Chromatic aberration and refractive index of a sample

[Laevsky, Gary S.]

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Hello All,

Chromatic aberration is an artifact produced by differing wavelengths passing through a medium with a refractive index, and that n affects different wavelengths differently.

In the example of a chromatically corrected objective, multiple wavelengths pass through a very specific combination of lenses to reduce the artifact/mis-alignment.

When imaging a sample, say a monolayer or a tissue sample, the excitation (and emission for that matter) wavelengths pass through multiple n's.  Will this induce chromatic aberration in the emission pathway?

How is this corrected for?  In the objective again?  Or is it not necessary, or insignificant within the realm of light microscopy resolution. If not necessary for "standard" light microscopy, what about super res?

Thank you for explaining.

Best,

Gary


Sent from my iPad

[mmodel]

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Hi Gary

Refractive index of water changes roughly by 0.01 every 200 nm in the visible range. That will (very roughly) change the imaging distance by 1% (assuming the objective is low-NA and is perfectly corrected). I would expect this to be the main consequence - if you do double imaging of DAPI  and TRITC 10 um into an aqueous sample, the emission will be coming from actual depths that differ by 0.1 um. In reality, blurring from spherical aberration is likely to be worse. (But I don't know the real answer to your question, I am just improvising)

Best -

Mike Model


____________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Laevsky, Gary S. <[hidden email]>
Sent: Thursday, February 27, 2014 7:27 PM
To: [hidden email]
Subject: Chromatic aberration and refractive index of a sample

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Hello All,

Chromatic aberration is an artifact produced by differing wavelengths passing through a medium with a refractive index, and that n affects different wavelengths differently.

In the example of a chromatically corrected objective, multiple wavelengths pass through a very specific combination of lenses to reduce the artifact/mis-alignment.

When imaging a sample, say a monolayer or a tissue sample, the excitation (and emission for that matter) wavelengths pass through multiple n's.  Will this induce chromatic aberration in the emission pathway?

How is this corrected for?  In the objective again?  Or is it not necessary, or insignificant within the realm of light microscopy resolution. If not necessary for "standard" light microscopy, what about super res?

Thank you for explaining.

Best,

Gary


Sent from my iPad

[Sergey Tauger]

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Hi Gary,

For any objective, aberrations are corrected only for first 0.5-1 micrometer and
only if you use coverglass of recommended thickness with accuracy of 1%. I.e.
if 0.17 cg is recommended, you will get identical images in terms of quality
analysis only for cg thickness range 168-172 micrometer.

However, chromatic aberrations are negligible if you image not deeper than 3
micrometer into specimen and use standart cg, i.e 170 +/- 15. For deeper
imaging  you can get false negative\positive in FISH.

If you want quantitative result for imaging depth up to 20-30 micrometer, it is
better to deconvolve images obtained. My favourite packages are Hyugens
Suite (commercial) and COSMOS (opensource), they perform most accurately
among the ones I tested.

Best,
Sergey Tauger

PhD student
Cell motility lab.
Dept. of Biology
Moscow State University

[Mark Cannell-2]

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Chromatic aberrations are highly objective/specimen dependent. The biggest concern for confocal is longitudinal chromatic aberrations as the pinhole mostly takes care of spherical aberration (by dumping photons).

My 2c

Mark



On 28/02/2014, at 8:27 am, Sergey Tauger <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Gary,
>
> For any objective, aberrations are corrected only for first 0.5-1 micrometer and
> only if you use coverglass of recommended thickness with accuracy of 1%. I.e.
> if 0.17 cg is recommended, you will get identical images in terms of quality
> analysis only for cg thickness range 168-172 micrometer.
>
> However, chromatic aberrations are negligible if you image not deeper than 3
> micrometer into specimen and use standart cg, i.e 170 +/- 15. For deeper
> imaging  you can get false negative\positive in FISH.
>
> If you want quantitative result for imaging depth up to 20-30 micrometer, it is
> better to deconvolve images obtained. My favourite packages are Hyugens
> Suite (commercial) and COSMOS (opensource), they perform most accurately
> among the ones I tested.
>
> Best,
> Sergey Tauger
>
> PhD student
> Cell motility lab.
> Dept. of Biology
> Moscow State University

Mark  B. Cannell Ph.D. FRSNZ
Professor of Cardiac Cell Biology
School of Physiology &  Pharmacology
Medical Sciences Building
University of Bristol
Bristol
BS8 1TD UK

[hidden email]

[Laevsky, Gary S.]

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Hi Mark,

I would think spherical aberration is mostly a longitudinal artifact.  And would affect chromatic similarly, meaning it would dump photons not in the same plane, thereby affecting the image (“cutting” out the blue (hypothetical) when it “should” be colocalized with the red.).

But my main question is, is there a correction for chromatic aberration after the excitation beam is cleaned up by the objective?  Or is it necessary?  Sounds like in thin “er” samples, less than 10 um, it is not necessary?

Greater than that and fiducially with a warping algorithm?

Thanks again



Best,


Gary



On Feb 28, 2014, at 3:43 AM, Mark Cannell <[hidden email]<mailto:[hidden email]>> wrote:

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Chromatic aberrations are highly objective/specimen dependent. The biggest concern for confocal is longitudinal chromatic aberrations as the pinhole mostly takes care of spherical aberration (by dumping photons).

My 2c

Mark



On 28/02/2014, at 8:27 am, Sergey Tauger <[hidden email]> wrote:

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Hi Gary,

For any objective, aberrations are corrected only for first 0.5-1 micrometer and
only if you use coverglass of recommended thickness with accuracy of 1%. I.e.
if 0.17 cg is recommended, you will get identical images in terms of quality
analysis only for cg thickness range 168-172 micrometer.

However, chromatic aberrations are negligible if you image not deeper than 3
micrometer into specimen and use standart cg, i.e 170 +/- 15. For deeper
imaging  you can get false negative\positive in FISH.

If you want quantitative result for imaging depth up to 20-30 micrometer, it is
better to deconvolve images obtained. My favourite packages are Hyugens
Suite (commercial) and COSMOS (opensource), they perform most accurately
among the ones I tested.

Best,
Sergey Tauger

PhD student
Cell motility lab.
Dept. of Biology
Moscow State University

Mark  B. Cannell Ph.D. FRSNZ
Professor of Cardiac Cell Biology
School of Physiology &  Pharmacology
Medical Sciences Building
University of Bristol
Bristol
BS8 1TD UK

[hidden email]

[Laevsky, Gary S.]

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I meant to say the pinhole would affect chromatic aberration similar to spherical, but cutting out the out of focal plane signal.

Best,

Gary





On Feb 28, 2014, at 5:59 AM, Laevsky, Gary S. <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Mark,
>
> I would think spherical aberration is mostly a longitudinal artifact.  And would affect chromatic similarly, meaning it would dump photons not in the same plane, thereby affecting the image (“cutting” out the blue (hypothetical) when it “should” be colocalized with the red.).
>
> But my main question is, is there a correction for chromatic aberration after the excitation beam is cleaned up by the objective?  Or is it necessary?  Sounds like in thin “er” samples, less than 10 um, it is not necessary?
>
> Greater than that and fiducially with a warping algorithm?
>
> Thanks again
>
>
>
> Best,
>
>
> Gary
>
>
>
> On Feb 28, 2014, at 3:43 AM, Mark Cannell <[hidden email]<mailto:[hidden email]>> wrote:
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Chromatic aberrations are highly objective/specimen dependent. The biggest concern for confocal is longitudinal chromatic aberrations as the pinhole mostly takes care of spherical aberration (by dumping photons).
>
> My 2c
>
> Mark
>
>
>
> On 28/02/2014, at 8:27 am, Sergey Tauger <[hidden email]> wrote:
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Gary,
>
> For any objective, aberrations are corrected only for first 0.5-1 micrometer and
> only if you use coverglass of recommended thickness with accuracy of 1%. I.e.
> if 0.17 cg is recommended, you will get identical images in terms of quality
> analysis only for cg thickness range 168-172 micrometer.
>
> However, chromatic aberrations are negligible if you image not deeper than 3
> micrometer into specimen and use standart cg, i.e 170 +/- 15. For deeper
> imaging  you can get false negative\positive in FISH.
>
> If you want quantitative result for imaging depth up to 20-30 micrometer, it is
> better to deconvolve images obtained. My favourite packages are Hyugens
> Suite (commercial) and COSMOS (opensource), they perform most accurately
> among the ones I tested.
>
> Best,
> Sergey Tauger
>
> PhD student
> Cell motility lab.
> Dept. of Biology
> Moscow State University
>
> Mark  B. Cannell Ph.D. FRSNZ
> Professor of Cardiac Cell Biology
> School of Physiology &  Pharmacology
> Medical Sciences Building
> University of Bristol
> Bristol
> BS8 1TD UK
>
> [hidden email]

[mmodel]

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For a zero Stokes shift, longitudinal blurring should be insignificant because longitudinal shift in excitation should be corrected by the same shift in emission and all emitted photons will arrive at the same place. But for a non-zero shift things may happen of course.

Mike
________________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Laevsky, Gary S. <[hidden email]>
Sent: Friday, February 28, 2014 5:59 AM
To: [hidden email]
Subject: Re: Chromatic aberration and refractive index of a sample

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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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Hi Mark,

I would think spherical aberration is mostly a longitudinal artifact.  And would affect chromatic similarly, meaning it would dump photons not in the same plane, thereby affecting the image (“cutting” out the blue (hypothetical) when it “should” be colocalized with the red.).

But my main question is, is there a correction for chromatic aberration after the excitation beam is cleaned up by the objective?  Or is it necessary?  Sounds like in thin “er” samples, less than 10 um, it is not necessary?

Greater than that and fiducially with a warping algorithm?

Thanks again



Best,


Gary



On Feb 28, 2014, at 3:43 AM, Mark Cannell <[hidden email]<mailto:[hidden email]>> wrote:

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

Chromatic aberrations are highly objective/specimen dependent. The biggest concern for confocal is longitudinal chromatic aberrations as the pinhole mostly takes care of spherical aberration (by dumping photons).

My 2c

Mark



On 28/02/2014, at 8:27 am, Sergey Tauger <[hidden email]> wrote:

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

Hi Gary,

For any objective, aberrations are corrected only for first 0.5-1 micrometer and
only if you use coverglass of recommended thickness with accuracy of 1%. I.e.
if 0.17 cg is recommended, you will get identical images in terms of quality
analysis only for cg thickness range 168-172 micrometer.

However, chromatic aberrations are negligible if you image not deeper than 3
micrometer into specimen and use standart cg, i.e 170 +/- 15. For deeper
imaging  you can get false negative\positive in FISH.

If you want quantitative result for imaging depth up to 20-30 micrometer, it is
better to deconvolve images obtained. My favourite packages are Hyugens
Suite (commercial) and COSMOS (opensource), they perform most accurately
among the ones I tested.

Best,
Sergey Tauger

PhD student
Cell motility lab.
Dept. of Biology
Moscow State University

Mark  B. Cannell Ph.D. FRSNZ
Professor of Cardiac Cell Biology
School of Physiology &  Pharmacology
Medical Sciences Building
University of Bristol
Bristol
BS8 1TD UK

[hidden email]

[Mark Cannell-2]

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

Hi Gary

Chromatic longitudinal focal shift is not the same as spherical aberration. The former can be much more than the nominal diffraction limit (depending on difference of wavelengths of course and objective correction). The longitudinal focal shift arises from the change in power of the system with wavelength while spherical aberration is due to the centre of curvature of the wavefronts not coinciding with the focus (hence the longitudinal asymmetry in PSF). Provided you know the focal shift (think about teraspec beads embedded with sample) the longitudinal shift may be fixed in 3D images later.

Cheers

On 28/02/2014, at 10:59 am, Laevsky, Gary S. <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Mark,
>
> I would think spherical aberration is mostly a longitudinal artifact.  And would affect chromatic similarly, meaning it would dump photons not in the same plane, thereby affecting the image (“cutting” out the blue (hypothetical) when it “should” be colocalized with the red.).
>
> But my main question is, is there a correction for chromatic aberration after the excitation beam is cleaned up by the objective?  Or is it necessary?  Sounds like in thin “er” samples, less than 10 um, it is not necessary?
>
> Greater than that and fiducially with a warping algorithm?
>
> Thanks again
>
>
>
> Best,
>
>
> Gary
>
>
>
> On Feb 28, 2014, at 3:43 AM, Mark Cannell <[hidden email]<mailto:[hidden email]>> wrote:
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Chromatic aberrations are highly objective/specimen dependent. The biggest concern for confocal is longitudinal chromatic aberrations as the pinhole mostly takes care of spherical aberration (by dumping photons).
>
> My 2c
>
> Mark
>
>
>
> On 28/02/2014, at 8:27 am, Sergey Tauger <[hidden email]> wrote:
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Gary,
>
> For any objective, aberrations are corrected only for first 0.5-1 micrometer and
> only if you use coverglass of recommended thickness with accuracy of 1%. I.e.
> if 0.17 cg is recommended, you will get identical images in terms of quality
> analysis only for cg thickness range 168-172 micrometer.
>
> However, chromatic aberrations are negligible if you image not deeper than 3
> micrometer into specimen and use standart cg, i.e 170 +/- 15. For deeper
> imaging  you can get false negative\positive in FISH.
>
> If you want quantitative result for imaging depth up to 20-30 micrometer, it is
> better to deconvolve images obtained. My favourite packages are Hyugens
> Suite (commercial) and COSMOS (opensource), they perform most accurately
> among the ones I tested.
>
> Best,
> Sergey Tauger
>
> PhD student
> Cell motility lab.
> Dept. of Biology
> Moscow State University
>
> Mark  B. Cannell Ph.D. FRSNZ
> Professor of Cardiac Cell Biology
> School of Physiology &  Pharmacology
> Medical Sciences Building
> University of Bristol
> Bristol
> BS8 1TD UK
>
> [hidden email]

Mark  B. Cannell Ph.D. FRSNZ
Professor of Cardiac Cell Biology
School of Physiology &  Pharmacology
Medical Sciences Building
University of Bristol
Bristol
BS8 1TD UK

[hidden email]

[Laevsky, Gary S.]

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Hi Mark,

I knew that, but forgot!  Thanks.

Mike did make an interesting point, I think.  Does the correction occur in both directions?  Assuming a blue to red correction for excitation, a blue to green emission would be corrected for?

So the only real way to correct for chromatic aberration would be to put tetraspeck beads in the sample and then apply a warping correction algorithm (an Olympus rep did suggest this (not an Olympus system).  This would seem logistically impractical for many applications though.  Trade offs ...



Best,


Gary



On Feb 28, 2014, at 6:56 AM, Mark Cannell <[hidden email]<mailto:[hidden email]>> wrote:

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Hi Gary

Chromatic longitudinal focal shift is not the same as spherical aberration. The former can be much more than the nominal diffraction limit (depending on difference of wavelengths of course and objective correction). The longitudinal focal shift arises from the change in power of the system with wavelength while spherical aberration is due to the centre of curvature of the wavefronts not coinciding with the focus (hence the longitudinal asymmetry in PSF). Provided you know the focal shift (think about teraspec beads embedded with sample) the longitudinal shift may be fixed in 3D images later.

Cheers

On 28/02/2014, at 10:59 am, Laevsky, Gary S. <[hidden email]<mailto:[hidden email]>> wrote:

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Hi Mark,

I would think spherical aberration is mostly a longitudinal artifact.  And would affect chromatic similarly, meaning it would dump photons not in the same plane, thereby affecting the image (“cutting” out the blue (hypothetical) when it “should” be colocalized with the red.).

But my main question is, is there a correction for chromatic aberration after the excitation beam is cleaned up by the objective?  Or is it necessary?  Sounds like in thin “er” samples, less than 10 um, it is not necessary?

Greater than that and fiducially with a warping algorithm?

Thanks again



Best,


Gary



On Feb 28, 2014, at 3:43 AM, Mark Cannell <[hidden email]<mailto:[hidden email]><mailto:[hidden email]>> wrote:

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Chromatic aberrations are highly objective/specimen dependent. The biggest concern for confocal is longitudinal chromatic aberrations as the pinhole mostly takes care of spherical aberration (by dumping photons).

My 2c

Mark



On 28/02/2014, at 8:27 am, Sergey Tauger <[hidden email]> wrote:

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

Hi Gary,

For any objective, aberrations are corrected only for first 0.5-1 micrometer and
only if you use coverglass of recommended thickness with accuracy of 1%. I.e.
if 0.17 cg is recommended, you will get identical images in terms of quality
analysis only for cg thickness range 168-172 micrometer.

However, chromatic aberrations are negligible if you image not deeper than 3
micrometer into specimen and use standart cg, i.e 170 +/- 15. For deeper
imaging  you can get false negative\positive in FISH.

If you want quantitative result for imaging depth up to 20-30 micrometer, it is
better to deconvolve images obtained. My favourite packages are Hyugens
Suite (commercial) and COSMOS (opensource), they perform most accurately
among the ones I tested.

Best,
Sergey Tauger

PhD student
Cell motility lab.
Dept. of Biology
Moscow State University

Mark  B. Cannell Ph.D. FRSNZ
Professor of Cardiac Cell Biology
School of Physiology &  Pharmacology
Medical Sciences Building
University of Bristol
Bristol
BS8 1TD UK

[hidden email]

Mark  B. Cannell Ph.D. FRSNZ
Professor of Cardiac Cell Biology
School of Physiology &  Pharmacology
Medical Sciences Building
University of Bristol
Bristol
BS8 1TD UK

[hidden email]<mailto:[hidden email]>