Working distance

[Stéphane Pagès]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear List,

I wonder about the exact definition of working distance. As I
understood, in the case were the objective is not corrected for
coverslip, this distance is the between the front of the objective and
the specimen to be imaged.
In the case were I use a a coverslip corrected objective, what does it
mean?. For example I use a 63X water objective from Olympus with a
working distance of 300 um. The coverslip thickness is 150 um. What is
the effective distance between the front of the objective and the
specimen ?? 300 um ??? Here
www.microscopyu.com/articles/ formulas/formulasworkingparfocal.htm
I saw that the distance is between the objective and the upper side of
the coverslip ??
I don t know if I misunderstood something or not

Thanks, Stephane
--
Stéphane Pages PhD
Neurobiologie cellulaire
Centre de Recherche Université Laval Robert-Giffard (CRULRG)
Bureau/office: F-5570
2601, Chemin de la Canardière
Beauport (Québec) G1J 2G3
Canada
Tél: [phone] ext. 4712 (bur./office)
Fax: [phone]
Courriel/email: [hidden email]

[Edelmann, Richard E. Dr.]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Stéphane:

        To my understanding the cover slip is considered an optical element
of the lens itself - which is why coverslip thickness is so critical,
and the working distance is the distance "beyond" the  optical
elements of the lens.  So if your lens calls for a 0.17mm coverslip
and has a WD or 300um, you get 300um beyond the 170um coverslip.
HOWEVER, if your coverslip is really 190um ( 0.160-0.190mm) you only
get 280um.  If you are using a correction collar then you can correct
the optics to include the thicker 190um coverslip, and thereby
recover your "lost" 20um.




On 30 Sep 2007 at 12:50, Stéphane Pagès wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear List,
>
> I wonder about the exact definition of working distance. As I
> understood, in the case were the objective is not corrected for
> coverslip, this distance is the between the front of the objective and
> the specimen to be imaged.
> In the case were I use a a coverslip corrected objective, what does it
> mean?. For example I use a 63X water objective from Olympus with a
> working distance of 300 um. The coverslip thickness is 150 um. What is
> the effective distance between the front of the objective and the
> specimen ?? 300 um ??? Here
> www.microscopyu.com/articles/ formulas/formulasworkingparfocal.htm
> I saw that the distance is between the objective and the upper side of
> the coverslip ??
> I don t know if I misunderstood something or not
>
> Thanks, Stephane
> --
> Stéphane Pages PhD
> Neurobiologie cellulaire
> Centre de Recherche Université Laval Robert-Giffard (CRULRG)
> Bureau/office: F-5570
> 2601, Chemin de la Canardière
> Beauport (Québec) G1J 2G3
> Canada
> Tél: [phone] ext. 4712 (bur./office)
> Fax: [phone]
> Courriel/email: [hidden email]

Richard E. Edelmann, Ph.D.
Electron Microscopy Facility Director
364 Pearson Hall
Miami University, Oxford, OH 45056
Ph: 513.529.5712        Fax: [phone]
E-mail: [hidden email]
http://www.emf.muohio.edu

[Barbara Foster]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear Stephane,

The working distance is defined simply as the distance between the front element of the objective (OR condenser, since it has a WD also) to the top of the specimen.  For the objective, the coverslip is considered the top of the objectivev..

Richard has an important point regarding the coverslip being an optical element in this system.  Image formation has two components: refraction and diffraction. Both involve the bending of light as it interacts with the sample ... or anything else it encounters. Most of us don't need a PhD in optics, so I won't go into all the fine detail, but I strongly recommend checking out a simple physics review book (one of those paperback summaries) and looking up Snell's law.  It will really help you to understand the impact, especially of refraction, as light passes through various boundaries, from slide to the mountant to the sample to the coverslip through the air or oil or water and into the objective.  The basic thought here:  if light crosses a boundary at an angle, it will change direction.  If it is going from higher refractive index (where its electric field has more interaction with the electric field, as in glass or oil), it will bend toward an imaginary reference line which is perpenidcular to the interface.  In the microscope, that is typically the optical axis (with the caveat of a few assumptions).  If it moves into a material of lower refractive index, such as air, it will bend away from that reference line.  Light carries information, so if it bends enough so that it escapes being collected by the objective, that information is lost.  That happens with either very fine detail, which causes light to diffract (bend) strongly and/or or with a big difference in refractive index (ex: moving from coverslip into air).  Also, as has been mentioned, if you don't use the right coverslip thickness (0.17mm = # 1-1/2), it is like having a lens out of position and your image will suffer from spherical aberration (it will be soft, mushy, and difficult to focus).  Simple experiment:  Use a thin, well-stained sample.  Observe it with an objective that has a coverslip rating of 0.17 (see the engravings on the barrel).  Then observe it (a) without a coverslip and (b) with a double coverslip. 

There's lots more to this story, but them's the bare bones.  Hope it was helpful.

Best regards,
Barbara Foster
We've moved!
Microscopy/Microscopy Education
7101 Royal Glen Trail, Suite A
McKinney TX 75070
P: [phone]
Skype: fostermme
W: www.MicroscopyEducation.com


MME is now scheduling customized, on-site courses through December.  Call us today for details.

P. S.
Need a good general reference or light microscopy text for next semester? Call us today to learn more about "Optimizing LIght Microscopy".  Copies still available through MME... even for class-room lots ... and we give quantity discounts. Just call us here in the MME office for details.



At 01:42 PM 10/1/2007, Richard E. Edelmann wrote:

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Stéphane:

        To my understanding the cover slip is considered an optical element
of the lens itself - which is why coverslip thickness is so critical,
and the working distance is the distance "beyond" the  optical
elements of the lens.  So if your lens calls for a 0.17mm coverslip
and has a WD or 300um, you get 300um beyond the 170um coverslip. 
HOWEVER, if your coverslip is really 190um ( 0.160-0.190mm) you only
get 280um.  If you are using a correction collar then you can correct
the optics to include the thicker 190um coverslip, and thereby
recover your "lost" 20um.




On 30 Sep 2007 at 12:50, Stéphane Pagès wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear List,
>
> I wonder about the exact definition of working distance. As I
> understood, in the case were the objective is not corrected for
> coverslip, this distance is the between the front of the objective and
> the specimen to be imaged.
> In the case were I use a a coverslip corrected objective, what does it
> mean?. For example I use a 63X water objective from Olympus with a
> working distance of 300 um. The coverslip thickness is 150 um. What is
> the effective distance between the front of the objective and the
> specimen ?? 300 um ??? Here
> www.microscopyu.com/articles/ formulas/formulasworkingparfocal.htm
> I saw that the distance is between the objective and the upper side of
> the coverslip ??
> I don t know if I misunderstood something or not
>
> Thanks, Stephane
> --
> Stéphane Pages PhD
> Neurobiologie cellulaire
> Centre de Recherche Université Laval Robert-Giffard (CRULRG)
> Bureau/office: F-5570
> 2601, Chemin de la Canardière
> Beauport (Québec) G1J 2G3
> Canada
> Tél: [phone] ext. 4712 (bur./office)
> Fax: [phone]
> Courriel/email: [hidden email]


Richard E. Edelmann, Ph.D.
Electron Microscopy Facility Director
364 Pearson Hall
Miami University, Oxford, OH 45056
Ph: 513.529.5712        Fax: [phone]
E-mail: [hidden email]
http://www.emf.muohio.edu

[Peterson, Lauren M.]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Barbara – Is there a typo?  Did you mean the coverslip is considered the top of the SPECIMEN ?

lauren M. Peterson, PhD     Lead Physicist - Photonics   
Michigan R&D Center  (formerly ERIM - Veridian)   
General Dynamics - Advanced Information Systems    ISS-SIS-ATE-EPA  
1200 Joe Hall Dr.,  P.O. Box 990       Ypsilanti, MI  48197   
734-480-5303              -480-5252 FAX       
[hidden email]     

 

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Barbara Foster
Sent: Monday, October 01, 2007 4:50 PM
To: [hidden email]
Subject: Re: Working distance

 

Dear Stephane,

The working distance is defined simply as the distance between the front element of the objective (OR condenser, since it has a WD also) to the top of the specimen.  For the objective, the coverslip is considered the top of the objectivev..

Richard has an important point regarding the coverslip being an optical element in this system.  Image formation has two components: refraction and diffraction. Both involve the bending of light as it interacts with the sample ... or anything else it encounters. Most of us don't need a PhD in optics, so I won't go into all the fine detail, but I strongly recommend checking out a simple physics review book (one of those paperback summaries) and looking up Snell's law.  It will really help you to understand the impact, especially of refraction, as light passes through various boundaries, from slide to the mountant to the sample to the coverslip through the air or oil or water and into the objective.  The basic thought here:  if light crosses a boundary at an angle, it will change direction.  If it is going from higher refractive index (where its electric field has more interaction with the electric field, as in glass or oil), it will bend toward an imaginary reference line which is perpenidcular to the interface.  In the microscope, that is typically the optical axis (with the caveat of a few assumptions).  If it moves into a material of lower refractive index, such as air, it will bend away from that reference line.  Light carries information, so if it bends enough so that it escapes being collected by the objective, that information is lost.  That happens with either very fine detail, which causes light to diffract (bend) strongly and/or or with a big difference in refractive index (ex: moving from coverslip into air).  Also, as has been mentioned, if you don't use the right coverslip thickness (0.17mm = # 1-1/2), it is like having a lens out of position and your image will suffer from spherical aberration (it will be soft, mushy, and difficult to focus).  Simple experiment:  Use a thin, well-stained sample.  Observe it with an objective that has a coverslip rating of 0.17 (see the engravings on the barrel).  Then observe it (a) without a coverslip and (b) with a double coverslip. 

There's lots more to this story, but them's the bare bones.  Hope it was helpful.

Best regards,
Barbara Foster
We've moved!
Microscopy/Microscopy Education
7101 Royal Glen Trail, Suite A
McKinney TX 75070
P: [phone]
Skype: fostermme
W: www.MicroscopyEducation.com


MME is now scheduling customized, on-site courses through December.  Call us today for details.

P. S.
Need a good general reference or light microscopy text for next semester? Call us today to learn more about "Optimizing LIght Microscopy".  Copies still available through MME... even for class-room lots ... and we give quantity discounts. Just call us here in the MME office for details.



At 01:42 PM 10/1/2007, Richard E. Edelmann wrote:

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Stéphane:

        To my understanding the cover slip is considered an optical element
of the lens itself - which is why coverslip thickness is so critical,
and the working distance is the distance "beyond" the  optical
elements of the lens.  So if your lens calls for a 0.17mm coverslip
and has a WD or 300um, you get 300um beyond the 170um coverslip. 
HOWEVER, if your coverslip is really 190um ( 0.160-0.190mm) you only
get 280um.  If you are using a correction collar then you can correct
the optics to include the thicker 190um coverslip, and thereby
recover your "lost" 20um.




On 30 Sep 2007 at 12:50, Stéphane Pagès wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear List,
>
> I wonder about the exact definition of working distance. As I
> understood, in the case were the objective is not corrected for
> coverslip, this distance is the between the front of the objective and
> the specimen to be imaged.
> In the case were I use a a coverslip corrected objective, what does it
> mean?. For example I use a 63X water objective from Olympus with a
> working distance of 300 um. The coverslip thickness is 150 um. What is
> the effective distance between the front of the objective and the
> specimen ?? 300 um ??? Here
> www.microscopyu.com/articles/ formulas/formulasworkingparfocal.htm
> I saw that the distance is between the objective and the upper side of
> the coverslip ??
> I don t know if I misunderstood something or not
>
> Thanks, Stephane
> --
> Stéphane Pages PhD
> Neurobiologie cellulaire
> Centre de Recherche Université Laval Robert-Giffard (CRULRG)
> Bureau/office: F-5570
> 2601, Chemin de la Canardière
> Beauport (Québec) G1J 2G3
> Canada
> Tél: [phone] ext. 4712 (bur./office)
> Fax: [phone]
> Courriel/email: [hidden email]


Richard E. Edelmann, Ph.D.
Electron Microscopy Facility Director
364 Pearson Hall
Miami University, Oxford, OH 45056
Ph: 513.529.5712        Fax: [phone]
E-mail: [hidden email]
http://www.emf.muohio.edu

[Martin Wessendorf]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Barbara Foster wrote:

> The working distance is defined simply as the distance between the front
> element of the objective (OR condenser, since it has a WD also) to the
> top of the specimen.  For the objective, the coverslip is considered the
> top of the objectivev..

Barbara--

Did you mean to say "For the objective, the coverslip is considered the
top of the objective," or "For the objective, the coverslip is
considered the top of the SPECIMEN."

Martin
--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455             E-mail: martinw[at]med.umn.edu

[Barbara Foster]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Nope.  For a conventionally prepared specimen, the working distance is the distance between the front element of the objective and the coverslip.

B
At 03:33 PM 10/1/2007, you wrote:

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Barbara – Is there a typo?  Did you mean the coverslip is considered the top of the SPECIMEN ?

lauren M. Peterson, PhD     Lead Physicist - Photonics   
Michigan R&D Center  (formerly ERIM - Veridian)   
General Dynamics - Advanced Information Systems    ISS-SIS-ATE-EPA  
1200 Joe Hall Dr.,  P.O. Box 990       Ypsilanti, MI  48197   
734-480-5303              -480-5252 FAX       
[hidden email]    

 

---

From: Confocal Microscopy List [[hidden email]] On Behalf Of Barbara Foster
Sent: Monday, October 01, 2007 4:50 PM
To: [hidden email]
Subject: Re: Working distance
 
Dear Stephane,

The working distance is defined simply as the distance between the front element of the objective (OR condenser, since it has a WD also) to the top of the specimen.  For the objective, the coverslip is considered the top of the objectivev..

Richard has an important point regarding the coverslip being an optical element in this system.  Image formation has two components: refraction and diffraction. Both involve the bending of light as it interacts with the sample ... or anything else it encounters. Most of us don't need a PhD in optics, so I won't go into all the fine detail, but I strongly recommend checking out a simple physics review book (one of those paperback summaries) and looking up Snell's law.  It will really help you to understand the impact, especially of refraction, as light passes through various boundaries, from slide to the mountant to the sample to the coverslip through the air or oil or water and into the objective.  The basic thought here:  if light crosses a boundary at an angle, it will change direction.  If it is going from higher refractive index (where its electric field has more interaction with the electric field, as in glass or oil), it will bend toward an imaginary reference line which is perpenidcular to the interface.  In the microscope, that is typically the optical axis (with the caveat of a few assumptions).  If it moves into a material of lower refractive index, such as air, it will bend away from that reference line.  Light carries information, so if it bends enough so that it escapes being collected by the objective, that information is lost.  That happens with either very fine detail, which causes light to diffract (bend) strongly and/or or with a big difference in refractive index (ex: moving from coverslip into air).  Also, as has been mentioned, if you don't use the right coverslip thickness (0.17mm = # 1-1/2), it is like having a lens out of position and your image will suffer from spherical aberration (it will be soft, mushy, and difficult to focus).  Simple experiment:  Use a thin, well-stained sample.  Observe it with an objective that has a coverslip rating of 0.17 (see the engravings on the barrel).  Then observe it (a) without a coverslip and (b) with a double coverslip. 

There's lots more to this story, but them's the bare bones.  Hope it was helpful.

Best regards,
Barbara Foster
We've moved!
Microscopy/Microscopy Education
7101 Royal Glen Trail, Suite A
McKinney TX 75070
P: [phone]
Skype: fostermme
W: www.MicroscopyEducation.com


MME is now scheduling customized, on-site courses through December.  Call us today for details.

P. S.
Need a good general reference or light microscopy text for next semester? Call us today to learn more about "Optimizing LIght Microscopy".  Copies still available through MME... even for class-room lots ... and we give quantity discounts. Just call us here in the MME office for details.



At 01:42 PM 10/1/2007, Richard E. Edelmann wrote:

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Stéphane:

        To my understanding the cover slip is considered an optical element
of the lens itself - which is why coverslip thickness is so critical,
and the working distance is the distance "beyond" the  optical
elements of the lens.  So if your lens calls for a 0.17mm coverslip
and has a WD or 300um, you get 300um beyond the 170um coverslip. 
HOWEVER, if your coverslip is really 190um ( 0.160-0.190mm) you only
get 280um.  If you are using a correction collar then you can correct
the optics to include the thicker 190um coverslip, and thereby
recover your "lost" 20um.




On 30 Sep 2007 at 12:50, Stéphane Pagès wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear List,
>
> I wonder about the exact definition of working distance. As I
> understood, in the case were the objective is not corrected for
> coverslip, this distance is the between the front of the objective and
> the specimen to be imaged.
> In the case were I use a a coverslip corrected objective, what does it
> mean?. For example I use a 63X water objective from Olympus with a
> working distance of 300 um. The coverslip thickness is 150 um. What is
> the effective distance between the front of the objective and the
> specimen ?? 300 um ??? Here
> www.microscopyu.com/articles/ formulas/formulasworkingparfocal.htm
> I saw that the distance is between the objective and the upper side of
> the coverslip ??
> I don t know if I misunderstood something or not
>
> Thanks, Stephane
> --
> Stéphane Pages PhD
> Neurobiologie cellulaire
> Centre de Recherche Université Laval Robert-Giffard (CRULRG)
> Bureau/office: F-5570
> 2601, Chemin de la Canardière
> Beauport (Québec) G1J 2G3
> Canada
> Tél: [phone] ext. 4712 (bur./office)
> Fax: [phone]
> Courriel/email: [hidden email]


Richard E. Edelmann, Ph.D.
Electron Microscopy Facility Director
364 Pearson Hall
Miami University, Oxford, OH 45056
Ph: 513.529.5712        Fax: [phone]
E-mail: [hidden email]
http://www.emf.muohio.edu

[Barbara Foster]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

At 04:33 PM 10/1/2007, Martin Wessendorf wrote:
Sorry, folks.  I did mean the top of the SPECIMEN (busy Monday afternoon!!!).

B

>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Barbara Foster wrote:
>
>>The working distance is defined simply as the distance between the
>>front element of the objective (OR condenser, since it has a WD
>>also) to the top of the specimen.  For the objective, the coverslip
>>is considered the top of the objectivev..
>
>Barbara--
>
>Did you mean to say "For the objective, the coverslip is considered the
>top of the objective," or "For the objective, the coverslip is
>considered the top of the SPECIMEN."
>
>Martin
>--
>Martin Wessendorf, Ph.D.                   office: [phone]
>Assoc Prof, Dept Neuroscience                 lab: [phone]
>University of Minnesota             Preferred FAX: [phone]
>6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
>Minneapolis, MN  55455             E-mail: martinw[at]med.umn.edu

[Steffen Dietzel]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

I have just noted that I sent my reply to
Stéphane directly instead of to the list. Richard
and Barbara have meanwhile explained most of in
in much more detail, but maybe the aspect of the
AFP versus NFP is worth it to be reposted.

For those who want to look in the details of this
aspect, I recommend chapter 20: "Lens aberrations
in confocal fluorescent microscopy" by Stefan
Hell and Ernst Stelzer in the second edition of
the "Handbook of Biological Confocal Microscopy."
There is a respective chapter in the 3rd edition
but I haven't read this one yet.

Steffen
----------------
Stéphane,

for coverslip-objectives (were the coverslip
should usually be 0.17 mm thick, as marked on the
barrel) the working distance is the distance
between the focal plane and the
coverslip/mounting medium interface when the
objective hits the coverslip. In other words, in
your case with 63x? (probably 60x when from
Olympus) you can image at most a total height of
300 um, the coverslip/mounting medium interface is then at 0 um.

Of course, this is all calculated for a perfect
optical system (sample embedded in immersion
medium). In real microscopy you often have a
refractive index mismatch (in particular with oil
immersion objectives) which causes the "nominal
focal point" to diverge from the "actual focal
point". This means: In a system with an automated
z-drive, when you move the stage down or up for
100 um, the focal point moves in the specimen by
a different height. With the water immersion lens
and an aqueous medium (and a coverslip of the
right thickness) you should do ok in this respect.

Steffen


At 18:50 30.09.2007, you wrote:

>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Dear List,
>
>I wonder about the exact definition of working distance. As I
>understood, in the case were the objective is not corrected for
>coverslip, this distance is the between the front of the objective and
>the specimen to be imaged.
>In the case were I use a a coverslip corrected objective, what does it
>mean?. For example I use a 63X water objective from Olympus with a
>working distance of 300 um. The coverslip thickness is 150 um. What is
>the effective distance between the front of the objective and the
>specimen ?? 300 um ??? Here
>www.microscopyu.com/articles/ formulas/formulasworkingparfocal.htm
>I saw that the distance is between the objective and the upper side of
>the coverslip ??
>I don t know if I misunderstood something or not
>
>Thanks, Stephane
>--
>Stéphane Pages PhD
>Neurobiologie cellulaire
>Centre de Recherche Université Laval Robert-Giffard (CRULRG)
>Bureau/office: F-5570
>2601, Chemin de la Canardière
>Beauport (Québec) G1J 2G3
>Canada
>Tél: [phone] ext. 4712 (bur./office)
>Fax: [phone]
>Courriel/email: [hidden email]

--
---------------------------------------------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum (WBZ)
Head of light microscopy

Building location and address for courier, parcel services etc:
Marchioninistr. 27, D-81366 München (Großhadern)

Mail room (for letters etc.):
Marchioninistr. 15, D-81366 München

Phone: [phone]
Fax:   [phone] (please anounce incoming fax by e-mail)
skype: steffendietzel
e-mail: [hidden email] (for everything university related)
          or [hidden email]

[Peterson, Lauren M.]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Richard - I believe that your 'HOWEVER' is a bit more complicated than you suggest and also not quite as bad as you say in that you do not loose as much working distance.  I did a simple ray tracing based upon Snell's Law for the rays going through the cover slip to find that for large NA the "lost" working distance in your example is not 20um but 20/n = 20/1.523 = 13.1um.  This is the case for NA < 0.3 and assumes that objective to coverslip is air.  For larger NA, WD lost is even less than 20/n and goes to zero for the largest NA = 1 in air.
      Here are the details of the formula if you are interested.  If x is the coverslip mismatch thickness (20um in your example) and d is the actual lost WD then

d = x * sqrt { [(1/NA)^2 - 1] / [(n/NA)^2 - 1] }

    You can see that when NA is very small, the 1/NA terms dominate and the -1 is negligible.  We have sqrt{1/n^2} = 1/n. 
    In the limit as NA goes to 1, the numerator goes to 1-1=0 such that d goes toward 0 -- almost no loss of WD when NA is large.
    If you plot d/x, you get a quadrant of an ellipse with a low NA asymptote of 1/n = 0.657 and a high NA asymptote of 0.

Lauren M. Peterson, PhD     Lead Physicist - Photonics   
Michigan R&D Center  (formerly ERIM - Veridian)   
General Dynamics - Advanced Information Systems
1200 Joe Hall Dr.,  P.O. Box 990       Ypsilanti, MI  48197   
734-480-5303              -480-5252 FAX       
[hidden email]     

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Richard E. Edelmann
Sent: Monday, October 01, 2007 3:34 PM
To: [hidden email]
Subject: Re: Working distance

Stéphane:
      To my understanding the cover slip is considered an optical element
of the lens itself - which is why coverslip thickness is so critical,
and the working distance is the distance "beyond" the  optical
elements of the lens.  So if your lens calls for a 0.17mm coverslip
and has a WD or 300um, you get 300um beyond the 170um coverslip. 
HOWEVER, if your coverslip is really 190um ( 0.160-0.190mm) you only
get 280um.  If you are using a correction collar then you can correct
the optics to include the thicker 190um coverslip, and thereby
recover your "lost" 20um.

On 30 Sep 2007 at 12:50, Stéphane Pagès wrote:

>
> Dear List,
> I wonder about the exact definition of working distance. As I
> understood, in the case were the objective is not corrected for
> coverslip, this distance is the between the front of the objective and
> the specimen to be imaged.
> In the case were I use a a coverslip corrected objective, what does it
> mean?. For example I use a 63X water objective from Olympus with a
> working distance of 300 um. The coverslip thickness is 150 um. What is
> the effective distance between the front of the objective and the
> specimen ?? 300 um ??? Here
> www.microscopyu.com/articles/ formulas/formulasworkingparfocal.htm
> I saw that the distance is between the objective and the upper side of
> the coverslip ??
> I don t know if I misunderstood something or not
>
> Thanks, Stephane
> --
> Stéphane Pages PhD
> Neurobiologie cellulaire
> Centre de Recherche Université Laval Robert-Giffard (CRULRG)
> Bureau/office: F-5570
> 2601, Chemin de la Canardière
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> Canada
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> Fax: [phone]
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Richard E. Edelmann, Ph.D.
Electron Microscopy Facility Director
364 Pearson Hall
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T

[Mark Cannell]

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Dear Lauren

My intuition tells me that that the working distance loss due to a
thicker coverslip cannot fall to zero with higher NA lenses.... (else I
would be able to use a coverslip thicker than the WD!!!).

Cheers Mark

Peterson, Lauren M. wrote:
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Richard - I believe that your 'HOWEVER' is a bit more complicated than you suggest and also not quite as bad as you say in that you do not loose as much working distance.  I did a simple ray tracing based upon Snell's Law for the rays going through the cover slip to find that for large NA the "lost" working distance in your example is not 20um but 20/n = 20/1.523 = 13.1um.  This is the case for NA < 0.3 and assumes that objective to coverslip is air.  For larger NA, WD lost is even less than 20/n and goes to zero for the largest NA = 1 in air.
>  
>

[Donnelly, Tom]

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Working Distance change with wrong coverslip is minor compared with the havoc spherical aberration wreaks on the image.

TD

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]On
Behalf Of Mark Cannell
Sent: Tuesday, October 02, 2007 1:31 PM
To: [hidden email]
Subject: Re: Working distance


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Dear Lauren

My intuition tells me that that the working distance loss due to a
thicker coverslip cannot fall to zero with higher NA lenses.... (else I
would be able to use a coverslip thicker than the WD!!!).

Cheers Mark

Peterson, Lauren M. wrote:
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Richard - I believe that your 'HOWEVER' is a bit more complicated than you suggest and also not quite as bad as you say in that you do not loose as much working distance.  I did a simple ray tracing based upon Snell's Law for the rays going through the cover slip to find that for large NA the "lost" working distance in your example is not 20um but 20/n = 20/1.523 = 13.1um.  This is the case for NA < 0.3 and assumes that objective to coverslip is air.  For larger NA, WD lost is even less than 20/n and goes to zero for the largest NA = 1 in air.
>  
>