Using Nomarski DIC (on a Zeiss ICM 405), is it correct for the field
background to have a dark diagonal bar running across it under normal
viewing conditions? Everything looks right. The faux 3d effect is
"text book" perfect and optional sectioning works great (and the
resolution is nothing short of incredible).

The DIC sections of Molecular Expressions (excellent) site shows this
to be the case, but it's not clear this is how the field should look
when you're using the scope to observe a specimen; it implies it's
only like this during setup (though I see no setup step that would
remove it).

With the de Sarnmont filter added to the analyzer, the dark bar is
replaced with a diagonal rainbow gradient across the field instead.
Neither prevents observation of (magnificently) small and low-contrast
details, and it's easy to ignore - so no big deal really.

But what has raised my doubts is that all the references (and pictures)
I've found on the web either imply, or show, a relatively uniform
background. Also, when DIC and phase contrast are compared, the halos
of phase contrast are cited as a drawback. I would have thought the
gradient background in DIC (that I see) would be cited as an artefact
too, but it isn't.

Have I missed something?

Thanks
Beats

From:	justbeats
Subject:	Re: Nomarski DIC - field background appearance
Date:	17 Jan 2005 08:09:43 -0800

....and if it shows colours in polarised light. Beats.

From:	Dr. Georg N.Nyman
Subject:	Re: Nomarski DIC - field background appearance
Date:	Wed, 12 Jan 2005 23:22:31 +0100

Hi,
no that is not normal. The field background of a DIC image should be
more or less homogenous - yes some inhomogenity is existing due to the
position of the Nomarski prims (not exactly in the pupil) but a bar
tells me that something is very misaligned. If you add the Senarmont
compensator, the field becomes "colored" but again, it has to be more or
less even. Check among the general setup if you are using the right
prism for the selected lens, if you are using a DIC-capable lens, if the
vertical position of the prism is correct and if the entire illumination
setup (position of the condensor etc) is correct.
If you go to my website, there were I show photomicrographs, you can see
at least four which were taken in DIC. Here is the link for that page:
http://www.gnyman.com/Personal/photo1.htm
Ask me if you need more help -
rgds George

justbeats wrote:
> Using Nomarski DIC (on a Zeiss ICM 405), is it correct for the field
> background to have a dark diagonal bar running across it under normal
> viewing conditions? Everything looks right. The faux 3d effect is
> "text book" perfect and optional sectioning works great (and the
> resolution is nothing short of incredible).
>
> The DIC sections of Molecular Expressions (excellent) site shows this
> to be the case, but it's not clear this is how the field should look
> when you're using the scope to observe a specimen; it implies it's
> only like this during setup (though I see no setup step that would
> remove it).
>
> With the de Sarnmont filter added to the analyzer, the dark bar is
> replaced with a diagonal rainbow gradient across the field instead.
> Neither prevents observation of (magnificently) small and low-contrast
> details, and it's easy to ignore - so no big deal really.
>
> But what has raised my doubts is that all the references (and pictures)
> I've found on the web either imply, or show, a relatively uniform
> background. Also, when DIC and phase contrast are compared, the halos
> of phase contrast are cited as a drawback. I would have thought the
> gradient background in DIC (that I see) would be cited as an artefact
> too, but it isn't.
>
> Have I missed something?
>
> Thanks
> Beats
>

From:	Aaron
Subject:	Re: Nomarski DIC - field background appearance
Date:	12 Jan 2005 23:32:08 -0600

When the Wolsaton prisms create the correct shear for the objective,
the field is illuminated evenly and there is not dark diagonal. If
the shear is not exactly correct there is the darkened bar you
described. This is particularly a problem with the fist design
iteration of Zeiss transmitted DIC components.

The first design itteration was intended for use with Plan Achromat
objectives. It consisted of a turret condenser with several prisms
identified with the roman mumeratls I, II, III (sometimes IV) and a
single slider... In this design the turret condenser is rotated to
find the best prism to work with the other components.

There were different sliders marked with roman numerals II and III
which were designed one for the Universal series and the other for the
Standard series microscope stands.. With this design iteration, if
the slider, objective and condenser are not exactly compatible the
diagonal dark bar occurs. This system did not work with some
Apocharomats.but worked with Neofluars and pol achromats. That was
the motivation for the second design.

The second design iteration used small sliders that are specific for
each single objective. The sliders are inserted in little cages
installed on the nosepiece just behind the objective. The turret
condenser remained. This design provides a very even background.

The entire DIC system relies on polarized light. Any glass components
in the system that have not been annealed will also cause problems.
This goes for the top lens of the condenser which com in pol and
standard forms.

The sliders have screw drives to control displacement of the Wolaston
prisms to tune the system. With the deSenatmont compensatior in the
light path the seame tuning requires rotating the polarizing filter.

Aaron

On 12 Jan 2005 01:52:27 -0800, "justbeats"
wrote:

>Using Nomarski DIC (on a Zeiss ICM 405), is it correct for the field
>background to have a dark diagonal bar running across it under normal
>viewing conditions? Everything looks right. The faux 3d effect is
>"text book" perfect and optional sectioning works great (and the
>resolution is nothing short of incredible).
>
>The DIC sections of Molecular Expressions (excellent) site shows this
>to be the case, but it's not clear this is how the field should look
>when you're using the scope to observe a specimen; it implies it's
>only like this during setup (though I see no setup step that would
>remove it).
>
>With the de Sarnmont filter added to the analyzer, the dark bar is
>replaced with a diagonal rainbow gradient across the field instead.
>Neither prevents observation of (magnificently) small and low-contrast
>details, and it's easy to ignore - so no big deal really.
>
>But what has raised my doubts is that all the references (and pictures)
>I've found on the web either imply, or show, a relatively uniform
>background. Also, when DIC and phase contrast are compared, the halos
>of phase contrast are cited as a drawback. I would have thought the
>gradient background in DIC (that I see) would be cited as an artefact
>too, but it isn't.
>
>Have I missed something?
>
> Thanks
> Beats

From:	Repeating Rifle
Subject:	Re: Nomarski DIC - field background appearance
Date:	Wed, 12 Jan 2005 20:54:51 GMT

Where can I find a simple explanation of the essence of the Nomarski
technique?

Bill

From:	Dr. Georg N.Nyman
Subject:	Re: Nomarski DIC - field background appearance
Date:	Wed, 12 Jan 2005 23:24:47 +0100

Repeating Rifle wrote:
> Where can I find a simple explanation of the essence of the Nomarski
> technique?
>
> Bill
>
Hi,
any textbook on microscopy should talk about DIC, if you want to dig
into the topic more in detail, I recommend M. Pluta's Advanced Light
Microscopy.
If you like know more, ask me -
Rgds George

From:	GTO
Subject:	Re: Nomarski DIC - field background appearance
Date:	Thu, 13 Jan 2005 04:01:39 GMT

Pluta's books are expensive and hard to get. Especially volume 2 (chapter
7). In the meantime, try Douglas B. Murphy, Fundamentals of Light Microscopy
and Electronic Imaging, Wiley-Liss, 2001. It's cheaper and very easy to
read. Chapter 10 is about DIC. After this, you may want to get Pluta's $200+
volume 2.

Gregor

"Dr. Georg N.Nyman" wrote in message
news:BZedncUgvesvPnjcRVn-tg@comcast.com...
> Repeating Rifle wrote:
>> Where can I find a simple explanation of the essence of the Nomarski
>> technique?
>>
>> Bill
>>
> Hi,
> any textbook on microscopy should talk about DIC, if you want to dig into
> the topic more in detail, I recommend M. Pluta's Advanced Light
> Microscopy.
> If you like know more, ask me -
> Rgds George

From:	Aaron
Subject:	Re: Nomarski DIC - field background appearance
Date:	13 Jan 2005 01:41:04 -0600

Most light microscope specimen are fixed and stained. The eye is very
sensitive to the contrating colors and intensities.

Unstained, transparent "water white" specimen have details that are
difficult to detect because the eye is not sensitive to minute
differences in refractive index that affect the light transmitted
through the specimen. For these types of specimen interference
techniques are used to make the details visible.

Mechanically this is accomplised by splitting the beam of light
directed at the specimen into two parts before passing through the
specimen and recombining them before reaching the eye.. Any slight
differences in refrative index in the specimen cause phase changes in
the light which upon recombination causes some light waves to be
reinforced and othes to be cancelled. By using the interfernce
technique the refrative index diferences are converted to differences
in brightness for which the eye is very sensitive.

Two types of interference microscopy have become popular although
there are others that have been developed. Phase contrast uses an
annulus pattern in the condenser and the objective to divide the light
paths. DIC takes advantage of specially designed birefingent crystals
and polarized light to divide the light paths.

For illustarionions of the mechanics and additional information see:

http://micro.magnet.fsu.edu/primer/techniques/dic/dicconfiguration.html

On Wed, 12 Jan 2005 20:54:51 GMT, Repeating Rifle
wrote:

>Where can I find a simple explanation of the essence of the Nomarski
>technique?
>
>Bill

From:	Repeating Rifle
Subject:	Re: Nomarski DIC - field background appearance
Date:	Fri, 14 Jan 2005 05:59:54 GMT

in article 4e7cu0917tl4rnqqsbaat2abu0hs07um24@4ax.com, Aaron at
nghy@comcast.net wrote on 1/12/05 11:41 PM:

> Two types of interference microscopy have become popular although
> there are others that have been developed. Phase contrast uses an
> annulus pattern in the condenser and the objective to divide the light
> paths. DIC takes advantage of specially designed birefingent crystals
> and polarized light to divide the light paths.
>
> For illustarionions of the mechanics and additional information see:
>
>
> http://micro.magnet.fsu.edu/primer/techniques/dic/dicconfiguration.html
>
>
> On Wed, 12 Jan 2005 20:54:51 GMT, Repeating Rifle
> wrote:
>
>> Where can I find a simple explanation of the essence of the Nomarski
>> technique?

Thank you for the information. I am going to read the reference you cited
above again and more carefully

I know what shear interferometry is. I also know what polarization
interferometry is (Such as you get in a polariscope.) Quick perusal of the
information makes me think striaghtforward polarization interferometry, but
I do not know if I am right.

Does the Nomarski technique work primarily using birefringence from foreign
material or possible strain birefringence? I can understand how the
Wollaston provide polarization separation and shear. It is not clear to me
how having slightly different paths for Wollaston's outputs. So, to see if I
am getting a better grasp of the process:

Which is more important; birefirngence or shear?

Bill

From:	justbeats
Subject:	Re: Nomarski DIC - field background appearance
Date:	13 Jan 2005 00:35:50 -0800

Fixed it!

The Wollaston sliders (that go behind the objectives) were upside down!
Turned 'em over and voila, a flat background field. Not sure how THAT
works, (a spacing thing?), but it's perfect now. I've dialled the
sliders for a nice "baby blue" background with the deSenarmont
compensator in place - but for many specimens, I think I prefer the
more natural appearance without the compensator. It is s-o-o-o cool.
I've completely run out of superlatives...

Cheers
Beats

From:	Aaron
Subject:	Re: Nomarski DIC - field background appearance
Date:	14 Jan 2005 00:12:07 -0600

So you have the second design with the individual sliders for each
objective. Neat.

Aaron

On 13 Jan 2005 00:35:50 -0800, "justbeats"
wrote:

>Fixed it!
>
>The Wollaston sliders (that go behind the objectives) were upside down!
>Turned 'em over and voila, a flat background field. Not sure how THAT
>works, (a spacing thing?), but it's perfect now. I've dialled the
>sliders for a nice "baby blue" background with the deSenarmont
>compensator in place - but for many specimens, I think I prefer the
>more natural appearance without the compensator. It is s-o-o-o cool.
>I've completely run out of superlatives...
>
> Cheers
> Beats

From:	rene
Subject:	Re: Nomarski DIC - field background appearance
Date:	17 Jan 2005 02:00:41 -0800

>In white light, are some images colored?
If the specimen is coloured in normal BF, yes.

Rene.

From:	justbeats
Subject:	Re: Nomarski DIC - field background appearance
Date:	12 Jan 2005 17:18:10 -0800

Thanks for the reply George,

I'll keep the response in the public domain as the discussion or (more
precisely) your tuition may be of general interest.

look at
http://micro.magnet.fsu.edu/primer/techniques/dic/dicconfiguration.html

Note to "Repeating Rifle": this site has the best descriptions
available on the web (IMO).

Anyway, figs 7b and 9a represent exactly what I see as "normal" field
backgrounds (without and with de Sarnemont compensator respectively).
The DIC effect is exactly as it should be - contrast, resolution and
the 3d effect are as described in every bit of online documentation I
can find. But the background is as pictured in the figures cited above.

Maybe this is the significant point - I do not own a phase telescope or
Bertrand lens. The statement "Refocus the phase telescope or Bertrand
lens to observe the interference fringe that appears in the objective
rear focal plane" makes me wonder if I've (somehow) managed to focus
this at the image plane instead? Is it possible to set up Nomarski DIC
without these accessories?

I know for sure I have all the relevant pieces in the optical train
since the Zeiss part numbers are clear, and I know the DIC sliders,
Wollaston prisms (in the turret "rotator") and polarisation fillters
are all "the right thing" and in perfect condition.

Objectives are Zeiss NeoFluar 10x and 40x and the prisms all contain
the relevant matching NA and mag values - so I don't think it's that.

Let's leave it at that for now - if there are no mistakes embedded in
the observations above, I'll list the exact equipment I'm using and see
if the problem is there...
Thanks for the willingness to help.

Cheers
Beats

From:	rene
Subject:	Re: Nomarski DIC - field background appearance
Date:	14 Jan 2005 02:25:27 -0800

> Which is more important; birefirngence or shear?

The wollaston prisms produces a shear BECAUSE they are birefringent (I
hope I'm right in saying this). Birefringence (in the sample) is
actually not wanted as it obscures the structures by 'overlighting'.

It is not clear to me
> how having slightly different paths for Wollaston's outputs. So, to
see if I
> am getting a better grasp of the process:

Hmm, I hope I can be clear enough. Imagine a cell with a transparent
but dense nucleus in the shape of a ball. The system produces numerous
images, which differ very very slightly from eachother. Not enough to
see them different in BF when separate. For the nucleus in the cell
however, there's a refractive index gradient from one image to the
next. Because of the interference this translates into a gradient of
light intensity(contrast).

HTH, Rene.
>
> Bill

From:	Repeating Rifle
Subject:	Re: Nomarski DIC - field background appearance
Date:	Fri, 14 Jan 2005 19:46:40 GMT

in article 1105698327.878967.131600@z14g2000cwz.googlegroups.com, rene at
renevanwezel@hotmail.com wrote on 1/14/05 2:25 AM:

> Hmm, I hope I can be clear enough. Imagine a cell with a transparent
> but dense nucleus in the shape of a ball. The system produces numerous
> images, which differ very very slightly from eachother. Not enough to
> see them different in BF when separate. For the nucleus in the cell
> however, there's a refractive index gradient from one image to the
> next. Because of the interference this translates into a gradient of
> light intensity(contrast).

I think I understand. I realize that the Wollastin prism sends the two
polarizations in slightly differfent directions.

I am used to using shear interferometey for measuring error of a wavefront.
The interferometer consists of a slightly wedged plate with flat faces. In
that case, two partially reflected "images," one from the front surface and
one from the rear surfaces are overlapped but displaced. Thus one gets a
double image of the object (wavefront rather than something material) but
not a single sharp image. For examining almost perfect wavefronts, this is
not really a problem. We do not look for fine detail in the wavefront.

In Nomarski, do you superimpose two images accurately over each other but as
if the object were rotated a bit from one image to the other? I can see how
that would lead to polarization interference.

Bill

From:	Aaron
Subject:	Re: Nomarski DIC - field background appearance
Date:	14 Jan 2005 23:35:04 -0600

Hi,

There are two Wolaston ( also called Nomarski) prisms in the light
path. The first prism,usually located in the condenser shears the
beam the second usually located above the objective combines the two
beams. .The effect of the specimen structures on the light beams is
very complex. There are very small refection, diffraction, and
absorbtions components. That is why these specimen often look water
white with few features. Retardation of the transmitted portions of
the light is the key.. The retardation is determined both by the
refractive index gradients and the thickness gradients. It is during
the recombination of the light beams that the light and dark details
are generated by the process of addition or subtraction of the light
waves.

On Fri, 14 Jan 2005 19:46:40 GMT, Repeating Rifle
wrote:

>in article 1105698327.878967.131600@z14g2000cwz.googlegroups.com, rene at
>renevanwezel@hotmail.com wrote on 1/14/05 2:25 AM:
>
>> Hmm, I hope I can be clear enough. Imagine a cell with a transparent
>> but dense nucleus in the shape of a ball. The system produces numerous
>> images, which differ very very slightly from eachother. Not enough to
>> see them different in BF when separate. For the nucleus in the cell
>> however, there's a refractive index gradient from one image to the
>> next. Because of the interference this translates into a gradient of
>> light intensity(contrast).
>
>I think I understand. I realize that the Wollastin prism sends the two
>polarizations in slightly differfent directions.
>
>I am used to using shear interferometey for measuring error of a wavefront.
>The interferometer consists of a slightly wedged plate with flat faces. In
>that case, two partially reflected "images," one from the front surface and
>one from the rear surfaces are overlapped but displaced. Thus one gets a
>double image of the object (wavefront rather than something material) but
>not a single sharp image. For examining almost perfect wavefronts, this is
>not really a problem. We do not look for fine detail in the wavefront.
>
>In Nomarski, do you superimpose two images accurately over each other but as
>if the object were rotated a bit from one image to the other? I can see how
>that would lead to polarization interference.
>
>Bill

From:	Repeating Rifle
Subject:	Re: Nomarski DIC - field background appearance
Date:	Sat, 15 Jan 2005 07:03:03 GMT

in article o39hu0dvd9u4bdoq3m7h1a10rknb8v9aku@4ax.com, Aaron at
nghy@comcast.net wrote on 1/14/05 9:35 PM:

> Hi,
>
> There are two Wolaston ( also called Nomarski) prisms in the light
> path. The first prism,usually located in the condenser shears the
> beam the second usually located above the objective combines the two
> beams. .The effect of the specimen structures on the light beams is
> very complex. There are very small refection, diffraction, and
> absorbtions components. That is why these specimen often look water
> white with few features. Retardation of the transmitted portions of
> the light is the key.. The retardation is determined both by the
> refractive index gradients and the thickness gradients. It is during
> the recombination of the light beams that the light and dark details
> are generated by the process of addition or subtraction of the light
> waves.

Thanks for the response. The more I read, the more I think that I
understand.

If I do understand, the first Wollaston prism (I am sure that the name
preceded Nomarski by at least a century) sends beams of orthogonal
polarizations in slightly different directions through the sample. The
second prism recombines the two images except to the extent that the object
changes shape as viewed in the two slightly different viewing directions.
Differences in the optical path should show up as interference figures.

In white light, are some images colored?

Bill

From:	Aaron
Subject:	Re: Nomarski DIC - field background appearance
Date:	15 Jan 2005 03:47:12 -0600

Our eyes are very sensitive to very slight differences in color and
brightness. That is why most specimen for study with a light
microscope are stained. For unstained living specimen the
interference techniques (phast contrast or DIC) are very useful.

Aaron

On Sat, 15 Jan 2005 07:03:03 GMT, Repeating Rifle
wrote:

>in article o39hu0dvd9u4bdoq3m7h1a10rknb8v9aku@4ax.com, Aaron at
>nghy@comcast.net wrote on 1/14/05 9:35 PM:
>
>> Hi,
>>
>> There are two Wolaston ( also called Nomarski) prisms in the light
>> path. The first prism,usually located in the condenser shears the
>> beam the second usually located above the objective combines the two
>> beams. .The effect of the specimen structures on the light beams is
>> very complex. There are very small refection, diffraction, and
>> absorbtions components. That is why these specimen often look water
>> white with few features. Retardation of the transmitted portions of
>> the light is the key.. The retardation is determined both by the
>> refractive index gradients and the thickness gradients. It is during
>> the recombination of the light beams that the light and dark details
>> are generated by the process of addition or subtraction of the light
>> waves.
>
>Thanks for the response. The more I read, the more I think that I
>understand.
>
>If I do understand, the first Wollaston prism (I am sure that the name
>preceded Nomarski by at least a century) sends beams of orthogonal
>polarizations in slightly different directions through the sample. The
>second prism recombines the two images except to the extent that the object
>changes shape as viewed in the two slightly different viewing directions.
>Differences in the optical path should show up as interference figures.
>
>In white light, are some images colored?
>
>Bill