Using a mirror for axial resolution testing

[John Oreopoulos]

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I have a question regarding the use of a mirror for measuring the axial resolution of a confocal imaging system (laser scanning or spinning disk).

Jim Pawley's handbook has a chapter that describes a few useful tests that can be done across different wavelengths to assess your microscope's axial resolution using a mirror. I have wondered for quite some time if there is any difference between using a mirror and the excitation wavelengths or a fluorescent object (microsphere) to test axial resolution. I could only find one posting on this in the archive:

http://lists.umn.edu/cgi-bin/wa?A2=ind0106&L=CONFOCALMICROSCOPY&D=0&P=8438

So I'm curious, what is the consensus out there right now? Is there a difference or not? I haven't actually had a chance to test it yet and see if the results obtained with a mirror (with laser illumination) match those obtained with a sub-diffraction sized fluorescent microbead.

John Oreopoulos
Research Assistant
Spectral Applied Research
Richmond Hill, Ontario
Canada
www.spectral.ca

[Lutz Schaefer]

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John,

obviously there is a difference. Besides the different wavelengths, when
using a mirror the image formation in the detection path is almost coherent.
With a fluorescent object on the other hand, it is incoherent. The
difference between these two forward problems lies in the way how the final
intensities on the camera are produced. In the coherent case, (complex)
amplitudes of elementary sample waves add up (causing the typical
constructive/destructive interference) before they become subject to
convolution with the observation PSF. Finally, the camera will sense the
absolute intensity. In the incoherent case, no interference takes place. The
forward problem can be modeled as the sum of the intensities before
convolution. This is the fundamental difference! In practice you will also
see that the mirror images will show depth varying transverse fringe
patterns when your mirror has a good quality, indicating partial coherence.
This is clearly not the case when you are using a SIP chart. To me, adding
amplitudes and adding intensities are different quantities after convolution
that you can't really compare against each other better than approximately.
On that subject is plentiful literature available, one contribution that
comes immediately to mind is Norbert Streibl's thesis from 1984.

Best Regards
Lutz

__________________________________
L u t z   S c h a e f e r
Sen. Scientist
Mathematical modeling / Image processing
Advanced Imaging Methodology Consultation
16-715 Doon Village Rd.
Kitchener, ON, N2P 2A2, Canada
Phone/Fax: [phone]
Email:     [hidden email]
Website: http://home.golden.net/~lschafer/
___________________________________



--------------------------------------------------
From: "John Oreopoulos" <[hidden email]>
Sent: Sunday, July 03, 2011 12:25
To: <[hidden email]>
Subject: Using a mirror for axial resolution testing

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I have a question regarding the use of a mirror for measuring the axial
> resolution of a confocal imaging system (laser scanning or spinning disk).
>
> Jim Pawley's handbook has a chapter that describes a few useful tests that
> can be done across different wavelengths to assess your microscope's axial
> resolution using a mirror. I have wondered for quite some time if there is
> any difference between using a mirror and the excitation wavelengths or a
> fluorescent object (microsphere) to test axial resolution. I could only
> find one posting on this in the archive:
>
> http://lists.umn.edu/cgi-bin/wa?A2=ind0106&L=CONFOCALMICROSCOPY&D=0&P=8438
>
> So I'm curious, what is the consensus out there right now? Is there a
> difference or not? I haven't actually had a chance to test it yet and see
> if the results obtained with a mirror (with laser illumination) match
> those obtained with a sub-diffraction sized fluorescent microbead.
>
> John Oreopoulos
> Research Assistant
> Spectral Applied Research
> Richmond Hill, Ontario
> Canada
> www.spectral.ca=

[John Oreopoulos]

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

Alright, suppose the light source is incoherent then. There are a few ways to achieve this. Would then the mirror test be equivalent to the fluorescent microbead test?

Also, I should have mentioned that the handbook is very clear about using a coverslipped mirror surface with the correct immersion medium between the coverslip and the mirror to avoid spherical aberration.

John Oreopoulos

On 2011-07-03, at 1:15 PM, Lutz Schaefer wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> John,
>
> obviously there is a difference. Besides the different wavelengths, when using a mirror the image formation in the detection path is almost coherent. With a fluorescent object on the other hand, it is incoherent. The difference between these two forward problems lies in the way how the final intensities on the camera are produced. In the coherent case, (complex) amplitudes of elementary sample waves add up (causing the typical constructive/destructive interference) before they become subject to convolution with the observation PSF. Finally, the camera will sense the absolute intensity. In the incoherent case, no interference takes place. The forward problem can be modeled as the sum of the intensities before convolution. This is the fundamental difference! In practice you will also see that the mirror images will show depth varying transverse fringe patterns when your mirror has a good quality, indicating partial coherence. This is clearly not the case when you are using a SIP chart. To me, adding amplitudes and adding intensities are different quantities after convolution that you can't really compare against each other better than approximately. On that subject is plentiful literature available, one contribution that comes immediately to mind is Norbert Streibl's thesis from 1984.
>
> Best Regards
> Lutz
>
> __________________________________
> L u t z   S c h a e f e r
> Sen. Scientist
> Mathematical modeling / Image processing
> Advanced Imaging Methodology Consultation
> 16-715 Doon Village Rd.
> Kitchener, ON, N2P 2A2, Canada
> Phone/Fax: [phone]
> Email:     [hidden email]
> Website: http://home.golden.net/~lschafer/
> ___________________________________
>
>
>
> --------------------------------------------------
> From: "John Oreopoulos" <[hidden email]>
> Sent: Sunday, July 03, 2011 12:25
> To: <[hidden email]>
> Subject: Using a mirror for axial resolution testing
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> I have a question regarding the use of a mirror for measuring the axial resolution of a confocal imaging system (laser scanning or spinning disk).
>>
>> Jim Pawley's handbook has a chapter that describes a few useful tests that can be done across different wavelengths to assess your microscope's axial resolution using a mirror. I have wondered for quite some time if there is any difference between using a mirror and the excitation wavelengths or a fluorescent object (microsphere) to test axial resolution. I could only find one posting on this in the archive:
>>
>> http://lists.umn.edu/cgi-bin/wa?A2=ind0106&L=CONFOCALMICROSCOPY&D=0&P=8438
>>
>> So I'm curious, what is the consensus out there right now? Is there a difference or not? I haven't actually had a chance to test it yet and see if the results obtained with a mirror (with laser illumination) match those obtained with a sub-diffraction sized fluorescent microbead.
>>
>> John Oreopoulos
>> Research Assistant
>> Spectral Applied Research
>> Richmond Hill, Ontario
>> Canada
>> www.spectral.ca=

[Lutz Schaefer]

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

John,

an incoherent light source would not change the fact of a partially coherent
forward problem! The illumination light still has a chance to interfere with
the observation light, which is not the case with fluorescence. If, and only
if you can guarantee that any observation light emanating from the mirror is
indeed incoherent and would never interfere with the oncoming illumination
light and with itself (e.g. due to mirror imperfections), then both results
should be identical. I can see this condition easily violated. Though, for
approximate axial response estimation it may be sufficient, at best. Using
the cover slip is required to impose lens design conditions for a 3 layer
refractive system (in your case the thickness of the 3rd embedding layer
approaches zero length).

regards
Lutz

__________________________________
L u t z   S c h a e f e r
Sen. Scientist
Mathematical modeling / Image processing
Advanced Imaging Methodology Consultation
16-715 Doon Village Rd.
Kitchener, ON, N2P 2A2, Canada
Phone/Fax: [phone]
Email:     [hidden email]
Website: http://home.golden.net/~lschafer/
___________________________________

--------------------------------------------------
From: "John Oreopoulos" <[hidden email]>
Sent: Sunday, July 03, 2011 13:30
To: <[hidden email]>
Subject: Re: Using a mirror for axial resolution testing

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Alright, suppose the light source is incoherent then. There are a few ways
> to achieve this. Would then the mirror test be equivalent to the
> fluorescent microbead test?
>
> Also, I should have mentioned that the handbook is very clear about using
> a coverslipped mirror surface with the correct immersion medium between
> the coverslip and the mirror to avoid spherical aberration.
>
> John Oreopoulos
>
> On 2011-07-03, at 1:15 PM, Lutz Schaefer wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> John,
>>
>> obviously there is a difference. Besides the different wavelengths, when
>> using a mirror the image formation in the detection path is almost
>> coherent. With a fluorescent object on the other hand, it is incoherent.
>> The difference between these two forward problems lies in the way how the
>> final intensities on the camera are produced. In the coherent case,
>> (complex) amplitudes of elementary sample waves add up (causing the
>> typical constructive/destructive interference) before they become subject
>> to convolution with the observation PSF. Finally, the camera will sense
>> the absolute intensity. In the incoherent case, no interference takes
>> place. The forward problem can be modeled as the sum of the intensities
>> before convolution. This is the fundamental difference! In practice you
>> will also see that the mirror images will show depth varying transverse
>> fringe patterns when your mirror has a good quality, indicating partial
>> coherence. This is clearly not the case when you are using a SIP chart.
>> To me, adding amplitudes and adding intensities are different quantities
>> after convolution that you can't really compare against each other better
>> than approximately. On that subject is plentiful literature available,
>> one contribution that comes immediately to mind is Norbert Streibl's
>> thesis from 1984.
>>
>> Best Regards
>> Lutz
>>
>> __________________________________
>> L u t z   S c h a e f e r
>> Sen. Scientist
>> Mathematical modeling / Image processing
>> Advanced Imaging Methodology Consultation
>> 16-715 Doon Village Rd.
>> Kitchener, ON, N2P 2A2, Canada
>> Phone/Fax: [phone]
>> Email:     [hidden email]
>> Website: http://home.golden.net/~lschafer/
>> ___________________________________
>>
>>
>>
>> --------------------------------------------------
>> From: "John Oreopoulos" <[hidden email]>
>> Sent: Sunday, July 03, 2011 12:25
>> To: <[hidden email]>
>> Subject: Using a mirror for axial resolution testing
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> I have a question regarding the use of a mirror for measuring the axial
>>> resolution of a confocal imaging system (laser scanning or spinning
>>> disk).
>>>
>>> Jim Pawley's handbook has a chapter that describes a few useful tests
>>> that can be done across different wavelengths to assess your
>>> microscope's axial resolution using a mirror. I have wondered for quite
>>> some time if there is any difference between using a mirror and the
>>> excitation wavelengths or a fluorescent object (microsphere) to test
>>> axial resolution. I could only find one posting on this in the archive:
>>>
>>> http://lists.umn.edu/cgi-bin/wa?A2=ind0106&L=CONFOCALMICROSCOPY&D=0&P=8438
>>>
>>> So I'm curious, what is the consensus out there right now? Is there a
>>> difference or not? I haven't actually had a chance to test it yet and
>>> see if the results obtained with a mirror (with laser illumination)
>>> match those obtained with a sub-diffraction sized fluorescent microbead.
>>>
>>> John Oreopoulos
>>> Research Assistant
>>> Spectral Applied Research
>>> Richmond Hill, Ontario
>>> Canada
>>> www.spectral.ca=

[John Oreopoulos]

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

I'd be curious to know from anyone on this listserver who's tried the mirror test with a spinning disk confocal system what axial resolution they've measured (FWHM of the transverse profile line taken through the reflected surface as seen in the XZ or YZ plane). What are the typical values for the various excitation wavelengths that you've found when using a high NA oil objective (~1.4)?

John Oreopoulos
Research Assistant
Spectral Applied Research
Richmond Hill, Ontario
Canada
www.spectral.ca

On 2011-07-03, at 4:17 PM, Lutz Schaefer wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> John,
>
> an incoherent light source would not change the fact of a partially coherent forward problem! The illumination light still has a chance to interfere with the observation light, which is not the case with fluorescence. If, and only if you can guarantee that any observation light emanating from the mirror is indeed incoherent and would never interfere with the oncoming illumination light and with itself (e.g. due to mirror imperfections), then both results should be identical. I can see this condition easily violated. Though, for approximate axial response estimation it may be sufficient, at best. Using the cover slip is required to impose lens design conditions for a 3 layer refractive system (in your case the thickness of the 3rd embedding layer approaches zero length).
>
> regards
> Lutz
>
> __________________________________
> L u t z   S c h a e f e r
> Sen. Scientist
> Mathematical modeling / Image processing
> Advanced Imaging Methodology Consultation
> 16-715 Doon Village Rd.
> Kitchener, ON, N2P 2A2, Canada
> Phone/Fax: [phone]
> Email:     [hidden email]
> Website: http://home.golden.net/~lschafer/
> ___________________________________
>
> --------------------------------------------------
> From: "John Oreopoulos" <[hidden email]>
> Sent: Sunday, July 03, 2011 13:30
> To: <[hidden email]>
> Subject: Re: Using a mirror for axial resolution testing
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Alright, suppose the light source is incoherent then. There are a few ways to achieve this. Would then the mirror test be equivalent to the fluorescent microbead test?
>>
>> Also, I should have mentioned that the handbook is very clear about using a coverslipped mirror surface with the correct immersion medium between the coverslip and the mirror to avoid spherical aberration.
>>
>> John Oreopoulos
>>
>> On 2011-07-03, at 1:15 PM, Lutz Schaefer wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> John,
>>>
>>> obviously there is a difference. Besides the different wavelengths, when using a mirror the image formation in the detection path is almost coherent. With a fluorescent object on the other hand, it is incoherent. The difference between these two forward problems lies in the way how the final intensities on the camera are produced. In the coherent case, (complex) amplitudes of elementary sample waves add up (causing the typical constructive/destructive interference) before they become subject to convolution with the observation PSF. Finally, the camera will sense the absolute intensity. In the incoherent case, no interference takes place. The forward problem can be modeled as the sum of the intensities before convolution. This is the fundamental difference! In practice you will also see that the mirror images will show depth varying transverse fringe patterns when your mirror has a good quality, indicating partial coherence. This is clearly not the case when you are using a SIP chart. To me, adding amplitudes and adding intensities are different quantities after convolution that you can't really compare against each other better than approximately. On that subject is plentiful literature available, one contribution that comes immediately to mind is Norbert Streibl's thesis from 1984.
>>>
>>> Best Regards
>>> Lutz
>>>
>>> __________________________________
>>> L u t z   S c h a e f e r
>>> Sen. Scientist
>>> Mathematical modeling / Image processing
>>> Advanced Imaging Methodology Consultation
>>> 16-715 Doon Village Rd.
>>> Kitchener, ON, N2P 2A2, Canada
>>> Phone/Fax: [phone]
>>> Email:     [hidden email]
>>> Website: http://home.golden.net/~lschafer/
>>> ___________________________________
>>>
>>>
>>>
>>> --------------------------------------------------
>>> From: "John Oreopoulos" <[hidden email]>
>>> Sent: Sunday, July 03, 2011 12:25
>>> To: <[hidden email]>
>>> Subject: Using a mirror for axial resolution testing
>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> I have a question regarding the use of a mirror for measuring the axial resolution of a confocal imaging system (laser scanning or spinning disk).
>>>>
>>>> Jim Pawley's handbook has a chapter that describes a few useful tests that can be done across different wavelengths to assess your microscope's axial resolution using a mirror. I have wondered for quite some time if there is any difference between using a mirror and the excitation wavelengths or a fluorescent object (microsphere) to test axial resolution. I could only find one posting on this in the archive:
>>>>
>>>> http://lists.umn.edu/cgi-bin/wa?A2=ind0106&L=CONFOCALMICROSCOPY&D=0&P=8438
>>>>
>>>> So I'm curious, what is the consensus out there right now? Is there a difference or not? I haven't actually had a chance to test it yet and see if the results obtained with a mirror (with laser illumination) match those obtained with a sub-diffraction sized fluorescent microbead.
>>>>
>>>> John Oreopoulos
>>>> Research Assistant
>>>> Spectral Applied Research
>>>> Richmond Hill, Ontario
>>>> Canada
>>>> www.spectral.ca=