Rejected posting to [e-mail]

[Hanna_SN]

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Dear all,

For a couple of months we are struggling with a serious problem with
the field illumination in our Olympus Scan^R Scanning station. The
image of the specimen, when observed on the computer screen is much
darker on one side than on the other. We thought it is the lamp but
the lamp is new at properly inserted. Then we tried calibrating the
optic fiber, but it didn't help at all. Our third guess was the
filters.We checked all of the filters  -  the coating has lots of
small dark spots with biggest concentration of the spots in the middle
of the filters, but I am concerned if that can  produce uneven
illumination of the field so that only one part (let say left corner)
is underilluminated? So, I turn to you and your experience: did may
anyone have the similar problem? Do you have any suggestion what can
be the cause of the problem?

Best regards,
Hanna

--
Department of Plant Anatomy and Cytology
Faculty of Biology and Environmental Protection
University of Silesia
Jagiellonska Str. 28
40-032 Katowice
Poland

[tineke vendrig]

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Have you optimised you microscope ?? Kohler illumination and so on..??

Tineke Vendrig, ing
technical engeneer optical microscopy
Delft University of Technology
Bionano Science
Kavli Institute of Nanoscience
Lorentzweg 1
2628LJ Delft
room F185
Tel: [phone]
Fax:[phone]
email: [hidden email]
mobile phone: [phone]


2011/10/6 Hanna Sas Nowosielska <[hidden email]>

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear all,
>
> For a couple of months we are struggling with a serious problem with
> the field illumination in our Olympus Scan^R Scanning station. The
> image of the specimen, when observed on the computer screen is much
> darker on one side than on the other. We thought it is the lamp but
> the lamp is new at properly inserted. Then we tried calibrating the
> optic fiber, but it didn't help at all. Our third guess was the
> filters.We checked all of the filters  -  the coating has lots of
> small dark spots with biggest concentration of the spots in the middle
> of the filters, but I am concerned if that can  produce uneven
> illumination of the field so that only one part (let say left corner)
> is underilluminated? So, I turn to you and your experience: did may
> anyone have the similar problem? Do you have any suggestion what can
> be the cause of the problem?
>
> Best regards,
> Hanna
>
> --
> Department of Plant Anatomy and Cytology
> Faculty of Biology and Environmental Protection
> University of Silesia
> Jagiellonska Str. 28
> 40-032 Katowice
> Poland
>

--
Tineke Vendrig, ing
technical engeneer optical microscopy
Delft University of Technology
Bionano Science
Kavli Institute of Nanoscience
Lorentzweg 1
2628LJ Delft
room F185
Tel: [phone]
Fax:[phone]
email: [hidden email]
mobile phone: [phone]

[Hanna_SN]

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Yes, at least Olymus  claims everything with the optical path is ok.

Hanna

2011/10/6 tineke vendrig <[hidden email]>:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Have you optimised you microscope ?? Kohler illumination and so on..??
>
> Tineke Vendrig, ing
> technical engeneer optical microscopy
> Delft University of Technology
> Bionano Science
> Kavli Institute of Nanoscience
> Lorentzweg 1
> 2628LJ Delft
> room F185
> Tel: [phone]
> Fax:[phone]
> email: [hidden email]
> mobile phone: [phone]
>
>
> 2011/10/6 Hanna Sas Nowosielska <[hidden email]>
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear all,
>>
>> For a couple of months we are struggling with a serious problem with
>> the field illumination in our Olympus Scan^R Scanning station. The
>> image of the specimen, when observed on the computer screen is much
>> darker on one side than on the other. We thought it is the lamp but
>> the lamp is new at properly inserted. Then we tried calibrating the
>> optic fiber, but it didn't help at all. Our third guess was the
>> filters.We checked all of the filters  -  the coating has lots of
>> small dark spots with biggest concentration of the spots in the middle
>> of the filters, but I am concerned if that can  produce uneven
>> illumination of the field so that only one part (let say left corner)
>> is underilluminated? So, I turn to you and your experience: did may
>> anyone have the similar problem? Do you have any suggestion what can
>> be the cause of the problem?
>>
>> Best regards,
>> Hanna
>>
>> --
>> Department of Plant Anatomy and Cytology
>> Faculty of Biology and Environmental Protection
>> University of Silesia
>> Jagiellonska Str. 28
>> 40-032 Katowice
>> Poland
>>
>
>
>
> --
> Tineke Vendrig, ing
> technical engeneer optical microscopy
> Delft University of Technology
> Bionano Science
> Kavli Institute of Nanoscience
> Lorentzweg 1
> 2628LJ Delft
> room F185
> Tel: [phone]
> Fax:[phone]
> email: [hidden email]
> mobile phone: [phone]
>

--
Department of Plant Anatomy and Cytology
Faculty of Biology and Environmental Protection
University of Silesia
Jagiellonska Str. 28
40-032 Katowice
Poland

[Arne Seitz]

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If the beam path is ok then you do not have uneven illumination.
Or the other way round: Uneven illumination is a perfect sign that the beam path is not ok (but a service technician should be able to fix it).

Cheers Arne

> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of Hanna Sas
> Nowosielska
> Sent: jeudi 6 octobre 2011 16:37
> To: [hidden email]
> Subject: Re: Rejected posting to [hidden email]
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Yes, at least Olymus  claims everything with the optical path is ok.
>
> Hanna
>
> 2011/10/6 tineke vendrig <[hidden email]>:
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Have you optimised you microscope ?? Kohler illumination and so on..??
> >
> > Tineke Vendrig, ing
> > technical engeneer optical microscopy
> > Delft University of Technology
> > Bionano Science
> > Kavli Institute of Nanoscience
> > Lorentzweg 1
> > 2628LJ Delft
> > room F185
> > Tel: [phone]
> > Fax:[phone]
> > email: [hidden email]
> > mobile phone: [phone]
> >
> >
> > 2011/10/6 Hanna Sas Nowosielska <[hidden email]>
> >
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> *****
> >>
> >> Dear all,
> >>
> >> For a couple of months we are struggling with a serious problem with
> >> the field illumination in our Olympus Scan^R Scanning station. The
> >> image of the specimen, when observed on the computer screen is much
> >> darker on one side than on the other. We thought it is the lamp but
> >> the lamp is new at properly inserted. Then we tried calibrating the
> >> optic fiber, but it didn't help at all. Our third guess was the
> >> filters.We checked all of the filters  -  the coating has lots of
> >> small dark spots with biggest concentration of the spots in the
> >> middle of the filters, but I am concerned if that can  produce uneven
> >> illumination of the field so that only one part (let say left corner)
> >> is underilluminated? So, I turn to you and your experience: did may
> >> anyone have the similar problem? Do you have any suggestion what can
> >> be the cause of the problem?
> >>
> >> Best regards,
> >> Hanna
> >>
> >> --
> >> Department of Plant Anatomy and Cytology Faculty of Biology and
> >> Environmental Protection University of Silesia Jagiellonska Str. 28
> >> 40-032 Katowice
> >> Poland
> >>
> >
> >
> >
> > --
> > Tineke Vendrig, ing
> > technical engeneer optical microscopy
> > Delft University of Technology
> > Bionano Science
> > Kavli Institute of Nanoscience
> > Lorentzweg 1
> > 2628LJ Delft
> > room F185
> > Tel: [phone]
> > Fax:[phone]
> > email: [hidden email]
> > mobile phone: [phone]
> >
>
>
>
> --
> Department of Plant Anatomy and Cytology Faculty of Biology and
> Environmental Protection University of Silesia Jagiellonska Str. 28
> 40-032 Katowice
> Poland

[Julio Vazquez]

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When we see similar problems, it tends to be because someone inadvertently bumped a shutter.... start near the light source and work your way along the light path up to the objective, and move any lever or slider you encounter. Our Olympus IX70 has a manual shutter which is a horizontal rotating disk between the filter turret and the objectives. People often bump into the lever and close it partially. On other microscopes, the shutter may be between the lamp and the filter turret. Also, if you have any DIC components, make sure they are either in or out.... sometimes they are half way, and will block part of the illumination field....
--
Julio Vazquez
Fred Hutchinson Cancer Research Center
Seattle, WA

http://www.fhcrc.org/




On Oct 6, 2011, at 7:20 AM, Hanna Sas Nowosielska wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear all,
>
> For a couple of months we are struggling with a serious problem with
> the field illumination in our Olympus Scan^R Scanning station. The
> image of the specimen, when observed on the computer screen is much
> darker on one side than on the other. We thought it is the lamp but
> the lamp is new at properly inserted. Then we tried calibrating the
> optic fiber, but it didn't help at all. Our third guess was the
> filters.We checked all of the filters  -  the coating has lots of
> small dark spots with biggest concentration of the spots in the middle
> of the filters, but I am concerned if that can  produce uneven
> illumination of the field so that only one part (let say left corner)
> is underilluminated? So, I turn to you and your experience: did may
> anyone have the similar problem? Do you have any suggestion what can
> be the cause of the problem?
>
> Best regards,
> Hanna
>
> --
> Department of Plant Anatomy and Cytology
> Faculty of Biology and Environmental Protection
> University of Silesia
> Jagiellonska Str. 28
> 40-032 Katowice
> Poland

[Guy Cox-2]

*****
To join, leave or search the confocal microscopy listserv, go to:
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*****

I got a rather left field enquiry today, as to whether there were
calibration standards for quantum yield.  It seems that the person wants
to measure quantum yield under the microscope.  My immediate response
was that this is impossible.  Quantum yield is easy enough to measure in
a cuvette but would it be possible in a microscope?  You could make a
standard of a known concentration of fluorescein in a cell made by a
spacer under the coverslip, but where do you go from there, if   both
quantum yield and extinction coefficient of the test sample are unknown?

Any bright ideas?

                                          Guy  


 

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net
 

[Craig Brideau]

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If you knew the focal volume being illuminated, you could guess at how many
dye molecules were present based on concentration.  Then based on input
power figure out the photon flux rate through the volume.  The tricky part
would be figuring out what the chances are of any one dye molecule being
struck by a photon.  If you had a photon counting detection system you could
then compare detected photons vs inputted photons.  This would have to be
modified by the probability of a photon hitting a dye molecule in the first
place and the QE of your detector (so for every detected emitted photon
roughly how many do you fail to detected, and of course photon density in
the illuminated volume.

These are just my first thoughts.  I'd have to give it more consideration,
especially regarding the likelihood of a photon hitting a dye molecule and
how that would modify the final value.

Craig



On Thu, Oct 6, 2011 at 7:43 PM, Guy Cox <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I got a rather left field enquiry today, as to whether there were
> calibration standards for quantum yield.  It seems that the person wants
> to measure quantum yield under the microscope.  My immediate response
> was that this is impossible.  Quantum yield is easy enough to measure in
> a cuvette but would it be possible in a microscope?  You could make a
> standard of a known concentration of fluorescein in a cell made by a
> spacer under the coverslip, but where do you go from there, if   both
> quantum yield and extinction coefficient of the test sample are unknown?
>
> Any bright ideas?
>
>                                          Guy
>
>
>
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor & Francis
>     http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Australian Centre for Microscopy & Microanalysis,
> Madsen Building F09, University of Sydney, NSW 2006
>
> Phone [phone]     Fax [phone]
>             Mobile [phone]
> ______________________________________________
>      http://www.guycox.net
>
>

[mmodel]

*****
To join, leave or search the confocal microscopy listserv, go to:
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*****

Is the test sample a biological tissue or a solution of a chemical?

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox
Sent: Thursday, October 06, 2011 9:44 PM
To: [hidden email]
Subject: Quantum yield

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

I got a rather left field enquiry today, as to whether there were
calibration standards for quantum yield.  It seems that the person wants
to measure quantum yield under the microscope.  My immediate response
was that this is impossible.  Quantum yield is easy enough to measure in
a cuvette but would it be possible in a microscope?  You could make a
standard of a known concentration of fluorescein in a cell made by a
spacer under the coverslip, but where do you go from there, if   both
quantum yield and extinction coefficient of the test sample are unknown?

Any bright ideas?

                                          Guy  


 

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net
 

[Unruh, Jay]

*****
To join, leave or search the confocal microscopy listserv, go to:
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In order to measure quantum yields by the reference method, one only needs a defined measurement volume, a standard dye of known absorbance (at the microscope excitation wavelength) and quantum yield, and the absorbance of your sample at the microscope excitation wavelength.  Quantum yield is then defined as QY=QYstandard*(A/Astandard) *(Fstandard/F)*(n^2/nstandard^2).  Here n is the refractive index, F is the measured fluorescence intensity and A is the absorbance at the microscope excitation wavelength.  Note that it is important that the standard and your unknown have similar emission spectra.  Otherwise you will have to correct for the wavelength dependence of your microscope detection efficiency.  Of course, if you have enough sample to measure absorbance, you can also measure fluorescence in a cuvette and then there is no point in going to the microscope.

Jay

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox
Sent: Thursday, October 06, 2011 8:44 PM
To: [hidden email]
Subject: Quantum yield

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

I got a rather left field enquiry today, as to whether there were calibration standards for quantum yield.  It seems that the person wants to measure quantum yield under the microscope.  My immediate response was that this is impossible.  Quantum yield is easy enough to measure in a cuvette but would it be possible in a microscope?  You could make a standard of a known concentration of fluorescein in a cell made by a
spacer under the coverslip, but where do you go from there, if   both
quantum yield and extinction coefficient of the test sample are unknown?

Any bright ideas?

                                          Guy  


 

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon) Australian Centre for Microscopy & Microanalysis, Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net
 

[Craig Brideau]

*****
To join, leave or search the confocal microscopy listserv, go to:
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*****

That's a pretty neat method!  It avoids having to worry about figuring out
photon flux by calibrating against a known.  Where would you get trustworthy
QE calibration standards though?

Craig

On Fri, Oct 7, 2011 at 10:14 AM, Unruh, Jay <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> In order to measure quantum yields by the reference method, one only needs
> a defined measurement volume, a standard dye of known absorbance (at the
> microscope excitation wavelength) and quantum yield, and the absorbance of
> your sample at the microscope excitation wavelength.  Quantum yield is then
> defined as QY=QYstandard*(A/Astandard) *(Fstandard/F)*(n^2/nstandard^2).
>  Here n is the refractive index, F is the measured fluorescence intensity
> and A is the absorbance at the microscope excitation wavelength.  Note that
> it is important that the standard and your unknown have similar emission
> spectra.  Otherwise you will have to correct for the wavelength dependence
> of your microscope detection efficiency.  Of course, if you have enough
> sample to measure absorbance, you can also measure fluorescence in a cuvette
> and then there is no point in going to the microscope.
>
> Jay
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Guy Cox
> Sent: Thursday, October 06, 2011 8:44 PM
> To: [hidden email]
> Subject: Quantum yield
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I got a rather left field enquiry today, as to whether there were
> calibration standards for quantum yield.  It seems that the person wants to
> measure quantum yield under the microscope.  My immediate response was that
> this is impossible.  Quantum yield is easy enough to measure in a cuvette
> but would it be possible in a microscope?  You could make a standard of a
> known concentration of fluorescein in a cell made by a
> spacer under the coverslip, but where do you go from there, if   both
> quantum yield and extinction coefficient of the test sample are unknown?
>
> Any bright ideas?
>
>                                          Guy
>
>
>
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor & Francis
>     http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon) Australian Centre for
> Microscopy & Microanalysis, Madsen Building F09, University of Sydney, NSW
> 2006
>
> Phone [phone]     Fax [phone]
>             Mobile [phone]
> ______________________________________________
>      http://www.guycox.net
>
>

[mmodel]

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

We used to create a defined measurement volume by placing a liquid sample between a slide and a spherical lens - then you get a range of depths that increase with the distance from the center. Cytometry 75A, 874-881 (2009)  

Mike

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Craig Brideau
Sent: Friday, October 07, 2011 12:53 PM
To: [hidden email]
Subject: Re: Quantum yield


On Fri, Oct 7, 2011 at 10:14 AM, Unruh, Jay <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> In order to measure quantum yields by the reference method, one only needs
> a defined measurement volume, a standard dye of known absorbance (at the
> microscope excitation wavelength) and quantum yield, and the absorbance of
> your sample at the microscope excitation wavelength.  Quantum yield is then
> defined as QY=QYstandard*(A/Astandard) *(Fstandard/F)*(n^2/nstandard^2).
>  Here n is the refractive index, F is the measured fluorescence intensity
> and A is the absorbance at the microscope excitation wavelength.  Note that
> it is important that the standard and your unknown have similar emission
> spectra.  Otherwise you will have to correct for the wavelength dependence
> of your microscope detection efficiency.  Of course, if you have enough
> sample to measure absorbance, you can also measure fluorescence in a cuvette
> and then there is no point in going to the microscope.
>
> Jay
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Guy Cox
> Sent: Thursday, October 06, 2011 8:44 PM
> To: [hidden email]
> Subject: Quantum yield
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I got a rather left field enquiry today, as to whether there were
> calibration standards for quantum yield.  It seems that the person wants to
> measure quantum yield under the microscope.  My immediate response was that
> this is impossible.  Quantum yield is easy enough to measure in a cuvette
> but would it be possible in a microscope?  You could make a standard of a
> known concentration of fluorescein in a cell made by a
> spacer under the coverslip, but where do you go from there, if   both
> quantum yield and extinction coefficient of the test sample are unknown?
>
> Any bright ideas?
>
>                                          Guy
>
>
>
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor & Francis
>     http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon) Australian Centre for
> Microscopy & Microanalysis, Madsen Building F09, University of Sydney, NSW
> 2006
>
> Phone [phone]     Fax [phone]
>             Mobile [phone]
> ______________________________________________
>      http://www.guycox.net
>
>

[Unruh, Jay]

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

Oops--I accidentally inverted the equation.  It should be QY=QYstandard*(F/Fstandard) *(Astandard/A)*(n^2/nstandard^2).

For quantum yield standards, see Magde et al photochemistry and photobiology 2002 vol 75 p 327.  It gives a thorough study for fluorescein and rhodamine 6G.  Other wavelengths are a bit more difficult.  Lakowicz principles of fluorescence spectroscopy gives more details and a few standards from the literature.

Jay

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Craig Brideau
Sent: Friday, October 07, 2011 11:53 AM
To: [hidden email]
Subject: Re: Quantum yield

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

That's a pretty neat method!  It avoids having to worry about figuring out photon flux by calibrating against a known.  Where would you get trustworthy QE calibration standards though?

Craig

On Fri, Oct 7, 2011 at 10:14 AM, Unruh, Jay <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> In order to measure quantum yields by the reference method, one only
> needs a defined measurement volume, a standard dye of known absorbance
> (at the microscope excitation wavelength) and quantum yield, and the
> absorbance of your sample at the microscope excitation wavelength.  
> Quantum yield is then defined as QY=QYstandard*(A/Astandard) *(Fstandard/F)*(n^2/nstandard^2).
>  Here n is the refractive index, F is the measured fluorescence
> intensity and A is the absorbance at the microscope excitation
> wavelength.  Note that it is important that the standard and your
> unknown have similar emission spectra.  Otherwise you will have to
> correct for the wavelength dependence of your microscope detection
> efficiency.  Of course, if you have enough sample to measure
> absorbance, you can also measure fluorescence in a cuvette and then there is no point in going to the microscope.
>
> Jay
>
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]]
> On Behalf Of Guy Cox
> Sent: Thursday, October 06, 2011 8:44 PM
> To: [hidden email]
> Subject: Quantum yield
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I got a rather left field enquiry today, as to whether there were
> calibration standards for quantum yield.  It seems that the person
> wants to measure quantum yield under the microscope.  My immediate
> response was that this is impossible.  Quantum yield is easy enough to
> measure in a cuvette but would it be possible in a microscope?  You
> could make a standard of a known concentration of fluorescein in a cell made by a
> spacer under the coverslip, but where do you go from there, if   both
> quantum yield and extinction coefficient of the test sample are unknown?
>
> Any bright ideas?
>
>                                          Guy
>
>
>
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor & Francis
>     http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon) Australian Centre for
> Microscopy & Microanalysis, Madsen Building F09, University of Sydney,
> NSW
> 2006
>
> Phone [phone]     Fax [phone]
>             Mobile [phone]
> ______________________________________________
>      http://www.guycox.net
>
>

[Guy Cox-2]

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It's quantum dots, just to compound the problem.

                                           Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net
 


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of MODEL, MICHAEL
Sent: Saturday, 8 October 2011 12:19 AM
To: [hidden email]
Subject: Re: Quantum yield

*****
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Is the test sample a biological tissue or a solution of a chemical?

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Guy Cox
Sent: Thursday, October 06, 2011 9:44 PM
To: [hidden email]
Subject: Quantum yield

*****
To join, leave or search the confocal microscopy listserv, go to:
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I got a rather left field enquiry today, as to whether there were
calibration standards for quantum yield.  It seems that the person wants
to measure quantum yield under the microscope.  My immediate response
was that this is impossible.  Quantum yield is easy enough to measure in
a cuvette but would it be possible in a microscope?  You could make a
standard of a known concentration of fluorescein in a cell made by a
spacer under the coverslip, but where do you go from there, if   both
quantum yield and extinction coefficient of the test sample are unknown?

Any bright ideas?

                                          Guy  


 

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net
 

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[mmodel]

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If the extinction coefficient of their suspension is known at a wavelength where some well-characterized fluorophore also absorbs, then their fluorescence intensities could be compared under identical conditions... But then, there is an issue of detector's spectral sensitivity, and even for some well-charactrized fluorophores, different people report very different values of quantum yield, I don't know why. I hope others would have something more helpful to say...

Mike
________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Guy Cox [[hidden email]]
Sent: Saturday, October 08, 2011 2:21 AM
To: [hidden email]
Subject: Re: Quantum yield

*****
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It's quantum dots, just to compound the problem.

                                           Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of MODEL, MICHAEL
Sent: Saturday, 8 October 2011 12:19 AM
To: [hidden email]
Subject: Re: Quantum yield

*****
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*****

Is the test sample a biological tissue or a solution of a chemical?

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Guy Cox
Sent: Thursday, October 06, 2011 9:44 PM
To: [hidden email]
Subject: Quantum yield

*****
To join, leave or search the confocal microscopy listserv, go to:
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*****

I got a rather left field enquiry today, as to whether there were
calibration standards for quantum yield.  It seems that the person wants
to measure quantum yield under the microscope.  My immediate response
was that this is impossible.  Quantum yield is easy enough to measure in
a cuvette but would it be possible in a microscope?  You could make a
standard of a known concentration of fluorescein in a cell made by a
spacer under the coverslip, but where do you go from there, if   both
quantum yield and extinction coefficient of the test sample are unknown?

Any bright ideas?

                                          Guy




Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net


-----
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[Iain Johnson]

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Any of the conventional macroscopic methods of quantum yield determination
will be susceptible to large errors if implemented under a microscope as the
optical pathlength is so short.  Even in a cuvette, the precision of the
macroscopic methods is not so good (generally about +/- 5%).  Particularly
in the case of quantum dots, single molecule approaches make more sense and
are more physically informative than ensemble measurements:
http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=PubMed&dopt=AbstractPlus&list_uids=16169907

Iain

Iain Johnson Consulting
Eugene, OR

On Fri, Oct 7, 2011 at 11:21 PM, Guy Cox <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> It's quantum dots, just to compound the problem.
>
>                                           Guy
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor & Francis
>     http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Australian Centre for Microscopy & Microanalysis,
> Madsen Building F09, University of Sydney, NSW 2006
>
> Phone [phone]     Fax [phone]
>             Mobile [phone]
> ______________________________________________
>      http://www.guycox.net
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of MODEL, MICHAEL
> Sent: Saturday, 8 October 2011 12:19 AM
> To: [hidden email]
> Subject: Re: Quantum yield
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Is the test sample a biological tissue or a solution of a chemical?
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Guy Cox
> Sent: Thursday, October 06, 2011 9:44 PM
> To: [hidden email]
> Subject: Quantum yield
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I got a rather left field enquiry today, as to whether there were
> calibration standards for quantum yield.  It seems that the person wants
> to measure quantum yield under the microscope.  My immediate response
> was that this is impossible.  Quantum yield is easy enough to measure in
> a cuvette but would it be possible in a microscope?  You could make a
> standard of a known concentration of fluorescein in a cell made by a
> spacer under the coverslip, but where do you go from there, if   both
> quantum yield and extinction coefficient of the test sample are unknown?
>
> Any bright ideas?
>
>                                          Guy
>
>
>
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor & Francis
>     http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Australian Centre for Microscopy & Microanalysis,
> Madsen Building F09, University of Sydney, NSW 2006
>
> Phone [phone]     Fax [phone]
>             Mobile [phone]
> ______________________________________________
>      http://www.guycox.net
>
>
> -----
> No virus found in this message.
> Checked by AVG - www.avg.com
> Version: 10.0.1410 / Virus Database: 1520/3942 - Release Date: 10/06/11
>

[Axel Kurt Preuss]

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Dear Hanna
Koehler comes to mind as already suggested.
I m not familiar with scanning station

We had similar problem on an Olympus scope and found that the glass in front of the   transmission lamp had a crack (heat?)

So, this  glass  in the transmission housing, which directly faces the microscope stage , had a crack and that produced a shadow in the image pretending to be a Koehler problem

cheers

Axel                    6-19B ,   92715622



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Hanna Sas Nowosielska
Sent: Thursday, 6 October, 2011 10:37 PM
To: [hidden email]
Subject: Re: Rejected posting to [hidden email]

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Yes, at least Olymus  claims everything with the optical path is ok.

Hanna

2011/10/6 tineke vendrig <[hidden email]>:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Have you optimised you microscope ?? Kohler illumination and so on..??
>
> Tineke Vendrig, ing
> technical engeneer optical microscopy
> Delft University of Technology
> Bionano Science
> Kavli Institute of Nanoscience
> Lorentzweg 1
> 2628LJ Delft
> room F185
> Tel: [phone]
> Fax:[phone]
> email: [hidden email]
> mobile phone: [phone]
>
>
> 2011/10/6 Hanna Sas Nowosielska <[hidden email]>
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear all,
>>
>> For a couple of months we are struggling with a serious problem with
>> the field illumination in our Olympus Scan^R Scanning station. The
>> image of the specimen, when observed on the computer screen is much
>> darker on one side than on the other. We thought it is the lamp but
>> the lamp is new at properly inserted. Then we tried calibrating the
>> optic fiber, but it didn't help at all. Our third guess was the
>> filters.We checked all of the filters  -  the coating has lots of
>> small dark spots with biggest concentration of the spots in the
>> middle of the filters, but I am concerned if that can  produce uneven
>> illumination of the field so that only one part (let say left corner)
>> is underilluminated? So, I turn to you and your experience: did may
>> anyone have the similar problem? Do you have any suggestion what can
>> be the cause of the problem?
>>
>> Best regards,
>> Hanna
>>
>> --
>> Department of Plant Anatomy and Cytology Faculty of Biology and
>> Environmental Protection University of Silesia Jagiellonska Str. 28
>> 40-032 Katowice
>> Poland
>>
>
>
>
> --
> Tineke Vendrig, ing
> technical engeneer optical microscopy
> Delft University of Technology
> Bionano Science
> Kavli Institute of Nanoscience
> Lorentzweg 1
> 2628LJ Delft
> room F185
> Tel: [phone]
> Fax:[phone]
> email: [hidden email]
> mobile phone: [phone]
>

--
Department of Plant Anatomy and Cytology Faculty of Biology and Environmental Protection University of Silesia Jagiellonska Str. 28
40-032 Katowice
Poland

Note: This message may contain confidential information. If this Email/Fax has been sent to you by mistake, please notify the sender and delete it immediately. Thank you.

[Zac Arrac Atelaz]

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I was thinking on this one, the uneveness of the field is more visible in a specific wavelength or in all of them? usually UV is the most uneven. A different case might be the DIC ilumination being in the wrong position (usually it has 2 positions), the other one would be a crack in the fiber (hopefully this one is just a wild guess) Best luck Gabriel OH
 > Date: Thu, 3 Nov 2011 11:46:58 +0800

> From: [hidden email]
> Subject: Re: Rejected posting to [hidden email]
> To: [hidden email]
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Hanna
> Koehler comes to mind as already suggested.
> I m not familiar with scanning station
>
> We had similar problem on an Olympus scope and found that the glass in front of the   transmission lamp had a crack (heat?)
>
> So, this  glass  in the transmission housing, which directly faces the microscope stage , had a crack and that produced a shadow in the image pretending to be a Koehler problem
>
> cheers
>
> Axel                    6-19B ,   92715622
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Hanna Sas Nowosielska
> Sent: Thursday, 6 October, 2011 10:37 PM
> To: [hidden email]
> Subject: Re: Rejected posting to [hidden email]
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Yes, at least Olymus  claims everything with the optical path is ok.
>
> Hanna
>
> 2011/10/6 tineke vendrig <[hidden email]>:
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Have you optimised you microscope ?? Kohler illumination and so on..??
> >
> > Tineke Vendrig, ing
> > technical engeneer optical microscopy
> > Delft University of Technology
> > Bionano Science
> > Kavli Institute of Nanoscience
> > Lorentzweg 1
> > 2628LJ Delft
> > room F185
> > Tel: [phone]
> > Fax:[phone]
> > email: [hidden email]
> > mobile phone: [phone]
> >
> >
> > 2011/10/6 Hanna Sas Nowosielska <[hidden email]>
> >
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> *****
> >>
> >> Dear all,
> >>
> >> For a couple of months we are struggling with a serious problem with
> >> the field illumination in our Olympus Scan^R Scanning station. The
> >> image of the specimen, when observed on the computer screen is much
> >> darker on one side than on the other. We thought it is the lamp but
> >> the lamp is new at properly inserted. Then we tried calibrating the
> >> optic fiber, but it didn't help at all. Our third guess was the
> >> filters.We checked all of the filters  -  the coating has lots of
> >> small dark spots with biggest concentration of the spots in the
> >> middle of the filters, but I am concerned if that can  produce uneven
> >> illumination of the field so that only one part (let say left corner)
> >> is underilluminated? So, I turn to you and your experience: did may
> >> anyone have the similar problem? Do you have any suggestion what can
> >> be the cause of the problem?
> >>
> >> Best regards,
> >> Hanna
> >>
> >> --
> >> Department of Plant Anatomy and Cytology Faculty of Biology and
> >> Environmental Protection University of Silesia Jagiellonska Str. 28
> >> 40-032 Katowice
> >> Poland
> >>
> >
> >
> >
> > --
> > Tineke Vendrig, ing
> > technical engeneer optical microscopy
> > Delft University of Technology
> > Bionano Science
> > Kavli Institute of Nanoscience
> > Lorentzweg 1
> > 2628LJ Delft
> > room F185
> > Tel: [phone]
> > Fax:[phone]
> > email: [hidden email]
> > mobile phone: [phone]
> >
>
>
>
> --
> Department of Plant Anatomy and Cytology Faculty of Biology and Environmental Protection University of Silesia Jagiellonska Str. 28
> 40-032 Katowice
> Poland
>
> Note: This message may contain confidential information. If this Email/Fax has been sent to you by mistake, please notify the sender and delete it immediately. Thank you.

     

[Guy Cox-2]

*****
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I was doing a bit of historical research today and I looked up Jan
Wichmann, of the historic first paper on STED (Hell & Wichmann, 1994,
Optics Letters 19: 780-782).  To my surprise the Scopus database showed
just one paper for Wichmann - that one, with more than 500 citations.
This must be a pretty rare situation.  Does anyone know who Wichmann
(from Turku in Finland) was and what became of him?

                                                        Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net
 

[Sudipta Maiti]

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According to Prof. Jurgen Wolfrum, Wichmann's Ph.D. advisor and currently a
visitor here, Wichmann left science without finishing his Ph.D. for personal
reasons. He had taken a leave for a few months during his Ph.D. period to
pursue the STED idea with Stefan Hell.
Sudipta

On Mon, 7 Nov 2011 21:49:36 +1100, Guy Cox wrote

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I was doing a bit of historical research today and I looked up Jan
> Wichmann, of the historic first paper on STED (Hell & Wichmann, 1994,
> Optics Letters 19: 780-782).  To my surprise the Scopus database showed
> just one paper for Wichmann - that one, with more than 500 citations.
> This must be a pretty rare situation.  Does anyone know who Wichmann
> (from Turku in Finland) was and what became of him?
>
> Guy
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor & Francis
>      http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Australian Centre for Microscopy & Microanalysis,
> Madsen Building F09, University of Sydney, NSW 2006
>
> Phone [phone]     Fax [phone]
>              Mobile [phone]
> ______________________________________________
>       http://www.guycox.net

Dr. Sudipta Maiti
Dept. of Chemical Sciences
Tata Institute of Fundamental Research
Homi Bhabha Raod, Colaba, Mumbai 400005
Ph. 91-22-2278-2716 / 2539
Fax: [phone]
alternate e-mail: [hidden email]
url: biophotonics.weebly.com

[Guy Cox-2]

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Sudipta,

                  Thanks for this - it is very interesting.  You will
have noticed that Vitaly Boyko dug out a Jan Wichmann in Finland, and I
have contacted him.  He was not the Jan Wichmann of the Wichmann and
Hell paper.  So does Jurgen know what the 'real' Jan Wichmann ended up
doing?

                                           Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net
 

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Sudipta Maiti
Sent: Saturday, 12 November 2011 9:43 PM
To: [hidden email]
Subject: Re: Historical question - Jan Wichmann

*****
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According to Prof. Jurgen Wolfrum, Wichmann's Ph.D. advisor and
currently a
visitor here, Wichmann left science without finishing his Ph.D. for
personal
reasons. He had taken a leave for a few months during his Ph.D. period
to
pursue the STED idea with Stefan Hell.
Sudipta

On Mon, 7 Nov 2011 21:49:36 +1100, Guy Cox wrote
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I was doing a bit of historical research today and I looked up Jan
> Wichmann, of the historic first paper on STED (Hell & Wichmann, 1994,
> Optics Letters 19: 780-782).  To my surprise the Scopus database
showed

> just one paper for Wichmann - that one, with more than 500 citations.
> This must be a pretty rare situation.  Does anyone know who Wichmann
> (from Turku in Finland) was and what became of him?
>
> Guy
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor & Francis
>      http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Australian Centre for Microscopy & Microanalysis,
> Madsen Building F09, University of Sydney, NSW 2006
>
> Phone [phone]     Fax [phone]
>              Mobile [phone]
> ______________________________________________
>       http://www.guycox.net

Dr. Sudipta Maiti
Dept. of Chemical Sciences
Tata Institute of Fundamental Research
Homi Bhabha Raod, Colaba, Mumbai 400005
Ph. 91-22-2278-2716 / 2539
Fax: [phone]
alternate e-mail: [hidden email]
url: biophotonics.weebly.com

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