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Craig Brideau

Re: chromatic aberration

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The paper looks great! Thanks for pointing it out; it gives a good test
methodology.

Craig

On Thu, Dec 29, 2011 at 9:33 AM, Rich Cole <[hidden email]> wrote:

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> *****
>
> Aberrations within our optical systems are more common that most images
> think. This belief was confirmed in an international image performance
> study recently completed (results: Stack, R., Bayles, C., Girard, A.,
> Martin, K., Opansky, C., Schulz, K., and Cole, R. (2011) Quality Assurance
> Testing for Modern Optical Imaging Systems. Microscopy & Microanalysis
> 17(4):598-606).
>
>
>
> Not wanting to be redundant but, check all the objective that you image
> with
> regularly. It is well worth the time and energy!
>
>
>
>
>
>
>
>
>
> Richard Cole
> Research Scientist V
> Director: Advanced Light Microscopy & Image Analysis Core
> Wadsworth Center
> P.O. Box 509 Albany N.Y. 12201-0509
>
>
>
> Research Assistant Professor
>
> Dept. of Biomedical Sciences
>
> School of Public Health State University of New York Albany, New York
>
>
> ( 518-474-7048
> Ê 518-474-4430
>
> * <mailto:[hidden email]> [hidden email]
>
> Website www.wadsworth.org/cores/alm/index.htm
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mmodel

Re: chromatic aberration

In reply to this post by Johannes Helm

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Thanks to all who responded to my question. But I also wanted to mention that not only new objectives need to be tested. Same goes for Wollaston prisms! I found that quite often DIC images look better with Wollaston prisms designed for other objectives or without any condenser prism at all! Since the prisms cost around $1000 I wanted to share this observation with the microscopy community.

Mike Model

Stanislav Vitha

Re: chromatic aberration

In reply to this post by mmodel

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Mike,
that is really interesting. What microscope do you have? Does it have one a
common objective prism and specific condenser prisms (as in Olympus) or a
common condenser prism and specific objective prisms (Zeiss)? Are you saying
you get a true DIC image with just one Nomarski/Wollaston prism (behind the
objective)? I did not think this could work, except for a setup like the PlasDIC
from Zeiss (but I really never fully ondertood how it works).

Stan Vitha

On Thu, 5 Jan 2012 09:27:39 -0500, MODEL, MICHAEL <[hidden email]>
wrote:

>Thanks to all who responded to my question. But I also wanted to mention
that not only new objectives need to be tested. Same goes for Wollaston
prisms! I found that quite often DIC images look better with Wollaston prisms
designed for other objectives or without any condenser prism at all! Since the
prisms cost around $1000 I wanted to share this observation with the
microscopy community.
>
>Mike Model

mmodel

Re: chromatic aberration

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We have an Olympus with objective-specific splitting prisms on the condenser and a common combining prism on the objective side. My understanding is that the purpose of the splitting prism is to form two parallel rays that would be coherent. Parallel rays exist even without the prism and can be more or less coherent, depending on the separation between them. So I guess it is possible that a DIC image might form with only one combining prism.

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Stanislav Vitha
Sent: Friday, January 06, 2012 11:00 AM
To: [hidden email]
Subject: Re: chromatic aberration

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Mike,
that is really interesting. What microscope do you have? Does it have one a
common objective prism and specific condenser prisms (as in Olympus) or a
common condenser prism and specific objective prisms (Zeiss)? Are you saying
you get a true DIC image with just one Nomarski/Wollaston prism (behind the
objective)? I did not think this could work, except for a setup like the PlasDIC
from Zeiss (but I really never fully ondertood how it works).

Stan Vitha

On Thu, 5 Jan 2012 09:27:39 -0500, MODEL, MICHAEL <[hidden email]>
wrote:

>Thanks to all who responded to my question. But I also wanted to mention
that not only new objectives need to be tested. Same goes for Wollaston
prisms! I found that quite often DIC images look better with Wollaston prisms
designed for other objectives or without any condenser prism at all! Since the
prisms cost around $1000 I wanted to share this observation with the
microscopy community.
>
>Mike Model

James Pawley

Re: chromatic aberration: Weird DIC effects

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This all sounds very strange. I would like to know more about this
image "without a condenser prism". Are we still assuming crossed
polars? Does the image have a shear direction (i.e., more contrast
for features that change RI at, say,+45 deg than at -45deg in the x-y
plane? Is the condenser aperture set to about 2/3 of the NA of the
objective? Can you "invert the contrast" (change the dark features to
bright ones and vice-versa) by the normal DIC adjustments?

And just as a reminder, you can get a very DIC-like contrast using
anaxial illumination (illumination filling only half (or less) of the
condenser aperture, and a corresponding obstruction in the objective
BFP to prevent most of the undiffracted light from reaching the
intermediate image.

http://micro.magnet.fsu.edu/primer/techniques/oblique/obliqueintro.html
(go half-way down this long page)

This contrast really depends mostly on "misalignment" of the
illumination in conjunction with leaving say, a filter-holder still
obscuring part of the light bundle (or moving the objective-side, DIC
prism only halfway out of its proper position). It will occur without
reference to the settings of the polarizers or DIC prisms.

Hoffman Modulation Contrast works in a somewhat similar manner

http://micro.magnet.fsu.edu/primer/techniques/hoffman/hoffmanindex.html
Images at

http://micro.magnet.fsu.edu/primer/techniques/hoffman/hoffmangallery.html

As do other non-axial techniques

http://www.microscopyu.com/articles/stereomicroscopy/stereooblique.html

And for the record, in my opinion, "Real Nomarski DIC" is almost
impossible to diagram honestly (most diagrams imply negative
refractive indexes) but the effort to do so can unintentionally
convey the impression that the illumination involved is parallel to
the optical axis. In fact, nothing could be further from the truth.
The most notable feature of good DIC is it very shallow depth of
field, and this comes about because, unlike phase, both the
illumination and observation optics operate at full NA. Indeed, this
is why the best high-NA DIC requires "rectification"

Rectification was initially developed by Shinya Inoue to overcome the
problems of doing Pol microscopy at high NA. High NA implies that
some rays pass optical surfaces that are at close to glancing
incidence. As rays emerging from a spot on the specimen strike such
surfaces, one pol direction will be preferentially reflected and so
not arrive at the BFP (or the image plane). This is a problem for DIC
because the two ray bundles can only be kept discrete because they
have different pol directions and this reflection artifact causes the
high NA rays to become somewhat depolarized. (This depolarization is
why you can't do normal DIC on plastic specimens.)

The problem can be "rectified" by adding meniscus lenses, that have
low power but near-incidence (but in the reverse sense) paths for
high-NA rays, after the polarizer and before the condenser, as first
suggested here

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2224132/pdf/831.pdf

Later discussion here.
http://spiedigitallibrary.org/oe/resource/1/opegar/v41/i5/p943_s1?isAuthorized=no

Hope that this doesn't add to the confusion!

Sorry,

JP

***************************************************************************
Prof. James B. Pawley, Ph.
608-238-3953
21. N. Prospect Ave. Madison, WI 53726 USA
[hidden email]
3D Microscopy of Living Cells Course, June 9-21, 2012, UBC, Vancouver Canada
Info: http://www.3dcourse.ubc.ca/ Application
deadline 3/16/2012
"If it ain't diffraction, it must be statistics." Anon. 11/16/12

>-----Original Message-----
>From: Confocal Microscopy List
>[mailto:[hidden email]] On Behalf Of Stanislav
>Vitha
>Sent: Friday, January 06, 2012 11:00 AM
>To: [hidden email]
>Subject: Re: chromatic aberration
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Mike,
>that is really interesting. What microscope do you have? Does it have one a
>common objective prism and specific condenser prisms (as in Olympus) or a
>common condenser prism and specific objective prisms (Zeiss)? Are you saying
>you get a true DIC image with just one Nomarski/Wollaston prism (behind the
>objective)? I did not think this could work, except for a setup
>like the PlasDIC
>from Zeiss (but I really never fully ondertood how it works).
>
>Stan Vitha
>
>
>On Thu, 5 Jan 2012 09:27:39 -0500, MODEL, MICHAEL <[hidden email]>
>wrote:
>
>>Thanks to all who responded to my question. But I also wanted to mention
>that not only new objectives need to be tested. Same goes for Wollaston
>prisms! I found that quite often DIC images look better with Wollaston prisms
>designed for other objectives or without any condenser prism at all! Since the
>prisms cost around $1000 I wanted to share this observation with the
>microscopy community.
>>
>>Mike Model

--
***************************************************************************
Prof. James B. Pawley, Ph.
608-238-3953
21. N. Prospect Ave. Madison, WI 53726 USA
[hidden email]
3D Microscopy of Living Cells Course, June 9-21, 2012, UBC, Vancouver Canada
Info: http://www.3dcourse.ubc.ca/ Application
deadline 3/16/2012
"If it ain't diffraction, it must be statistics." Anon. 11/16/12

Craig Brideau

Re: chromatic aberration

In reply to this post by mmodel

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The second prism just realigns (co-linearizes?) the orthogonal components
so they can interfere at the detector. If they are diverted by the sample
it is possible they could be somewhat realigned 'naturally', but I think
you would get pretty poor contrast relying on this 'chance' method.

Craig

On Fri, Jan 6, 2012 at 9:26 AM, MODEL, MICHAEL <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> We have an Olympus with objective-specific splitting prisms on the
> condenser and a common combining prism on the objective side. My
> understanding is that the purpose of the splitting prism is to form two
> parallel rays that would be coherent. Parallel rays exist even without the
> prism and can be more or less coherent, depending on the separation between
> them. So I guess it is possible that a DIC image might form with only one
> combining prism.
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Stanislav Vitha
> Sent: Friday, January 06, 2012 11:00 AM
> To: [hidden email]
> Subject: Re: chromatic aberration
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Mike,
> that is really interesting. What microscope do you have? Does it have one
> a
> common objective prism and specific condenser prisms (as in Olympus) or a
> common condenser prism and specific objective prisms (Zeiss)? Are you
> saying
> you get a true DIC image with just one Nomarski/Wollaston prism (behind the
> objective)? I did not think this could work, except for a setup like the
> PlasDIC
> from Zeiss (but I really never fully ondertood how it works).
>
> Stan Vitha
>
>
> On Thu, 5 Jan 2012 09:27:39 -0500, MODEL, MICHAEL <[hidden email]>
> wrote:
>
> >Thanks to all who responded to my question. But I also wanted to mention
> that not only new objectives need to be tested. Same goes for Wollaston
> prisms! I found that quite often DIC images look better with Wollaston
> prisms
> designed for other objectives or without any condenser prism at all! Since
> the
> prisms cost around $1000 I wanted to share this observation with the
> microscopy community.
> >
> >Mike Model
>

mmodel

single-prism DIC

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Unfortunately I don't have an easy way to share files with the outside world. But I suggest that those who have Olympus microscopes verify the following results with cells on slides:

With x20/0.7 UPlanApo
Best results with DPO40S prism (unrelated); DPA20 (designed for this objective) and empty position give acceptable and comparable contrast/uniformity

20/0.75 UPlanSApo
Best results with unrelated DPO40S or DPO60S, while IX2-DIC20 (designed for this one) or empty position produce slightly inferior and comparable results

X60/1.4 PlaApo, oil
DPO60S (designed for this objective) and empty position produce DIC images of similar quality

The other prism and polarizers must still be in place

Mike

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Craig Brideau
Sent: Friday, January 06, 2012 2:15 PM
To: [hidden email]
Subject: Re: chromatic aberration

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

The second prism just realigns (co-linearizes?) the orthogonal components
so they can interfere at the detector. If they are diverted by the sample
it is possible they could be somewhat realigned 'naturally', but I think
you would get pretty poor contrast relying on this 'chance' method.

Craig

On Fri, Jan 6, 2012 at 9:26 AM, MODEL, MICHAEL <[hidden email]> wrote:

JOEL B. SHEFFIELD

Re: single-prism DIC

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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Mike,

Can you set up a picasa or similar site for photos? You could then link to
them.

Joel

On Fri, Jan 6, 2012 at 3:15 PM, MODEL, MICHAEL <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Unfortunately I don't have an easy way to share files with the outside
> world. But I suggest that those who have Olympus microscopes verify the
> following results with cells on slides:
>
> With x20/0.7 UPlanApo
> Best results with DPO40S prism (unrelated); DPA20 (designed for this
> objective) and empty position give acceptable and comparable
> contrast/uniformity
>
> 20/0.75 UPlanSApo
> Best results with unrelated DPO40S or DPO60S, while IX2-DIC20 (designed
> for this one) or empty position produce slightly inferior and comparable
> results
>
> X60/1.4 PlaApo, oil
> DPO60S (designed for this objective) and empty position produce DIC images
> of similar quality
>
> The other prism and polarizers must still be in place
>
> Mike
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Craig Brideau
> Sent: Friday, January 06, 2012 2:15 PM
> To: [hidden email]
> Subject: Re: chromatic aberration
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> The second prism just realigns (co-linearizes?) the orthogonal components
> so they can interfere at the detector. If they are diverted by the sample
> it is possible they could be somewhat realigned 'naturally', but I think
> you would get pretty poor contrast relying on this 'chance' method.
>
> Craig
>
>
> On Fri, Jan 6, 2012 at 9:26 AM, MODEL, MICHAEL <[hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > We have an Olympus with objective-specific splitting prisms on the
> > condenser and a common combining prism on the objective side. My
> > understanding is that the purpose of the splitting prism is to form two
> > parallel rays that would be coherent. Parallel rays exist even without
> the
> > prism and can be more or less coherent, depending on the separation
> between
> > them. So I guess it is possible that a DIC image might form with only one
> > combining prism.
> >
> > -----Original Message-----
> > From: Confocal Microscopy List [mailto:[hidden email]]
> > On Behalf Of Stanislav Vitha
> > Sent: Friday, January 06, 2012 11:00 AM
> > To: [hidden email]
> > Subject: Re: chromatic aberration
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Mike,
> > that is really interesting. What microscope do you have? Does it have
> one
> > a
> > common objective prism and specific condenser prisms (as in Olympus) or a
> > common condenser prism and specific objective prisms (Zeiss)? Are you
> > saying
> > you get a true DIC image with just one Nomarski/Wollaston prism (behind
> the
> > objective)? I did not think this could work, except for a setup like the
> > PlasDIC
> > from Zeiss (but I really never fully ondertood how it works).
> >
> > Stan Vitha
> >
> >
> > On Thu, 5 Jan 2012 09:27:39 -0500, MODEL, MICHAEL <[hidden email]>
> > wrote:
> >
> > >Thanks to all who responded to my question. But I also wanted to mention
> > that not only new objectives need to be tested. Same goes for Wollaston
> > prisms! I found that quite often DIC images look better with Wollaston
> > prisms
> > designed for other objectives or without any condenser prism at all!
> Since
> > the
> > prisms cost around $1000 I wanted to share this observation with the
> > microscopy community.
> > >
> > >Mike Model
> >
>

--

Joel B. Sheffield, Ph.D
Department of Biology
Temple University
Philadelphia, PA 19122
Voice: 215 204 8839
e-mail: [hidden email]
URL: http://astro.temple.edu/~jbs

mmodel

Re: single-prism DIC

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Will try. I should also add that I am using the correct 0.55 NA condenser and the mix-and-match works only for some objectives.

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of JOEL B. SHEFFIELD
Sent: Friday, January 06, 2012 3:21 PM
To: [hidden email]
Subject: Re: single-prism DIC

*****
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*****

Mike,

Can you set up a picasa or similar site for photos? You could then link to
them.

Joel

On Fri, Jan 6, 2012 at 3:15 PM, MODEL, MICHAEL <[hidden email]> wrote:

--

Joel B. Sheffield, Ph.D
Department of Biology
Temple University
Philadelphia, PA 19122
Voice: 215 204 8839
e-mail: [hidden email]
URL: http://astro.temple.edu/~jbs

mmodel

single prism DIC

In reply to this post by Craig Brideau

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These are two images of cultured cells taken on an Olympus microscope with a 60/1.4 oil objective. The first one was taken using standard setup, for the second one the objective-specific prism on the condenser was removed and an empty position was used. (Michael, thanks for suggesting dropbox!)

Mike Model

http://dl.dropbox.com/u/56997340/DIC%20Olympus%20x601.4%20oil/x60-1.4oil%202%20prism%20DIC.jpg

http://dl.dropbox.com/u/56997340/DIC%20Olympus%20x601.4%20oil/x60-1.4oil%201%20prism%20DIC.jpg

JOEL B. SHEFFIELD

Re: single prism DIC

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MIke,

I am curious. What were these cells mounted on, and what was the mounting
solution?

Joel

On Thu, Jan 12, 2012 at 5:19 PM, MODEL, MICHAEL <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> These are two images of cultured cells taken on an Olympus microscope with
> a 60/1.4 oil objective. The first one was taken using standard setup, for
> the second one the objective-specific prism on the condenser was removed
> and an empty position was used. (Michael, thanks for suggesting dropbox!)
>
> Mike Model
>
>
>
> http://dl.dropbox.com/u/56997340/DIC%20Olympus%20x601.4%20oil/x60-1.4oil%202%20prism%20DIC.jpg
>
>
> http://dl.dropbox.com/u/56997340/DIC%20Olympus%20x601.4%20oil/x60-1.4oil%201%20prism%20DIC.jpg
>

--

Joel B. Sheffield, Ph.D
Department of Biology
Temple University
Philadelphia, PA 19122
Voice: 215 204 8839
e-mail: [hidden email]
URL: http://astro.temple.edu/~jbs

mmodel

Re: single prism DIC

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I added one more file, which is a montage of two tiff images compressed from 12 to 8 bit with a scale bar. These are live HeLa cells grown on a coverslip and mounted on a slide in RPMI. The analyzer and the polarizer were in crossed positions and used as prescribed, without any custom modification of anything. As I said before, this does not work for every objective. But with one objective we found that by using a prism designed for a different objective we get results that are MUCH better than with the intended prism. Unfortunately, those image files got corrupted and I don't have the "correct" prism anymore (there was no point in buying it) to reproduce the results. But when I showed those the images to Olympus people (two with "wrong" prisms, one with the "right" prism and one without any) they all agreed that the "correctly" acquired image was the worst of the four.

http://dl.dropbox.com/u/56997340/DIC%20Olympus%20x601.4%20oil/montage.tif

________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of JOEL B. SHEFFIELD [[hidden email]]
Sent: Thursday, January 12, 2012 7:31 PM
To: [hidden email]
Subject: Re: single prism DIC

*****
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MIke,

I am curious. What were these cells mounted on, and what was the mounting
solution?

Joel

On Thu, Jan 12, 2012 at 5:19 PM, MODEL, MICHAEL <[hidden email]> wrote:

--

Joel B. Sheffield, Ph.D
Department of Biology
Temple University
Philadelphia, PA 19122
Voice: 215 204 8839
e-mail: [hidden email]
URL: http://astro.temple.edu/~jbs