréal QC  H3C 3J7
CANADA

Courier:
2900, boulevard Édouard-Montpetit
Pavillon Marcelle-Coutu, Quai 20
Montreal QC  H3T 1J4
CANADA

paul.maddox@...
Ph: 514-343-7894
Fax: [phone]




On 12/10/09 9:32 AM, "Keith Morris" <kjmorris@...> wrote:

Hi all,
 
The small glass DIC prism directly below the objective normally just pulls out to completely remove it [it does in all our inverted Zeiss/Nikon/Leica microscopes], but it always seemed to me to have minimal effect on imaging [and so we tend to leave it in]. There’s certainly no comment by Zeiss about removing it during confocal imaging [it is ‘clearly’ optically transparent]. I’ll have to try imaging with and without it next time to reassure myself that is the case – if others can see a difference with it out of the path, it’s no doubt something to look out for. Would the effect be more marked with a cheaper Plan Neofluar or an expensive Plan Apochromat I wonder. As John suggests I wouldn’t want our users removing this DIC prism on a regular basis or we might never see it again.
 
The DIC Polaroid [analyzer] filter further below* this in the laser path is a rather different matter as the laser light itself is also polarized, so it could therefore adversely affect the laser light [being similar to the effect of rotating twin Polaroid filters – i.e. a Polarizer].  So it is the bottom Polaroid filter that is only required when viewing down the microscope with the objectives [as the halogen light isn’t polarized], but this Polaroid filter isn’t needed with polarized laser light DIC transmission or fluorescence confocal imaging. Indeed our Zeiss 510 automatically withdraws this Polaroid filter from the light path when the laser is on - with our Leica SP2 you had to remember to manually withdraw this filter. You still leave the other Polaroid filter & prism in on the condenser if you want DIC transmission images, and naturally these has no effect at all on epi-fluorescence in any case]. Nothing to do with the ‘prism’ thread, but this Polaroid filter bit of the DIC optics is worth keeping out of the confocal laser path.
 
Regards
 
Keith
 
*on inverted microscopes
 

---------------------------------------------------------------------------
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:  [phone]
Email:  kjmorris@...
Web-pages: http://www.well.ox.ac.uk/molecular-cytogenetics-and-microscopy


From: Confocal Microscopy List [[hidden email]] On Behalf Of John Runions
Sent: 09 October 2009 09:14
To: CONFOCALMICROSCOPY@...
Subject: Re: PSF with DIC

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.

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.

Thanks for your help, John.


Ian Dobbie wrote:
Zucker.Robert@... writes:
 
 

4.23. Interference Contrast and Confocal
   

 

Interference contrast is a very useful parameter in microscopy and
it can be combined with fluorescence. However, because the
microscope system was designed for light to traverse through two
interference filters, when this optical system is applied to a
confocal microscope there is distortion in the fluorescence
signals. The fluorescent light traverses the interference contrast
filter and excites the sample, and then the emitted fluorescence
travels back down through the same interference contrast filter and
back through the scan head.  The resulting image shows a duplication
of very small particles (0.17 ? m, PSF beads) and a distortion of
larger particles. PSF beads show two spots and 0.5 ?m beads show an
egg shaped image instead of a round image. The same distortion that
is observed on beads will occur on biological structures in cells (
see Fig.  15). For optimum resolution of data that will be
deconvoluted later, it is recommended to remove the interference
filters when acquiring an image.
   

On my first reading of this I thought by interference contrast filter
Robert was referring to the polariser. On a second reading I realise
that it refers to the DIC prism. I wrote this extended reply before
realising that we are saying the same thing but I am posting this
anyway as a second description might help people understand what is
going on and why this happens.
 
DIC works by sheering the two polarizations relative to each
other with the condenser prism. The beams then pass through slightly
different sections of the sample, and are recombined with the second
(objective) prism. This produces an image of relative phase shift
between the two beams.
 
In epi-fluorescence the excitation beam passes through the DIC
(objective) prism and is split into two beams, offset relative to one
another. The fluorescence from these two regions is them shifted back
as the emission passes back through the (objective) DIC prism. This
produces a double image shifted by the sheer in the DIC prism. The
sheer tends to be a fraction of the resolution, say 1/3rd but varies
with lens, manufacture etc... In conventional wide field this is
generally not noticeable. On a properly set up confocal this leads to
a pronounced broadening of the PSF in the sheer direction, at 45
degrees to the x and y sample axis.
 
As Robert says, the take home message is it is best to remove any DIC
optics before taking confocal images.
 
Ian
 

[Ian Dobbie]

John Runions <[hidden email]> 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

[Keith Morris]

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:  [phone]
Email:  [hidden email]
Web-pages: http://www.well.ox.ac.uk/molecular-cytogenetics-and-microscopy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Ian Dobbie
Sent: 13 October 2009 02:08
To: [hidden email]
Subject: Re: PSF with DIC

John Runions <[hidden email]> 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

[James Pawley]

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:  [phone]
>Email:  [hidden email]
>Web-pages: http://www.well.ox.ac.uk/molecular-cytogenetics-and-microscopy
>
>-----Original Message-----
>From: Confocal Microscopy List [mailto:[hidden email]] On
>Behalf Of Ian Dobbie
>Sent: 13 October 2009 02:08
>To: [hidden email]
>Subject: Re: PSF with DIC
>
>John Runions <[hidden email]> 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  [phone]
1117 Johnson Ave., Madison, WI, 53706  
[hidden email]
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.

[mmodel]

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:[hidden email]] On Behalf Of James Pawley
Sent: Wednesday, October 14, 2009 8:50 AM
To: [hidden email]
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:  [phone]
>Email:  [hidden email]
>Web-pages: http://www.well.ox.ac.uk/molecular-cytogenetics-and-microscopy
>
>-----Original Message-----
>From: Confocal Microscopy List [mailto:[hidden email]] On
>Behalf Of Ian Dobbie
>Sent: 13 October 2009 02:08
>To: [hidden email]
>Subject: Re: PSF with DIC
>
>John Runions <[hidden email]> 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  [phone]
1117 Johnson Ave., Madison, WI, 53706
[hidden email]
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.

[Sylvie Le Guyader-2]

I have also taken very nice DIC images on our LSM510. Of course the
condenser needs to be Koehlered properly. I often find that the best images
are done with the T-PMT set to a rather low offset and even lower gain.

Med vänlig hälsning / Best regards
 
Sylvie
 
@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader
Dept of Biosciences and Nutrition
Karolinska Institutet
Novum
14157 Huddinge
Sweden
[phone]

> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of MODEL,
> MICHAEL
> Sent: 14 October 2009 15:08
> To: [hidden email]
> 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:[hidden email]] On Behalf Of James
> Pawley
> Sent: Wednesday, October 14, 2009 8:50 AM
> To: [hidden email]
> 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:  [phone]
> >Email:  [hidden email]
> >Web-pages: http://www.well.ox.ac.uk/molecular-cytogenetics-and-microscopy
> >
> >-----Original Message-----
> >From: Confocal Microscopy List
> [mailto:[hidden email]] On
> >Behalf Of Ian Dobbie
> >Sent: 13 October 2009 02:08
> >To: [hidden email]
> >Subject: Re: PSF with DIC
> >
> >John Runions <[hidden email]> 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  [phone]
> 1117 Johnson Ave., Madison, WI, 53706
> [hidden email]
> 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.

[James Pawley]

>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:[hidden email]] On Behalf Of James Pawley
>Sent: Wednesday, October 14, 2009 8:50 AM
>To: [hidden email]
>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:  [phone]
>>Email:  [hidden email]
>>Web-pages: http://www.well.ox.ac.uk/molecular-cytogenetics-and-microscopy
>>
>>-----Original Message-----
>>From: Confocal Microscopy List [mailto:[hidden email]] On
>>Behalf Of Ian Dobbie
>>Sent: 13 October 2009 02:08
>>To: [hidden email]
>>Subject: Re: PSF with DIC
>>
>>John Runions <[hidden email]> 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  [phone]
>1117 Johnson Ave., Madison, WI, 53706
>[hidden email]
>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  [phone]
1117 Johnson Ave., Madison, WI, 53706  
[hidden email]
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.

[Guy Cox]

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 [phone]     Fax [phone]
Mobile [phone]
______________________________________________
     http://www.guycox.net
-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of MODEL, MICHAEL
Sent: Wednesday, 14 October 2009 11:08 PM
To: [hidden email]
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:[hidden email]]
On Behalf Of James Pawley
Sent: Wednesday, October 14, 2009 8:50 AM
To: [hidden email]
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:  [phone]
>Email:  [hidden email]
>Web-pages:
>http://www.well.ox.ac.uk/molecular-cytogenetics-and-microscopy
>
>-----Original Message-----
>From: Confocal Microscopy List
>[mailto:[hidden email]] On Behalf Of Ian Dobbie
>Sent: 13 October 2009 02:08
>To: [hidden email]
>Subject: Re: PSF with DIC
>
>John Runions <[hidden email]> 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  [phone]
1117 Johnson Ave., Madison, WI, 53706
[hidden email]
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.

[mmodel]

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:[hidden email]] On Behalf Of Guy Cox
Sent: Wednesday, October 14, 2009 10:59 AM
To: [hidden email]
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 [phone]     Fax [phone]
Mobile [phone]
______________________________________________
     http://www.guycox.net
-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of MODEL, MICHAEL
Sent: Wednesday, 14 October 2009 11:08 PM
To: [hidden email]
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:[hidden email]]
On Behalf Of James Pawley
Sent: Wednesday, October 14, 2009 8:50 AM
To: [hidden email]
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:  [phone]
>Email:  [hidden email]
>Web-pages:
>http://www.well.ox.ac.uk/molecular-cytogenetics-and-microscopy
>
>-----Original Message-----
>From: Confocal Microscopy List
>[mailto:[hidden email]] On Behalf Of Ian Dobbie
>Sent: 13 October 2009 02:08
>To: [hidden email]
>Subject: Re: PSF with DIC
>
>John Runions <[hidden email]> 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  [phone]
1117 Johnson Ave., Madison, WI, 53706
[hidden email]
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.

[Guy Cox]

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 [phone]     Fax [phone]
Mobile [phone]
______________________________________________
     http://www.guycox.net
-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of MODEL, MICHAEL
Sent: Thursday, 15 October 2009 1:06 AM
To: [hidden email]
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:[hidden email]]
On Behalf Of Guy Cox
Sent: Wednesday, October 14, 2009 10:59 AM
To: [hidden email]
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 [phone]     Fax [phone]
Mobile [phone]
______________________________________________
     http://www.guycox.net
-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of MODEL, MICHAEL
Sent: Wednesday, 14 October 2009 11:08 PM
To: [hidden email]
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:[hidden email]]
On Behalf Of James Pawley
Sent: Wednesday, October 14, 2009 8:50 AM
To: [hidden email]
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:  [phone]
>Email:  [hidden email]
>Web-pages:
>http://www.well.ox.ac.uk/molecular-cytogenetics-and-microscopy
>
>-----Original Message-----
>From: Confocal Microscopy List
>[mailto:[hidden email]] On Behalf Of Ian Dobbie
>Sent: 13 October 2009 02:08
>To: [hidden email]
>Subject: Re: PSF with DIC
>
>John Runions <[hidden email]> 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  [phone]
1117 Johnson Ave., Madison, WI, 53706
[hidden email]
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.

[Rosemary.White]

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" <[hidden email]> 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 [phone]     Fax [phone]
> Mobile [phone]
> ______________________________________________
>      http://www.guycox.net
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of MODEL, MICHAEL
> Sent: Thursday, 15 October 2009 1:06 AM
> To: [hidden email]
> 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:[hidden email]]
> On Behalf Of Guy Cox
> Sent: Wednesday, October 14, 2009 10:59 AM
> To: [hidden email]
> 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 [phone]     Fax [phone]
> Mobile [phone]
> ______________________________________________
>      http://www.guycox.net
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of MODEL, MICHAEL
> Sent: Wednesday, 14 October 2009 11:08 PM
> To: [hidden email]
> 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:[hidden email]]
> On Behalf Of James Pawley
> Sent: Wednesday, October 14, 2009 8:50 AM
> To: [hidden email]
> 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:  [phone]
>> Email:  [hidden email]
>> Web-pages:
>> http://www.well.ox.ac.uk/molecular-cytogenetics-and-microscopy
>>
>> -----Original Message-----
>> From: Confocal Microscopy List
>> [mailto:[hidden email]] On Behalf Of Ian Dobbie
>> Sent: 13 October 2009 02:08
>> To: [hidden email]
>> Subject: Re: PSF with DIC
>>
>> John Runions <[hidden email]> 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  [phone]
> 1117 Johnson Ave., Madison, WI, 53706
> [hidden email]
> 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.