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PSF asymetry

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lechristophe

PSF asymetry

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Dear microscopists,

I am using a very nice TIRF microscope with a 100X, 1.49 NA objective. I
had the impression that there was a slight lateral shift when defocusing up
and down, so I checked the PSF with 100 nm beads on a HR #1.5 coverslip.
What appears on the attached image (three planes taken at -1, 0 and +1 um)
is that the PSF is not rotationnally symetric, i.e.more intense on the
left-bottom side :

https://drive.google.com/file/d/0B_JeGjE7nBHWWFViM0pwSy0xR3M/view

This asymetry is quite constant over the field of view (it is not radial
relative to the center of the field). It does not depend on the
illumination (it is the same under azimutal laser, TIRF laser,
epifluorescence lamp). It does not depend on the filter cube used. Finally
(and this is what surprises me the most), I got another brand new 100X,
1.49 objective for testing and it still shows up (the attached image is
taken with the new objective).

Do you have an idea if what could be wrong, and how to correct it? Could it
be caused by an internal lens? By the sample used?

Thanks for your help,

Christophe

Eric Marino

Re: PSF asymetry

> On Apr 21, 2015, at 4:10 AM, Christophe Leterrier <[hidden email]> wrote:
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear microscopists,
>
> I am using a very nice TIRF microscope with a 100X, 1.49 NA objective. I
> had the impression that there was a slight lateral shift when defocusing up
> and down, so I checked the PSF with 100 nm beads on a HR #1.5 coverslip.
> What appears on the attached image (three planes taken at -1, 0 and +1 um)
> is that the PSF is not rotationnally symetric, i.e.more intense on the
> left-bottom side :
>
> https://drive.google.com/file/d/0B_JeGjE7nBHWWFViM0pwSy0xR3M/view
>
> This asymetry is quite constant over the field of view (it is not radial
> relative to the center of the field). It does not depend on the
> illumination (it is the same under azimutal laser, TIRF laser,
> epifluorescence lamp). It does not depend on the filter cube used. Finally
> (and this is what surprises me the most), I got another brand new 100X,
> 1.49 objective for testing and it still shows up (the attached image is
> taken with the new objective).
>
> Do you have an idea if what could be wrong, and how to correct it? Could it
> be caused by an internal lens? By the sample used?
>
> Thanks for your help,
>
> Christophe

Eric Marino
[hidden email]

John Oreopoulos

Re: PSF asymetry

In reply to this post by lechristophe

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Just a guess here, maybe the nosepiece is not engaging properly (ie: objective is no co-linear with the optic axis). A mis-aligned tube lens? What happens if you take the objective(s) to another microscope?
What about the microscope stage? Could it be grossly tilted in some manner? Some stages come with an adjustable stage insert to make the sample coverslip perpendicular to the optic axis.

You mentioned it was a TIRF objective, so I imagine you use this with immersion oil and a sample embedded in water. However, your images do look reminiscent of the abberation mentioned in this paper:

Arimoto, R. and J.M. Murray, A common aberration with water-immersion objective lenses. Journal of Microscopy-Oxford, 2004. 216: p. 49-51.

John Oreopoulos
Staff Scientist
Spectral Applied Research Inc.
A Division of Andor Technology
Richmond Hill, Ontario
Canada
www.spectral.ca

On 2015-04-21, at 4:10 AM, Christophe Leterrier wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear microscopists,
>
> I am using a very nice TIRF microscope with a 100X, 1.49 NA objective. I
> had the impression that there was a slight lateral shift when defocusing up
> and down, so I checked the PSF with 100 nm beads on a HR #1.5 coverslip.
> What appears on the attached image (three planes taken at -1, 0 and +1 um)
> is that the PSF is not rotationnally symetric, i.e.more intense on the
> left-bottom side :
>
> https://drive.google.com/file/d/0B_JeGjE7nBHWWFViM0pwSy0xR3M/view
>
> This asymetry is quite constant over the field of view (it is not radial
> relative to the center of the field). It does not depend on the
> illumination (it is the same under azimutal laser, TIRF laser,
> epifluorescence lamp). It does not depend on the filter cube used. Finally
> (and this is what surprises me the most), I got another brand new 100X,
> 1.49 objective for testing and it still shows up (the attached image is
> taken with the new objective).
>
> Do you have an idea if what could be wrong, and how to correct it? Could it
> be caused by an internal lens? By the sample used?
>
> Thanks for your help,
>
> Christophe

Peng Xi-2

Re: PSF asymetry

In reply to this post by lechristophe

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Dear Christophe,
Is your TIRF rotational polarization or linear polarization? It seems
to me like a fluorescence dipole PSF.

--
Sincerely,
Peng Xi
Ph. D. Associate Professor
Dept. of Biomedical Engineering, College of Engineering
Peking University, Beijing, China
Tel: +86 10-6276 7155
Email: [hidden email]
http://bme.pku.edu.cn/~xipeng/

On Tue, Apr 21, 2015 at 4:10 PM, Christophe Leterrier <
[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear microscopists,
>
> I am using a very nice TIRF microscope with a 100X, 1.49 NA objective. I
> had the impression that there was a slight lateral shift when defocusing up
> and down, so I checked the PSF with 100 nm beads on a HR #1.5 coverslip.
> What appears on the attached image (three planes taken at -1, 0 and +1 um)
> is that the PSF is not rotationnally symetric, i.e.more intense on the
> left-bottom side :
>
> https://drive.google.com/file/d/0B_JeGjE7nBHWWFViM0pwSy0xR3M/view
>
> This asymetry is quite constant over the field of view (it is not radial
> relative to the center of the field). It does not depend on the
> illumination (it is the same under azimutal laser, TIRF laser,
> epifluorescence lamp). It does not depend on the filter cube used. Finally
> (and this is what surprises me the most), I got another brand new 100X,
> 1.49 objective for testing and it still shows up (the attached image is
> taken with the new objective).
>
> Do you have an idea if what could be wrong, and how to correct it? Could it
> be caused by an internal lens? By the sample used?
>
> Thanks for your help,
>
> Christophe
>

Smith, Benjamin E.

Re: PSF asymetry

In reply to this post by John Oreopoulos

*****
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*****

It definitely made my day to read, "Long after it should have been apparent, we realized that this aberration is a result of tilt of the coverslip." This is such a classic moment in science, to the point that every paper, if the authors were being brutally honest, would have a sentence to the effect of, "Long after it should have been apparent, we realized that...[insert discovery that seems obvious in hindsight, but no one initially anticipated]."

Carry on,
Ben Smith

Benjamin E. Smith, Ph.D.
Samuel Roberts Noble Microscopy Laboratory
Research Scientist, Confocal Facility Manager
University of Oklahoma
Norman, OK 73019
E-mail: [hidden email]
Voice 405-325-4391
FAX 405-325-7619
http://www.microscopy.ou.edu/

________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of John Oreopoulos [[hidden email]]
Sent: Tuesday, April 21, 2015 7:18 AM
To: [hidden email]
Subject: Re: PSF asymetry

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Just a guess here, maybe the nosepiece is not engaging properly (ie: objective is no co-linear with the optic axis). A mis-aligned tube lens? What happens if you take the objective(s) to another microscope?
What about the microscope stage? Could it be grossly tilted in some manner? Some stages come with an adjustable stage insert to make the sample coverslip perpendicular to the optic axis.

You mentioned it was a TIRF objective, so I imagine you use this with immersion oil and a sample embedded in water. However, your images do look reminiscent of the abberation mentioned in this paper:

Arimoto, R. and J.M. Murray, A common aberration with water-immersion objective lenses. Journal of Microscopy-Oxford, 2004. 216: p. 49-51.

John Oreopoulos
Staff Scientist
Spectral Applied Research Inc.
A Division of Andor Technology
Richmond Hill, Ontario
Canada
www.spectral.ca

On 2015-04-21, at 4:10 AM, Christophe Leterrier wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear microscopists,
>
> I am using a very nice TIRF microscope with a 100X, 1.49 NA objective. I
> had the impression that there was a slight lateral shift when defocusing up
> and down, so I checked the PSF with 100 nm beads on a HR #1.5 coverslip.
> What appears on the attached image (three planes taken at -1, 0 and +1 um)
> is that the PSF is not rotationnally symetric, i.e.more intense on the
> left-bottom side :
>
> https://drive.google.com/file/d/0B_JeGjE7nBHWWFViM0pwSy0xR3M/view
>
> This asymetry is quite constant over the field of view (it is not radial
> relative to the center of the field). It does not depend on the
> illumination (it is the same under azimutal laser, TIRF laser,
> epifluorescence lamp). It does not depend on the filter cube used. Finally
> (and this is what surprises me the most), I got another brand new 100X,
> 1.49 objective for testing and it still shows up (the attached image is
> taken with the new objective).
>
> Do you have an idea if what could be wrong, and how to correct it? Could it
> be caused by an internal lens? By the sample used?
>
> Thanks for your help,
>
> Christophe

Kyle Michael Douglass

Re: PSF asymetry

In reply to this post by Peng Xi-2

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*****

Hi Christophe and Professor Xi,

The images are of fluorescent beads, which should have many different
fluorescent labels attached to their surfaces with dipoles pointing in
many different directions. I don't think that any single dipolar
emission pattern should be obvious since the patterns from all the other
labels on the same sphere should average out, but I could be wrong about
this.

I've also seen this same aberration in images of 100 nm fluorescent
beads on a custom microscope with multiple NA >= 1.45, 100x oil
immersion objectives. No degree of alignment of the imaging pathway
seems to fully correct it, at least in my hands. In the end, I also
suspected coverslip tilt based on the Arimoto paper and this
dissertation from Stefan Hell's lab:
https://ediss.uni-goettingen.de/bitstream/handle/11858/00-1735-0000-000D-F0B1-E/berning.pdf?sequence=1

Best,
Kyle

On 04/21/2015 03:01 PM, Peng Xi wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear Christophe,
> Is your TIRF rotational polarization or linear polarization? It seems
> to me like a fluorescence dipole PSF.
>

--
Kyle M. Douglass, PhD
Post-doctoral researcher
The Laboratory of Experimental Biophysics
EPFL, Lausanne, Switzerland
http://kmdouglass.github.io
http://leb.epfl.ch

Weis, Michael-2

Re: PSF asymetry

In reply to this post by lechristophe

I recently had this same problem with one objective on a new confocal installation here. After acquiring a XYZ stack documenting the lateral shift of the PSF I rotated the objective 90 degrees and acquired another stack. The lateral shift rotates the same as the objective rotates therefore the defect is in the objective. The manufacturer is replacing the objective.

Cheers, Michael

Michael Weis
Supervisor,                                            Superviser,
Microscopy Facility                                Installation de microscopie
Science and Technology Branch        Direction générale des sciences et de la technologie
Pacific Agri-Food Research Centre    Centre de recherches agroalimentaire du Pacifique
P.O. Box 5000, 4200 Highway 97      Boîte postale 5000, 4200 Autoroute 97
Summerland, BC, V0H 1Z0, Canada    Summerland, CB, V0H 1Z0, Canada

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christophe Leterrier
Sent: April-21-15 1:10 AM
To: [hidden email]
Subject: PSF asymetry

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Dear microscopists,

I am using a very nice TIRF microscope with a 100X, 1.49 NA objective. I had the impression that there was a slight lateral shift when defocusing up and down, so I checked the PSF with 100 nm beads on a HR #1.5 coverslip.
What appears on the attached image (three planes taken at -1, 0 and +1 um) is that the PSF is not rotationnally symetric, i.e.more intense on the left-bottom side :

https://drive.google.com/file/d/0B_JeGjE7nBHWWFViM0pwSy0xR3M/view

This asymetry is quite constant over the field of view (it is not radial relative to the center of the field). It does not depend on the illumination (it is the same under azimutal laser, TIRF laser, epifluorescence lamp). It does not depend on the filter cube used. Finally (and this is what surprises me the most), I got another brand new 100X,
1.49 objective for testing and it still shows up (the attached image is taken with the new objective).

Do you have an idea if what could be wrong, and how to correct it? Could it be caused by an internal lens? By the sample used?

Thanks for your help,

Christophe

John Oreopoulos

Re: PSF asymetry

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

I suppose it's possible, but highly unlikely that Christophe was given two defective objective lenses here. Christophe, is the angle of asymmetry the same for both objective lenses?

John Oreopoulos

On 2015-04-21, at 11:42 AM, Weis, Michael wrote:

> I recently had this same problem with one objective on a new confocal installation here. After acquiring a XYZ stack documenting the lateral shift of the PSF I rotated the objective 90 degrees and acquired another stack. The lateral shift rotates the same as the objective rotates therefore the defect is in the objective. The manufacturer is replacing the objective.
>
> Cheers, Michael
>
>
> Michael Weis
> Supervisor, Superviser,
> Microscopy Facility Installation de microscopie
> Science and Technology Branch Direction générale des sciences et de la technologie
> Pacific Agri-Food Research Centre Centre de recherches agroalimentaire du Pacifique
> P.O. Box 5000, 4200 Highway 97 Boîte postale 5000, 4200 Autoroute 97
> Summerland, BC, V0H 1Z0, Canada Summerland, CB, V0H 1Z0, Canada
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christophe Leterrier
> Sent: April-21-15 1:10 AM
> To: [hidden email]
> Subject: PSF asymetry
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear microscopists,
>
> I am using a very nice TIRF microscope with a 100X, 1.49 NA objective. I had the impression that there was a slight lateral shift when defocusing up and down, so I checked the PSF with 100 nm beads on a HR #1.5 coverslip.
> What appears on the attached image (three planes taken at -1, 0 and +1 um) is that the PSF is not rotationnally symetric, i.e.more intense on the left-bottom side :
>
> https://drive.google.com/file/d/0B_JeGjE7nBHWWFViM0pwSy0xR3M/view
>
> This asymetry is quite constant over the field of view (it is not radial relative to the center of the field). It does not depend on the illumination (it is the same under azimutal laser, TIRF laser, epifluorescence lamp). It does not depend on the filter cube used. Finally (and this is what surprises me the most), I got another brand new 100X,
> 1.49 objective for testing and it still shows up (the attached image is taken with the new objective).
>
> Do you have an idea if what could be wrong, and how to correct it? Could it be caused by an internal lens? By the sample used?
>
> Thanks for your help,
>
> Christophe

Kyle Michael Douglass

Re: PSF asymetry

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Hi John and Christophe,
I have heard recent, albeit anecdotal, stories about the folks in deconvolution microscopy trying absurd numbers of high NA, 100x objectives to find ones that give clean, unaberrated axial PSF's. Since our lab has seen this aberration on multiple NA >= 1.45 objectives and Christophe and Michael have also seen it, I am wondering if the axial PSF is too difficult to tolerance at large NA's during manufacturing. This would lead to a large spread in performance.

I have also heard stories that some objective manufacturers will pull the very nice objectives from their production lines and sell them at a higher price under a different label, which also leads me to believe that the aberration we are discussing is actually quite common.

Everything I have said so far is admittedly conjecture. I know there are a lot of objective vendors on this list, so I'm hoping that one of them can offer their opinion on the matter.

Best,
Kyle
________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of John Oreopoulos [[hidden email]]
Sent: Tuesday, April 21, 2015 7:50 PM
To: [hidden email]
Subject: Re: PSF asymetry

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

I suppose it's possible, but highly unlikely that Christophe was given two defective objective lenses here. Christophe, is the angle of asymmetry the same for both objective lenses?

John Oreopoulos

On 2015-04-21, at 11:42 AM, Weis, Michael wrote:

lechristophe

Re: PSF asymetry

In reply to this post by lechristophe

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Dear all,

Thanks a lot for your advices and hypotheses. I think both 100X objective
give the same asymetry, so maybe the problem is somewhere else (or it is a
case of bad luck). I have realigned the laser, but it is not the source of
the problem since it also happens with epifluorescence illumination.
Attached is an uncropped image of 512x512 image from the EMCCD field of
view, taken with epifluorescence illumination:

https://drive.google.com/file/d/0B_JeGjE7nBHWMUFzZEJ0NDFWS1k/view

My impression is that the center of the optical axis is not in the center
of the field of view (does that makes sense?), so maybe a problem with
camera alignment?

Christophe

On Tue, Apr 21, 2015 at 10:10 AM, Christophe Leterrier <
[hidden email]> wrote:

> Dear microscopists,
>
> I am using a very nice TIRF microscope with a 100X, 1.49 NA objective. I
> had the impression that there was a slight lateral shift when defocusing up
> and down, so I checked the PSF with 100 nm beads on a HR #1.5 coverslip.
> What appears on the attached image (three planes taken at -1, 0 and +1 um)
> is that the PSF is not rotationnally symetric, i.e.more intense on the
> left-bottom side :
>
> https://drive.google.com/file/d/0B_JeGjE7nBHWWFViM0pwSy0xR3M/view
>
> This asymetry is quite constant over the field of view (it is not radial
> relative to the center of the field). It does not depend on the
> illumination (it is the same under azimutal laser, TIRF laser,
> epifluorescence lamp). It does not depend on the filter cube used. Finally
> (and this is what surprises me the most), I got another brand new 100X,
> 1.49 objective for testing and it still shows up (the attached image is
> taken with the new objective).
>
> Do you have an idea if what could be wrong, and how to correct it? Could
> it be caused by an internal lens? By the sample used?
>
> Thanks for your help,
>
> Christophe
>

John Oreopoulos

Re: PSF asymetry

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

Christophe,

Yes, it looks like you've got some severe distortion or field curvature in the lower right corner of that image as well. Your question about the camera alignment is a good one and pretty simple to answer. Does an object that appears centred in the eyepiece field of view centred in the camera field of view as well?

Cheers,

John Oreopoulos

On 2015-04-22, at 5:30 AM, Christophe Leterrier wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear all,
>
> Thanks a lot for your advices and hypotheses. I think both 100X objective
> give the same asymetry, so maybe the problem is somewhere else (or it is a
> case of bad luck). I have realigned the laser, but it is not the source of
> the problem since it also happens with epifluorescence illumination.
> Attached is an uncropped image of 512x512 image from the EMCCD field of
> view, taken with epifluorescence illumination:
>
> https://drive.google.com/file/d/0B_JeGjE7nBHWMUFzZEJ0NDFWS1k/view
>
> My impression is that the center of the optical axis is not in the center
> of the field of view (does that makes sense?), so maybe a problem with
> camera alignment?
>
> Christophe
>
> On Tue, Apr 21, 2015 at 10:10 AM, Christophe Leterrier <
> [hidden email]> wrote:
>
>> Dear microscopists,
>>
>> I am using a very nice TIRF microscope with a 100X, 1.49 NA objective. I
>> had the impression that there was a slight lateral shift when defocusing up
>> and down, so I checked the PSF with 100 nm beads on a HR #1.5 coverslip.
>> What appears on the attached image (three planes taken at -1, 0 and +1 um)
>> is that the PSF is not rotationnally symetric, i.e.more intense on the
>> left-bottom side :
>>
>> https://drive.google.com/file/d/0B_JeGjE7nBHWWFViM0pwSy0xR3M/view
>>
>> This asymetry is quite constant over the field of view (it is not radial
>> relative to the center of the field). It does not depend on the
>> illumination (it is the same under azimutal laser, TIRF laser,
>> epifluorescence lamp). It does not depend on the filter cube used. Finally
>> (and this is what surprises me the most), I got another brand new 100X,
>> 1.49 objective for testing and it still shows up (the attached image is
>> taken with the new objective).
>>
>> Do you have an idea if what could be wrong, and how to correct it? Could
>> it be caused by an internal lens? By the sample used?
>>
>> Thanks for your help,
>>
>> Christophe
>>