Re: Using a mirror for axial resolution testing

[Brad Amos]

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The equations for resolution in z are different for a plane reflector, a
thin fluorescent lamina and a subresolution bead. The commercial story on
this is a mess of unattributed equations of obscure provenance. You may find
useful information in the following article, which is a permitted preprint
of an article about confocal microscopy to be edited and published in the
Elsevier online 'Comprehensive Biophysics', where Tony Wilson, Gail
McConnell and I have tried to make sense of it all :
http://www2.mrc-lmb.cam.ac.uk/images/groupleaders/Confocal_microscopy_Amos_McConnell_Wilson.pdf

[John Oreopoulos]

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Hello again,

A few years ago I sent a question to the confocal listserver about the differences in measuring confocal axial resolution with sub-diffraction-sized beads or mirrors and fluorescent sheets (see messages attached below). At the time, one of the most informative responses came from Brad Amos (response message also below). Since then, I've been doing these types of measurements on various confocal instruments and can confirm with certainty that the two test specimens (fluorescent beads vs. fluorescent sheets) yield different results when measuring the FWHM of the axial profile. So my question now is: Which specimen should we be using to report instrument performance in terms of axial resolution? I've been to quite a few microscopy courses and the conventional wisdom seems to be that fluorescent beads are best, but Brad's book chapter that he linked to made an argument that fluorescent sheets are better and that it's incorrect to use beads to report axial resolution. In case anyone is interested, in my hands, fluorescent sheets consistently yield an axial resolution that is a few hundred nm bigger than what the bead measurement says for a given instrument with a particular set of optics. I'm hoping Brad might chime in again here or anyone else who has something to say about this.

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



On 2011-07-25, at 3:31 PM, Brad Amos wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> The equations for resolution in z are different for a plane reflector, a
> thin fluorescent lamina and a subresolution bead. The commercial story on
> this is a mess of unattributed equations of obscure provenance. You may find
> useful information in the following article, which is a permitted preprint
> of an article about confocal microscopy to be edited and published in the
> Elsevier online 'Comprehensive Biophysics', where Tony Wilson, Gail
> McConnell and I have tried to make sense of it all :
> http://www2.mrc-lmb.cam.ac.uk/images/groupleaders/Confocal_microscopy_Amos_McConnell_Wilson.pdf

*****
On 2011-07-03, at 12:25 PM, John Oreopoulos wrote:

> *****
> 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

[Craig Brideau]

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As your focus enters the sheet, the sheet itself will diffract the beam,
rather like focusing into a coverslip. At least that's my preliminary
thought.  A bead would disrupt the light path less.

Craig


On Fri, Oct 4, 2013 at 1:01 PM, John Oreopoulos <[hidden email]
> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello again,
>
> A few years ago I sent a question to the confocal listserver about the
> differences in measuring confocal axial resolution with
> sub-diffraction-sized beads or mirrors and fluorescent sheets (see messages
> attached below). At the time, one of the most informative responses came
> from Brad Amos (response message also below). Since then, I've been doing
> these types of measurements on various confocal instruments and can confirm
> with certainty that the two test specimens (fluorescent beads vs.
> fluorescent sheets) yield different results when measuring the FWHM of the
> axial profile. So my question now is: Which specimen should we be using to
> report instrument performance in terms of axial resolution? I've been to
> quite a few microscopy courses and the conventional wisdom seems to be that
> fluorescent beads are best, but Brad's book chapter that he linked to made
> an argument that fluorescent sheets are better and that it's incorrect to
> use beads to report axial resolution. In case anyone is interested, in my
> hands, fluorescent sheets consistently yield an axial resolution that is a
> few hundred nm bigger than what the bead measurement says for a given
> instrument with a particular set of optics. I'm hoping Brad might chime in
> again here or anyone else who has something to say about this.
>
> John Oreopoulos
> Staff Scientist
> Spectral Applied Research
> Richmond Hill, Ontario
> Canada
> www.spectral.ca
>
>
>
> On 2011-07-25, at 3:31 PM, Brad Amos wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > The equations for resolution in z are different for a plane reflector, a
> > thin fluorescent lamina and a subresolution bead. The commercial story on
> > this is a mess of unattributed equations of obscure provenance. You may
> find
> > useful information in the following article, which is a permitted
> preprint
> > of an article about confocal microscopy to be edited and published in the
> > Elsevier online 'Comprehensive Biophysics', where Tony Wilson, Gail
> > McConnell and I have tried to make sense of it all :
> >
> http://www2.mrc-lmb.cam.ac.uk/images/groupleaders/Confocal_microscopy_Amos_McConnell_Wilson.pdf
>
>
> *****
> On 2011-07-03, at 12:25 PM, John Oreopoulos wrote:
>
> > *****
> > 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]

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*****

So, I should clarify a bit. The fluorescent sheets I've been using are not actually sheets. I use the concentrated dye slides originally described by Mike Model:

Model, M.A. and J.L. Blank, Concentrated dyes as a source of two-dimensional fluorescent field for characterization of a confocal microscope. Journal of Microscopy-Oxford, 2008. 229(1): p. 12-16.

These specimens are all liquid, but they have the neat property that they only emit light from an diffraction-limited thick zone adjacent to the coverslip. Essentially, they act as a perfect fluorescent sheet that doesn't photobleach (not easily at least).

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


On 2013-10-04, at 3:12 PM, Craig Brideau wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> As your focus enters the sheet, the sheet itself will diffract the beam,
> rather like focusing into a coverslip. At least that's my preliminary
> thought.  A bead would disrupt the light path less.
>
> Craig
>
>
> On Fri, Oct 4, 2013 at 1:01 PM, John Oreopoulos <[hidden email]
>> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hello again,
>>
>> A few years ago I sent a question to the confocal listserver about the
>> differences in measuring confocal axial resolution with
>> sub-diffraction-sized beads or mirrors and fluorescent sheets (see messages
>> attached below). At the time, one of the most informative responses came
>> from Brad Amos (response message also below). Since then, I've been doing
>> these types of measurements on various confocal instruments and can confirm
>> with certainty that the two test specimens (fluorescent beads vs.
>> fluorescent sheets) yield different results when measuring the FWHM of the
>> axial profile. So my question now is: Which specimen should we be using to
>> report instrument performance in terms of axial resolution? I've been to
>> quite a few microscopy courses and the conventional wisdom seems to be that
>> fluorescent beads are best, but Brad's book chapter that he linked to made
>> an argument that fluorescent sheets are better and that it's incorrect to
>> use beads to report axial resolution. In case anyone is interested, in my
>> hands, fluorescent sheets consistently yield an axial resolution that is a
>> few hundred nm bigger than what the bead measurement says for a given
>> instrument with a particular set of optics. I'm hoping Brad might chime in
>> again here or anyone else who has something to say about this.
>>
>> John Oreopoulos
>> Staff Scientist
>> Spectral Applied Research
>> Richmond Hill, Ontario
>> Canada
>> www.spectral.ca
>>
>>
>>
>> On 2011-07-25, at 3:31 PM, Brad Amos wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> The equations for resolution in z are different for a plane reflector, a
>>> thin fluorescent lamina and a subresolution bead. The commercial story on
>>> this is a mess of unattributed equations of obscure provenance. You may
>> find
>>> useful information in the following article, which is a permitted
>> preprint
>>> of an article about confocal microscopy to be edited and published in the
>>> Elsevier online 'Comprehensive Biophysics', where Tony Wilson, Gail
>>> McConnell and I have tried to make sense of it all :
>>>
>> http://www2.mrc-lmb.cam.ac.uk/images/groupleaders/Confocal_microscopy_Amos_McConnell_Wilson.pdf
>>
>>
>> *****
>> On 2011-07-03, at 12:25 PM, John Oreopoulos wrote:
>>
>>> *****
>>> 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,
it appears to be straight forward, that the convolution of a point object
with the 3d PSF is something different than that of a plane object. Compared
to a point source, lateral contributions from the plane will widen the axial
response of the observation. When using as a measurement one should apply a
correction that could be determined analytically.

Regards
Lutz
__________________________________
L u t z   S c h a e f e r
Sen. Scientist
Mathematical modeling / Computational microscopy
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/
___________________________________


-----Original Message-----
From: John Oreopoulos
Sent: Friday, October 04, 2013 3:01 PM
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
*****

Hello again,

A few years ago I sent a question to the confocal listserver about the
differences in measuring confocal axial resolution with
sub-diffraction-sized beads or mirrors and fluorescent sheets (see messages
attached below). At the time, one of the most informative responses came
from Brad Amos (response message also below). Since then, I've been doing
these types of measurements on various confocal instruments and can confirm
with certainty that the two test specimens (fluorescent beads vs.
fluorescent sheets) yield different results when measuring the FWHM of the
axial profile. So my question now is: Which specimen should we be using to
report instrument performance in terms of axial resolution? I've been to
quite a few microscopy courses and the conventional wisdom seems to be that
fluorescent beads are best, but Brad's book chapter that he linked to made
an argument that fluorescent sheets are better and that it's incorrect to
use beads to report axial resolution. In case anyone is interested, in my
hands, fluorescent sheets consistently yield an axial resolution that is a
few hundred nm bigger than what the bead measurement says for a given
instrument with a particular set of optics. I'm hoping Brad might chime in
again here or anyone else who has something to say about this.

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



On 2011-07-25, at 3:31 PM, Brad Amos wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> The equations for resolution in z are different for a plane reflector, a
> thin fluorescent lamina and a subresolution bead. The commercial story on
> this is a mess of unattributed equations of obscure provenance. You may
> find
> useful information in the following article, which is a permitted preprint
> of an article about confocal microscopy to be edited and published in the
> Elsevier online 'Comprehensive Biophysics', where Tony Wilson, Gail
> McConnell and I have tried to make sense of it all :
> http://www2.mrc-lmb.cam.ac.uk/images/groupleaders/Confocal_microscopy_Amos_McConnell_Wilson.pdf

*****
On 2011-07-03, at 12:25 PM, John Oreopoulos wrote:

> *****
> 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

[Reto Fiolka]

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

I agree with Lutz that in a PSF measurement, the size of the bead has to be
considered (corrected for after determining FWHM), however, for sufficiently
small beads this correction becomes minor.

I am surprised that in confocal microscopy the axial direction should be
"treated" differently than the lateral dimensions in a beads based PSF
measurement. This does not correspond to my view of image formation in a
shift invariant system. Shift invariance is fulfilled in a 3D point scanning system
(unless the scanning mechanism is bad), thus 3D convolution with a PSF applies
for image formation.


There is a fourth way to measure the axial PSF that I know and have tried
once:

Measuring the axial step response, i.e.moving the focus from an empty space
into the fluorescence medium and recording the fluorescence. This requires a
sharp interface from the non-fluorescent medium to the "sea of fluorescence".

The axial PSF can be obtained by a numerical derivative in z or by fitting a
model.

This way at least one of the first 4Pi PSFs was measured in the axial direction.

Best,
Reto

[Lemasters, John J.]

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

This probably has been mentioned, but you want to be sure and use a front-reflecting mirror.

Good luck, John

--
John J. Lemasters, MD, PhD
Departments of Drug Discovery & Biomedical Sciences and Biochemistry & Molecular Biology
Medical University of South Carolina
DD504 Drug Discovery Building
70 President Street, MSC 140
Charleston, SC 29425
 
Office: 843-876-2360
Lab: 843-876-2354
Fax: [phone]
Email: [hidden email]
http://academicdepartments.musc.edu/ccdir


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Lutz Schaefer
Sent: Friday, October 04, 2013 7:28 PM
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
*****

John,
it appears to be straight forward, that the convolution of a point object with the 3d PSF is something different than that of a plane object. Compared to a point source, lateral contributions from the plane will widen the axial response of the observation. When using as a measurement one should apply a correction that could be determined analytically.

Regards
Lutz
__________________________________
L u t z   S c h a e f e r
Sen. Scientist
Mathematical modeling / Computational microscopy 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/ ___________________________________


-----Original Message-----
From: John Oreopoulos
Sent: Friday, October 04, 2013 3:01 PM
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
*****

Hello again,

A few years ago I sent a question to the confocal listserver about the differences in measuring confocal axial resolution with sub-diffraction-sized beads or mirrors and fluorescent sheets (see messages attached below). At the time, one of the most informative responses came from Brad Amos (response message also below). Since then, I've been doing these types of measurements on various confocal instruments and can confirm with certainty that the two test specimens (fluorescent beads vs.
fluorescent sheets) yield different results when measuring the FWHM of the axial profile. So my question now is: Which specimen should we be using to report instrument performance in terms of axial resolution? I've been to quite a few microscopy courses and the conventional wisdom seems to be that fluorescent beads are best, but Brad's book chapter that he linked to made an argument that fluorescent sheets are better and that it's incorrect to use beads to report axial resolution. In case anyone is interested, in my hands, fluorescent sheets consistently yield an axial resolution that is a few hundred nm bigger than what the bead measurement says for a given instrument with a particular set of optics. I'm hoping Brad might chime in again here or anyone else who has something to say about this.

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



On 2011-07-25, at 3:31 PM, Brad Amos wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> The equations for resolution in z are different for a plane reflector, a
> thin fluorescent lamina and a subresolution bead. The commercial story on
> this is a mess of unattributed equations of obscure provenance. You may
> find
> useful information in the following article, which is a permitted preprint
> of an article about confocal microscopy to be edited and published in the
> Elsevier online 'Comprehensive Biophysics', where Tony Wilson, Gail
> McConnell and I have tried to make sense of it all :
> http://www2.mrc-lmb.cam.ac.uk/images/groupleaders/Confocal_microscopy_Amos_McConnell_Wilson.pdf

*****
On 2011-07-03, at 12:25 PM, John Oreopoulos wrote:

> *****
> 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

[Pascal Weber]

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*****

In fact it is not sure that a mirror behaves as a fluorescent ball. But it is a sure
way of control, both in the power and your laser alignment. It is very easy to
control the numerical aperture of the lens with the formula of depth of field. I use
very often this option because it is constant.

[Guy Cox-2]

*****
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*****

OK, I'm probably walking into a minefield here but .....

Imaging a mirror in reflection is coherent imaging so different calculations apply vs fluorescence imaging which is incoherent.   In principle one would expect worse resolution but in practice, since one can close the pinhole right down, one will probably get better resolution.  Regardless of such considerations it is a really great test specimen, since it will show up spherical aberration, pinhole misalignment and other problems very effectively.  And it is absolutely, unequivocally planar.  Contrary to what John says, you do NOT want to use a front surface mirror, you need a silvered coverslip of the correct thickness to match the correction of your objective.   (Unless you are using a metallurgical or geological objective, which is corrected for use without a coverslip).

A fluorescent bulk sample which gives an axial diffraction-limited image is absolutely NOT the same as a fluorescent planar sheet, and you could not calculate axial resolution from it without correcting for the effective thickness of the excited layer.

Beads are always great but you need to use VERY small ones if you want an absolute resolution measurement without calculation.

Lutz is quite right that different calculations apply to a sheet rather than a point,  and all these tests as carried out in routine maintenance should be regarded as relative rather than absolute.  But in most cases the relative figure is what matters to us.  

                                                                                                              Guy



Guy Cox, Honorary Associate Professor
School of Medical Sciences

Australian Centre for Microscopy and Microanalysis,
Madsen, F09, University of Sydney, NSW 2006


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Lemasters, John J.
Sent: Saturday, 5 October 2013 10:31 AM
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
*****

This probably has been mentioned, but you want to be sure and use a front-reflecting mirror.

Good luck, John

--
John J. Lemasters, MD, PhD
Departments of Drug Discovery & Biomedical Sciences and Biochemistry & Molecular Biology Medical University of South Carolina
DD504 Drug Discovery Building
70 President Street, MSC 140
Charleston, SC 29425
 
Office: 843-876-2360
Lab: 843-876-2354
Fax: [phone]
Email: [hidden email]
http://academicdepartments.musc.edu/ccdir


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Lutz Schaefer
Sent: Friday, October 04, 2013 7:28 PM
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
*****

John,
it appears to be straight forward, that the convolution of a point object with the 3d PSF is something different than that of a plane object. Compared to a point source, lateral contributions from the plane will widen the axial response of the observation. When using as a measurement one should apply a correction that could be determined analytically.

Regards
Lutz
__________________________________
L u t z   S c h a e f e r
Sen. Scientist
Mathematical modeling / Computational microscopy 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/ ___________________________________


-----Original Message-----
From: John Oreopoulos
Sent: Friday, October 04, 2013 3:01 PM
To: [hidden email]
Subject: Re: Using a mirror for axial resolution testing

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Hello again,

A few years ago I sent a question to the confocal listserver about the differences in measuring confocal axial resolution with sub-diffraction-sized beads or mirrors and fluorescent sheets (see messages attached below). At the time, one of the most informative responses came from Brad Amos (response message also below). Since then, I've been doing these types of measurements on various confocal instruments and can confirm with certainty that the two test specimens (fluorescent beads vs.
fluorescent sheets) yield different results when measuring the FWHM of the axial profile. So my question now is: Which specimen should we be using to report instrument performance in terms of axial resolution? I've been to quite a few microscopy courses and the conventional wisdom seems to be that fluorescent beads are best, but Brad's book chapter that he linked to made an argument that fluorescent sheets are better and that it's incorrect to use beads to report axial resolution. In case anyone is interested, in my hands, fluorescent sheets consistently yield an axial resolution that is a few hundred nm bigger than what the bead measurement says for a given instrument with a particular set of optics. I'm hoping Brad might chime in again here or anyone else who has something to say about this.

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



On 2011-07-25, at 3:31 PM, Brad Amos wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> The equations for resolution in z are different for a plane reflector,
> a thin fluorescent lamina and a subresolution bead. The commercial
> story on this is a mess of unattributed equations of obscure
> provenance. You may find useful information in the following article,
> which is a permitted preprint of an article about confocal microscopy
> to be edited and published in the Elsevier online 'Comprehensive
> Biophysics', where Tony Wilson, Gail McConnell and I have tried to
> make sense of it all :
> http://www2.mrc-lmb.cam.ac.uk/images/groupleaders/Confocal_microscopy_
> Amos_McConnell_Wilson.pdf

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On 2011-07-03, at 12:25 PM, John Oreopoulos wrote:

> *****
> 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]

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On 2013-10-07, at 8:32 AM, Guy Cox wrote:

> A fluorescent bulk sample which gives an axial diffraction-limited image is absolutely NOT the same as a fluorescent planar sheet, and you could not calculate axial resolution from it without correcting for the effective thickness of the excited layer.

Guy, unless I'm reading it wrong, I believe the whole point of the paper I mentioned by Mike Model was to show that at sufficiently high concentration, the bulk sample of dye in liquid effectively acts as a 2D fluorescent sheet / planar layer because of it's high optical density, and under those conditions, these samples can be used to measure the axial resolution of a confocal instrument. The authors specifically say that the exact thickness of the excited layer is not important for this measurement so long as the concentration is above a certain threshold. They even show that the concentrated dye solutions can be used with Brakenhoff's Sectioning Imaging Property (SIP) charts. Am I mis-interpreting all this?

I use a concentration of 0.5 g/mL Na-FITC when measuring the axial response of a confocal microscope at 488 nm through a green emission filter.

Model, M.A. and J.L. Blank, Concentrated dyes as a source of two-dimensional fluorescent field for characterization of a confocal microscope. Journal of Microscopy-Oxford, 2008. 229(1): p. 12-16.

http://www.imaging-git.com/science/image-processing/sectioning-imaging-property-sip-charts-tool-characterisation-confocal-fluor

Again, the main point of my post to the listserver was to enquire about which specimen (fluorescent beads or fluorescent sheets - not mirrors) is the appropriate specimen to accurately report the sectioning ability (axial resolution) of a confocal microscope because I'm finding that I consistently get a few hundred nm difference when performing the measurement with sheets vs. beads. I understand now that both specimens will inherently yield different results because of their geometry and the way the light of the microscope interacts with them, but the question remains then, which one is more appropriate to use as a tool to report the absolute (not relative) axial resolution?

Here's the quote from Brad Amos' book chapter which has me all up in knots about this:

"The bead is a poor test object for measuring confocal stringency: the equation for non-confocal axial FWHM derived here (equation 1) suggests that a wide-field microscope with no confocal optical sectioning whatever (i.e. no pinhole) would have an axial resolution of 0.47 μ m (assuming n= 1.515 ,NA 1.4 and λ = 0.543)."

http://www2.mrc-lmb.cam.ac.uk/images/groupleaders/Confocal_microscopy_Amos_McConnell_Wilson.pdf

Isn't Brad basically saying here that fluorescent beads are not going to yield the correct measurement of axial resolution?

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

[Reto Fiolka]

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Hi Guy

if you take a 100nm bead and assuming your axial PSF is 700nm, then the
measured axial FWHM increases only by ~1% due to the convolution with the
bead  (  sqrt (700^2+100^2)  ). Not too terrible! This was a bit crude by
assuming the bead to be a gaussian blob, but nevertheless shows the scaling.

Measuring the step response can give you the axial profile, however at the
cost of some modeling/calculation. This is a very common way to determine
transfer functions in control theory (albeit with transfer functions in time and
they deal with Laplace transforms instead of our beloved Fourier transforms),
yet the general idea of measuring a step response and then fitting a model of
your response function is applicable to microscopy too.

A step means having a sharp boundary from non-fluorescent to uniform bulk
fluorescence. It also means you can determine your PSF profile in one
dimension only.
If I am not mistaken, Hell et al used however a finite difference scheme to
compute the derivative of their measured 4Pi step response.

It is worth looking into other fields how they do similar stuff. I learnt from the
control theory guys interesting stuff. For instance they don't use a discrete
Laplace transform to switch to frequency space (in contrast to us, who happily
use an FFT to switch from PSF to OTF). Instead they often try to model their
measurement with elementary function of which the Laplace transform is
known.

Best,
Reto

[mmodel]

I missed the beginning of the conversation but I think John is right - as long the thickness of the sheet (or the size of the bead) is below resolution, the exact numbers don't matter. Of course the sheet cannot be used with widefield microscopes to measure PSF and of course the axial resolution determined with a bead, a mirror or a fluorescent sheet made of concentrated fluorescent dye will be somewhat different. I suspect (don't know for sure) that directed reflection may be a problem with mirrors, for example at the edges of the field.

Mike Model

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of John Oreopoulos
Sent: Monday, October 07, 2013 10:21 AM
To: [hidden email]
Subject: Re: Using a mirror for axial resolution testing

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On 2013-10-07, at 8:32 AM, Guy Cox wrote:

> A fluorescent bulk sample which gives an axial diffraction-limited image is absolutely NOT the same as a fluorescent planar sheet, and you could not calculate axial resolution from it without correcting for the effective thickness of the excited layer.

Guy, unless I'm reading it wrong, I believe the whole point of the paper I mentioned by Mike Model was to show that at sufficiently high concentration, the bulk sample of dye in liquid effectively acts as a 2D fluorescent sheet / planar layer because of it's high optical density, and under those conditions, these samples can be used to measure the axial resolution of a confocal instrument. The authors specifically say that the exact thickness of the excited layer is not important for this measurement so long as the concentration is above a certain threshold. They even show that the concentrated dye solutions can be used with Brakenhoff's Sectioning Imaging Property (SIP) charts. Am I mis-interpreting all this?

I use a concentration of 0.5 g/mL Na-FITC when measuring the axial response of a confocal microscope at 488 nm through a green emission filter.

Model, M.A. and J.L. Blank, Concentrated dyes as a source of two-dimensional fluorescent field for characterization of a confocal microscope. Journal of Microscopy-Oxford, 2008. 229(1): p. 12-16.

http://www.imaging-git.com/science/image-processing/sectioning-imaging-property-sip-charts-tool-characterisation-confocal-fluor

Again, the main point of my post to the listserver was to enquire about which specimen (fluorescent beads or fluorescent sheets - not mirrors) is the appropriate specimen to accurately report the sectioning ability (axial resolution) of a confocal microscope because I'm finding that I consistently get a few hundred nm difference when performing the measurement with sheets vs. beads. I understand now that both specimens will inherently yield different results because of their geometry and the way the light of the microscope interacts with them, but the question remains then, which one is more appropriate to use as a tool to report the absolute (not relative) axial resolution?

Here's the quote from Brad Amos' book chapter which has me all up in knots about this:

"The bead is a poor test object for measuring confocal stringency: the equation for non-confocal axial FWHM derived here (equation 1) suggests that a wide-field microscope with no confocal optical sectioning whatever (i.e. no pinhole) would have an axial resolution of 0.47 μ m (assuming n= 1.515 ,NA 1.4 and λ = 0.543)."

http://www2.mrc-lmb.cam.ac.uk/images/groupleaders/Confocal_microscopy_Amos_McConnell_Wilson.pdf

Isn't Brad basically saying here that fluorescent beads are not going to yield the correct measurement of axial resolution?

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

[Reto Fiolka]

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Hi John

This is my humble opinion:

Measuring resolution with beads in a shift invariant imaging system (image is
convolution with PSF) in any dimension is considered legitimate provided you
are using sufficiently small beads.


Sufficiently small: if your bead is 10 times smaller than the PSF you are
measuring, then your error is below 1% (rough estimation assuming gaussian
profile for PSF and bead fluorophore distribution: sqrt(1^2+0.1^2)=1.005 ). So
a 50nm bead for axial resolution in confocal microscopy is safe.

Any peer reviewed journal that I am aware of accepts such a measurment
when you introduce a new technique.

Brad Amos probably means that having a finite FWHM in the axial PSF does not
mean that you have a good optical sectioning capability. That the widefield
microscope has no sectioning capability is included in its PSF: the out of focus
rings conserve the same energy as is found in the focal plane, hence there is
no sectioning. However just measuring the profile along the central axis will not
reveal this, it might actually look as the intensity would decrease as you go
away from the focal plane.

Best,
Reto

[Brad Amos]

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It is important to distinguish between resolution and optical sectioning.
Resolution in optics can be defined as the degree of overlap between images
derived from point objects. The Rayleigh resolution criterion is that points
are resolved if their separation causes the peak of one Airy pattern to fall
over the first minimum of the other. The resolution distance is then the
radius of an Airy disk at the first minimum.
A similar criterion can be applied to axial resolution. Adding confocal
optics changes resolution very little. Brakenhoff demonstrated the expected
factor of the square root of two ( 1.414) in improvement thirty years ago,
but it is often not seen in practical microscopy with non-ideal specimens
and lenses. The resolution can be measured with a sub-resolution diameter
fluorescent or reflective bead, and it will be very similar whether
wide-field or confocal optics are used.  This is why I wrote, in the article
cited here, that a subresolution bead was a poor specimen for comparing
confocal stringency:  a confocal microscope resolves scarcely better than a
wide-field one.
   When we move away from point objects, we start to see a quite different
effect. With an ideal uniform reflective or fluorescent planar specimen
there is no change in intensity with focus, so no optical sectioning effect.
But with confocal optics applied to this type of specimen, the plane of
fluorescence or reflection is clearly defined in XZ sections. This is an
ideal type of specimen to measure the confocal stringency or optical
sectioning ability of a microscope. Since it is rather difficult to make
thin fluorescent laminae, a 'lake' specimen, which is the interface between
a fluorescent volume and a non-fluorescent coverslip is a reasonable
substitute.
    In mathematical terms, a planar object gives a planar spread function,
which is the convolution of the point spread function with a plane, which is
a constant in three dimensions when the psf is that of a wide-field
microscope and an utterly different axial peak of intensity when the psf is
that of a confocal system. At a plane distant from focus, the tails of the
all the widefield psfs add together to give a constant intensity, but these
tails are suppressed in the confocal case.
     To summarise: resolution is defined by the psf close to the peak and
requires a point specimen, but optical sectioning is defined by the psf
tails, far from the peak, which cannot be measured with a single point
specimen, but can be measured in aggregate with a plane or a volume specimen.

[Lutz Schaefer]

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Reto,
you pointed out correctly that the integrated intensity in any 2d plane of a
widefield PSF is identical, in theory. Although, to the trained eye it gives
away the singularity in reciprocal space, but this by itself may not fully
explain the sectioning capabilities. The widefield microscope indeed has no
sectioning ability, but strictly only at the zero frequency component! In
other words, you can not focus on a plane object or fluorescent sheet
without detail. However you can focus on small dust particles on it that
exhibit higher frequency components. This all becomes clear when looking at
the OTF. There you can see that the "sectioning strength" actually depends
on the frequency and it has a maximum in about the middle of the radius of
this torus, while as said before, there is a singularity (Dirac) in its
origin.

Now having said that, when the bead used in the measurements approximates a
Dirac, its spectrum is a constant, leaving the product with the OTF
unchanged and what you see in the inverse FT simply is the PSF. It is
straight forward that the axial extend of that PSF corresponds to the
sectioning ability at around the reciprocal of the pass-band frequencies of
the OTF torus (... and YES of a widefield microscope).

I am not sure how helpful this information is to some, as for specimens with
varying frequency content, your sectioning will vary too. Lower frequencies
usually dominate giving the well known response and the notion of no
sectioning. Using deconvolution one can increase the sectioning
capabilities, but really only in the nonlinear (iterative with positivity
constraint) case where the lower pass band frequencies, that got lost lost
due to the inner part of the torus become restored, if indeed they were
present in the specimen to begin with.

Hope that helped
Regards
Lutz

__________________________________
L u t z   S c h a e f e r
Sen. Scientist
Mathematical modeling / Computational microscopy
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/
___________________________________

-----Original Message-----
From: Reto Fiolka
Sent: Monday, October 07, 2013 10:47 AM
To: [hidden email]
Subject: Re: Using a mirror for axial resolution testing

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Hi John

This is my humble opinion:

Measuring resolution with beads in a shift invariant imaging system (image
is
convolution with PSF) in any dimension is considered legitimate provided you
are using sufficiently small beads.


Sufficiently small: if your bead is 10 times smaller than the PSF you are
measuring, then your error is below 1% (rough estimation assuming gaussian
profile for PSF and bead fluorophore distribution: sqrt(1^2+0.1^2)=1.005 ).
So
a 50nm bead for axial resolution in confocal microscopy is safe.

Any peer reviewed journal that I am aware of accepts such a measurment
when you introduce a new technique.

Brad Amos probably means that having a finite FWHM in the axial PSF does not
mean that you have a good optical sectioning capability. That the widefield
microscope has no sectioning capability is included in its PSF: the out of
focus
rings conserve the same energy as is found in the focal plane, hence there
is
no sectioning. However just measuring the profile along the central axis
will not
reveal this, it might actually look as the intensity would decrease as you
go
away from the focal plane.

Best,
Reto

[John Oreopoulos]

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Okay, thank you all for the responses. I think Brad's explanation makes the most sense to me again. All these years I had always implicitly assumed the axial resolution and the optical sectioning thickness of a confocal were the same thing (the general literature definitely gives that impression).

Lutz, I usually have difficulty visualizing these sorts of things in the frequency domain, but I have some vague sense of what you're talking about here. I have to think and read about it more.

John Oreopoulos

> On Oct 7, 2013, at 5:04 PM, Brad Amos <[hidden email]> wrote:
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> It is important to distinguish between resolution and optical sectioning.
> Resolution in optics can be defined as the degree of overlap between images
> derived from point objects. The Rayleigh resolution criterion is that points
> are resolved if their separation causes the peak of one Airy pattern to fall
> over the first minimum of the other. The resolution distance is then the
> radius of an Airy disk at the first minimum.
> A similar criterion can be applied to axial resolution. Adding confocal
> optics changes resolution very little. Brakenhoff demonstrated the expected
> factor of the square root of two ( 1.414) in improvement thirty years ago,
> but it is often not seen in practical microscopy with non-ideal specimens
> and lenses. The resolution can be measured with a sub-resolution diameter
> fluorescent or reflective bead, and it will be very similar whether
> wide-field or confocal optics are used.  This is why I wrote, in the article
> cited here, that a subresolution bead was a poor specimen for comparing
> confocal stringency:  a confocal microscope resolves scarcely better than a
> wide-field one.
>   When we move away from point objects, we start to see a quite different
> effect. With an ideal uniform reflective or fluorescent planar specimen
> there is no change in intensity with focus, so no optical sectioning effect.
> But with confocal optics applied to this type of specimen, the plane of
> fluorescence or reflection is clearly defined in XZ sections. This is an
> ideal type of specimen to measure the confocal stringency or optical
> sectioning ability of a microscope. Since it is rather difficult to make
> thin fluorescent laminae, a 'lake' specimen, which is the interface between
> a fluorescent volume and a non-fluorescent coverslip is a reasonable
> substitute.
>    In mathematical terms, a planar object gives a planar spread function,
> which is the convolution of the point spread function with a plane, which is
> a constant in three dimensions when the psf is that of a wide-field
> microscope and an utterly different axial peak of intensity when the psf is
> that of a confocal system. At a plane distant from focus, the tails of the
> all the widefield psfs add together to give a constant intensity, but these
> tails are suppressed in the confocal case.
>     To summarise: resolution is defined by the psf close to the peak and
> requires a point specimen, but optical sectioning is defined by the psf
> tails, far from the peak, which cannot be measured with a single point
> specimen, but can be measured in aggregate with a plane or a volume specimen.  

[James Pawley]

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>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Hi John
>
>This is my humble opinion:
>
>Measuring resolution with beads in a shift invariant imaging system (image is
>convolution with PSF) in any dimension is considered legitimate provided you
>are using sufficiently small beads.
>
>
>Sufficiently small: if your bead is 10 times smaller than the PSF you are
>measuring, then your error is below 1% (rough estimation assuming gaussian
>profile for PSF and bead fluorophore distribution: sqrt(1^2+0.1^2)=1.005 ). So
>a 50nm bead for axial resolution in confocal microscopy is safe.
>
>Any peer reviewed journal that I am aware of accepts such a measurment
>when you introduce a new technique.
>
>Brad Amos probably means that having a finite FWHM in the axial PSF does not
>mean that you have a good optical sectioning capability. That the widefield
>microscope has no sectioning capability is
>included in its PSF: the out of focus
>rings conserve the same energy as is found in the focal plane, hence there is
>no sectioning. However just measuring the
>profile along the central axis will not
>reveal this, it might actually look as the intensity would decrease as you go
>away from the focal plane.
>
>Best,
>Reto

Dear Reto,

I think that your last point is explained by the
fact that what we call widefield, isn't quite
wide. The waist of the illuminated area in the
focus plane (set by the field diaphragm) is
smaller than the front element of the objective.
Therefore, you still have conical illumination,
just not as conical as with "proper" confocal
where a point light source is (you hope) focused
into a Airy figure at the focus plane. The
Agard/Sedat group published a paper long ago
about the partial confocal nature of WF
illumination, and pointed out that it was quite
strong if you made the field diaphragm small so
that only a single nucleus was illuminated (~6µm
diam.)

Would this explain your result?

Jim Pawley
--
James and Christine Pawley, 5446 Burley Place (PO
Box 2348), Sechelt, BC, Canada, V0N3A0,
Phone [phone], email <[hidden email]>
NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!) 1-604-989-6146

[James Pawley]

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Hi all,

May I add two points to this important discussion?

1. My usual point: Maybe it is relevant that when
looking at the refection from a mirror, one has
an essentially infinite level of signal (the
reflection of a mW laser beam) that is millions
if not billions of times larger than that
available from viewing a fluorescent bead that is
only 100 nm in diameter. Because of this, it is
common to cylindrically average the signal in the
case of the bead, a process that can mask the
effect of asymmetries in the OTF such as the
astigmatism caused by imperfect beam splitters or
improper alignment.

Apart from this, Poisson noise will have a much
greater effect in terms of introducing
uncertainty..

2. My other usual point: Is spherical aberration
involved? Beads are made of plastic and the RI of
plastic may or may not be the same as that of the
surrounding medium or that for which the
objective was designed. Of course, one assumes
that this has little effect when the diameter of
the bead is 50nm, but as such beads usually give
only about 1/8th the signal of 100nm beads made
of the same material and only about 1/64th the
signal of 200nm beads, point 1 (above) can drive
the user to larger beads (and larger pinhole
settings). I have no calculation to describe the
blurring effect of using any particular design of
bead in any specific mounting medium, however, I
would expect such blurring to be more pronounced
in the z than in the x-y direction because some
of the rays from focus planes on the far side
have to pass through the bead. This would be even
more important is the entrance pupil of the
objective is not fully and evenly filled by the
laser beam.

Perhaps one should also point out the
peculiarities of the light signal reflected from
a flat RI interface, such as that between
coverslip and water, compared to that produced by
reflection from a metallic mirror. The former
will contain proportionally more signal from
"high-NA" rays than from those approaching the
interface at closer to normal incidence and there
is also the fact that the amount reflected at an
RI interface varies strongly with the angle
between the polarization of a high-NA ray and the
orientation of the surface. The asymmetric
doughnut-shaped apodization resulting from these
effects can make the "z-resolution" look better
(or worse) than it would be otherwise.

I can see the
"thin-layer-of-fluorescent-immersion-oil"
specimen being useful for measuring the
performance of oil-immersion lenses, but how does
one use it to measure the performance of water or
glycerine objectives?

The
"thin-layer-of-fluorescent-molecules-deposited-on-glass"
specimens do not have this problem, but I would
guess that the signal levels might be lower and
there could be orientation effects related to the
alignment between the (possibly non-random)
dipole axes of the dye and the electric field
orientation of a convergent and polarized light
beam.

More to the point, who has compared the PSF of a
bead near the coverslip surface with one embedded
even a few µm inside a watery (living?)
biological specimen? My few attempts to do so
have revealed pronounced asymmetries that are
readily visible by eye (as long as the eye is
observing and the stored image collected from the
CCD). In other words, apart from its use as a
(very important) check on instrument performance,
do we really need to know the PSF or FWHM etc to
such precision?

Regards,

Jim Pawley

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Reto,
>you pointed out correctly that the integrated
>intensity in any 2d plane of a widefield PSF is
>identical, in theory. Although, to the trained
>eye it gives away the singularity in reciprocal
>space, but this by itself may not fully explain
>the sectioning capabilities. The widefield
>microscope indeed has no sectioning ability, but
>strictly only at the zero frequency component!
>In other words, you can not focus on a plane
>object or fluorescent sheet without detail.
>However you can focus on small dust particles on
>it that exhibit higher frequency components.
>This all becomes clear when looking at the OTF.
>There you can see that the "sectioning strength"
>actually depends on the frequency and it has a
>maximum in about the middle of the radius of
>this torus, while as said before, there is a
>singularity (Dirac) in its origin.
>
>Now having said that, when the bead used in the
>measurements approximates a Dirac, its spectrum
>is a constant, leaving the product with the OTF
>unchanged and what you see in the inverse FT
>simply is the PSF. It is straight forward that
>the axial extend of that PSF corresponds to the
>sectioning ability at around the reciprocal of
>the pass-band frequencies of the OTF torus (...
>and YES of a widefield microscope).
>
>I am not sure how helpful this information is to
>some, as for specimens with varying frequency
>content, your sectioning will vary too. Lower
>frequencies usually dominate giving the well
>known response and the notion of no sectioning.
>Using deconvolution one can increase the
>sectioning capabilities, but really only in the
>nonlinear (iterative with positivity constraint)
>case where the lower pass band frequencies, that
>got lost lost due to the inner part of the torus
>become restored, if indeed they were present in
>the specimen to begin with.
>
>Hope that helped
>Regards
>Lutz
>
>__________________________________
>L u t z   S c h a e f e r
>Sen. Scientist
>Mathematical modeling / Computational microscopy
>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/
>___________________________________
>
>-----Original Message----- From: Reto Fiolka
>Sent: Monday, October 07, 2013 10:47 AM
>To: [hidden email]
>Subject: Re: Using a mirror for axial resolution testing
>
>*****
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>
>Hi John
>
>This is my humble opinion:
>
>Measuring resolution with beads in a shift invariant imaging system (image is
>convolution with PSF) in any dimension is considered legitimate provided you
>are using sufficiently small beads.
>
>
>Sufficiently small: if your bead is 10 times smaller than the PSF you are
>measuring, then your error is below 1% (rough estimation assuming gaussian
>profile for PSF and bead fluorophore distribution: sqrt(1^2+0.1^2)=1.005 ). So
>a 50nm bead for axial resolution in confocal microscopy is safe.
>
>Any peer reviewed journal that I am aware of accepts such a measurment
>when you introduce a new technique.
>
>Brad Amos probably means that having a finite FWHM in the axial PSF does not
>mean that you have a good optical sectioning capability. That the widefield
>microscope has no sectioning capability is
>included in its PSF: the out of focus
>rings conserve the same energy as is found in the focal plane, hence there is
>no sectioning. However just measuring the
>profile along the central axis will not
>reveal this, it might actually look as the intensity would decrease as you go
>away from the focal plane.
>
>Best,
>Reto

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James and Christine Pawley, 5446 Burley Place (PO
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Phone [phone], email <[hidden email]>
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[Csúcs Gábor-2]

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Dear All,

Not a very scientific (though who knows) but still an important (in terms
of comfort) question about the climatization of microscopy rooms. We are
just in the middle of discussions about the arrangement of our new
microscopy rooms. Most of the things are clear, however the planning team
suggested a solution for the climatization that is new to me and I wanted
to enquire whether anyone of you had experience with similar systems. In
the "conventional" arrangement the cold air comes in somewhere at the
ceiling and I think there is a general consensus that it is better if it
is well distributed and not simply blown in into one direction. Our
planning team however suggests a new solution, where the cold air would be
blown in (through some canvas tubes) close to the bottom/floor. The warm
air (that goes anyway upward) is sucked at the ceiling. According to them,
although this systems creates a height-dependent temperature gradient
(cold bottom, stable 22 C at the microscope level and warmer at the
ceiling) but with this one can avoid the continuous mixing/turbulence
where both the blowing in and the sucking away happens on the ceiling
(conventional solution). Now, in theory this sounds good but we are
somewhat skeptical how well this system works in practice and what the
users say if their feet has colder (approx. 16-18 C) temperatures then
their body. In a few weeks we will have the opportunity to check a similar
installation, but I'd really appreciate if you could share your
experiences with us. Obviously this is a important decision for us so any
feedback is welcome.

Thanks     Gabor

[Rosemary.White]

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Dear Gabor,

In our experience, the main thing is to get a much better a/c system than is usually budgeted for. We insisted that our new confocal room maintain temperature +/- one degree, yes, a two degree range, and that it not blow on the instrument or user. They did it, but at considerable cost, but it's worth it if you have multiple lasers, etc. that prefer a stable environment. And is a fraction of the cost of the instrument.

Good luck!

cheers,
Rosemary

Dr Rosemary White
CSIRO Plant Industry
GPO Box 1600
Canberra, ACT 2601
Australia

T 61 2 6246 5475
F 61 2 6246 5334
________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Csúcs  Gábor [[hidden email]]
Sent: Tuesday, 8 October 2013 6:51 p.m.
To: [hidden email]
Subject: Room climatization

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

Dear All,

Not a very scientific (though who knows) but still an important (in terms
of comfort) question about the climatization of microscopy rooms. We are
just in the middle of discussions about the arrangement of our new
microscopy rooms. Most of the things are clear, however the planning team
suggested a solution for the climatization that is new to me and I wanted
to enquire whether anyone of you had experience with similar systems. In
the "conventional" arrangement the cold air comes in somewhere at the
ceiling and I think there is a general consensus that it is better if it
is well distributed and not simply blown in into one direction. Our
planning team however suggests a new solution, where the cold air would be
blown in (through some canvas tubes) close to the bottom/floor. The warm
air (that goes anyway upward) is sucked at the ceiling. According to them,
although this systems creates a height-dependent temperature gradient
(cold bottom, stable 22 C at the microscope level and warmer at the
ceiling) but with this one can avoid the continuous mixing/turbulence
where both the blowing in and the sucking away happens on the ceiling
(conventional solution). Now, in theory this sounds good but we are
somewhat skeptical how well this system works in practice and what the
users say if their feet has colder (approx. 16-18 C) temperatures then
their body. In a few weeks we will have the opportunity to check a similar
installation, but I'd really appreciate if you could share your
experiences with us. Obviously this is a important decision for us so any
feedback is welcome.

Thanks     Gabor