Coverslip thickness and correction collar

[Teemu Ihalainen]

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

Dear list,

I have a small question considering coverslip thickness correction when
using water immersion objectives and I was hoping that you could help me
out.

We have bought a water immersion objective from Olympus (UPLSAPO 60x, 1.2
NA) with a correction collar. The objective works nicely in our LSM510,
when imaging living cells it gives good images, the PSF is better compared
to oil immersion objectives, etc. So, everything goes according to the
theory.

There is only one problem. The objecive is very "picky" about the
coverslip thickness (again, like the theory predicts...). We are using
glass bottom dishes from MatTek which have the thickness in range of
160-190um. Is there some simple way to adjust the correction collar when
imaging your sample?

The adjustment is easy to do when you are imaging for example ps-specks
but when you have your real sample, it is trickier. I have thought/found a
couple of ways to do it (below) but Im not sure which is the best and are
there other ways to do it.

I
Measure of the glass thickness before you culture your cells. Then at the
microscope you just adjust the collar accordingly.

II
At the microscope you first try to find a really small detail(s) in your
sample and then adjust the collar for best image quality (image z-stacks
or something like that).

III
Image some cell adhering beads(?) what you can just add to your sample.
Unfortunately I dont know if such beads even exists.

IV
Buy some other dishes with more accurate glass thickness. Is there any?

Any suggestions and tips are warmly welcome.



Best Regards,
Teemu Ihalainen
-------------------------------------------------
M.Sc., Nanoscience
University of Jyväskylä
Department of Biological and Environmental science
Molecular Biology, room B 212.2
Survontie 9 B2, 40500 Jyväskylä, Finland
Tel. [phone]
Mobile [phone]
-------------------------------------------------

[Michael Weber-4]

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

Hey Teemu,

I tried several ways of adjusting the correction collar, and for me the
results are as follows:

- The numbers written at the collar are not very precise. However,
measuring the thickness of coverslip and then put it to the according
number brings you at least in the range of the correct value.

- The way which brings me the best results is to perform a line Z-stack
over the coverslip, and go for the thinnest representation of it at half
maximum. Big disadvantage: this technique is quite a pain without fast
Z-stage.

- An alternative way is to simply go for the highest intensity while
imaging a sample in XY and using a line profile. To my understanding,
when it comes to refractive index missmatches, you lose light due to
refraction. If it matches better, you get more light back into the
objective, thus the best correction collar position should give you the
highest intensity.


I would appreciate if somebody could comment on that.

cheers,
Michael



Teemu Ihalainen wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear list,
>
> I have a small question considering coverslip thickness correction when
> using water immersion objectives and I was hoping that you could help me
> out.
>
> We have bought a water immersion objective from Olympus (UPLSAPO 60x, 1.2
> NA) with a correction collar. The objective works nicely in our LSM510,
> when imaging living cells it gives good images, the PSF is better compared
> to oil immersion objectives, etc. So, everything goes according to the
> theory.
>
> There is only one problem. The objecive is very "picky" about the
> coverslip thickness (again, like the theory predicts...). We are using
> glass bottom dishes from MatTek which have the thickness in range of
> 160-190um. Is there some simple way to adjust the correction collar when
> imaging your sample?
>
> The adjustment is easy to do when you are imaging for example ps-specks
> but when you have your real sample, it is trickier. I have thought/found a
> couple of ways to do it (below) but Im not sure which is the best and are
> there other ways to do it.
>
> I
> Measure of the glass thickness before you culture your cells. Then at the
> microscope you just adjust the collar accordingly.
>
> II
> At the microscope you first try to find a really small detail(s) in your
> sample and then adjust the collar for best image quality (image z-stacks
> or something like that).
>
> III
> Image some cell adhering beads(?) what you can just add to your sample.
> Unfortunately I dont know if such beads even exists.
>
> IV
> Buy some other dishes with more accurate glass thickness. Is there any?
>
> Any suggestions and tips are warmly welcome.
>
>
>
> Best Regards,
> Teemu Ihalainen
> -------------------------------------------------
> M.Sc., Nanoscience
> University of Jyväskylä
> Department of Biological and Environmental science
> Molecular Biology, room B 212.2
> Survontie 9 B2, 40500 Jyväskylä, Finland
> Tel. [phone]
> Mobile [phone]
> -------------------------------------------------

[Armstrong, Brian]

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

Well, I think you should start with method I, and then use method II for fine tuning. After all it is the image quality you are after regardless of what numbers on the collar are used.
It is my understanding that Zeiss uses the tube lens for correction whereas Olympus corrects entirely in the objective (see Handbook of Biological Confocal Microscopy, Pawley J). Which begs the question, why are you putting an Olympus lens on your LSM510? Try the Zeiss 63x/1.2W Corr and see if you have the same issues.
Cheers,
 

Brian D Armstrong PhD
Light Microscopy Core Manager
Beckman Research Institute
City of Hope
1450 E Duarte Rd
Duarte, CA 91010
626-359-8111 x62872
http://www.cityofhope.org/SharedResources/LightMicroscopy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Teemu Ihalainen
Sent: Thursday, December 06, 2007 5:26 AM
To: [hidden email]
Subject: Coverslip thickness and correction collar

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

Dear list,

I have a small question considering coverslip thickness correction when
using water immersion objectives and I was hoping that you could help me
out.

We have bought a water immersion objective from Olympus (UPLSAPO 60x, 1.2
NA) with a correction collar. The objective works nicely in our LSM510,
when imaging living cells it gives good images, the PSF is better compared
to oil immersion objectives, etc. So, everything goes according to the
theory.

There is only one problem. The objecive is very "picky" about the
coverslip thickness (again, like the theory predicts...). We are using
glass bottom dishes from MatTek which have the thickness in range of
160-190um. Is there some simple way to adjust the correction collar when
imaging your sample?

The adjustment is easy to do when you are imaging for example ps-specks
but when you have your real sample, it is trickier. I have thought/found a
couple of ways to do it (below) but Im not sure which is the best and are
there other ways to do it.

I
Measure of the glass thickness before you culture your cells. Then at the
microscope you just adjust the collar accordingly.

II
At the microscope you first try to find a really small detail(s) in your
sample and then adjust the collar for best image quality (image z-stacks
or something like that).

III
Image some cell adhering beads(?) what you can just add to your sample.
Unfortunately I dont know if such beads even exists.

IV
Buy some other dishes with more accurate glass thickness. Is there any?

Any suggestions and tips are warmly welcome.



Best Regards,
Teemu Ihalainen
-------------------------------------------------
M.Sc., Nanoscience
University of Jyväskylä
Department of Biological and Environmental science
Molecular Biology, room B 212.2
Survontie 9 B2, 40500 Jyväskylä, Finland
Tel. [phone]
Mobile [phone]
-------------------------------------------------


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[Guy Cox]

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

If it wasn't 'picky' it wouldn't be doing its job!
Correct adjustment is crucial and without it you'll
be doing worse than using a lower NA lens.  Forget
looking for more accurate cover slips - at this level
sufficiently accurate ones don't exist so all it would
do is give you a closer starting point.

Method 2 is the way to go but the key is not to look
for best image quality (even though this is what you'll
obtain, it's hard to spot).  What you are looking for
is IDENTICAL IMAGES EITHER SIDE OF FOCUS.  If your spot
goes to a circle one side of focus and a spot with a
halo around it on the other side, you have SA.  Adjust
the collar (widefield, visual observation) till the image
looks identical either side of focus.  It will actually
go to a bit of a ring on either side, and this is normal
(a look at a diagram of the psf will show you why).  It's
easiest to do this in fluorescence but if fading is a
problem it can also be done in brightfield.

                                              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)
Electron Microscope Unit, 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 Teemu Ihalainen
Sent: Friday, 7 December 2007 12:26 AM
To: [hidden email]
Subject: Coverslip thickness and correction collar

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

Dear list,

I have a small question considering coverslip thickness correction when using water immersion objectives and I was hoping that you could help me out.

We have bought a water immersion objective from Olympus (UPLSAPO 60x, 1.2
NA) with a correction collar. The objective works nicely in our LSM510, when imaging living cells it gives good images, the PSF is better compared to oil immersion objectives, etc. So, everything goes according to the theory.

There is only one problem. The objecive is very "picky" about the coverslip thickness (again, like the theory predicts...). We are using glass bottom dishes from MatTek which have the thickness in range of 160-190um. Is there some simple way to adjust the correction collar when imaging your sample?

The adjustment is easy to do when you are imaging for example ps-specks but when you have your real sample, it is trickier. I have thought/found a couple of ways to do it (below) but Im not sure which is the best and are there other ways to do it.

I
Measure of the glass thickness before you culture your cells. Then at the microscope you just adjust the collar accordingly.

II
At the microscope you first try to find a really small detail(s) in your sample and then adjust the collar for best image quality (image z-stacks or something like that).

III
Image some cell adhering beads(?) what you can just add to your sample.
Unfortunately I dont know if such beads even exists.

IV
Buy some other dishes with more accurate glass thickness. Is there any?

Any suggestions and tips are warmly welcome.



Best Regards,
Teemu Ihalainen
-------------------------------------------------
M.Sc., Nanoscience
University of Jyväskylä
Department of Biological and Environmental science Molecular Biology, room B 212.2 Survontie 9 B2, 40500 Jyväskylä, Finland Tel. [phone] Mobile [phone]
-------------------------------------------------

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[George McNamara]

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

option Ib. Measure of the glass thickness before you culture your cells. Then, for this lens, use dishes whose coverglasses are 170 um (or whatever thickness works best for the lens at its current "optimal" setting).

If one in five are the right thickness, than the cost of that dish, for this experiment, has gone from about $2 to $10. Should take less than a minute to measure a dish using a 20x, decent NA, dry lens. Compared to the cost of the confocal time (or of an LSM510), or of your time to do manual image analysis, or cost of the analysis computer and image analysis software, the extra cost is trivial. Plus, you can sort the "rejects" and use them with lenses each thickness is optimized for.



At 08:25 AM 12/6/2007, you wrote:

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

Dear list,

I have a small question considering coverslip thickness correction when
using water immersion objectives and I was hoping that you could help me
out.

We have bought a water immersion objective from Olympus (UPLSAPO 60x, 1.2
NA) with a correction collar. The objective works nicely in our LSM510,
when imaging living cells it gives good images, the PSF is better compared
to oil immersion objectives, etc. So, everything goes according to the
theory.

There is only one problem. The objecive is very "picky" about the
coverslip thickness (again, like the theory predicts...). We are using
glass bottom dishes from MatTek which have the thickness in range of
160-190um. Is there some simple way to adjust the correction collar when
imaging your sample?

The adjustment is easy to do when you are imaging for example ps-specks
but when you have your real sample, it is trickier. I have thought/found a
couple of ways to do it (below) but Im not sure which is the best and are
there other ways to do it.

I
Measure of the glass thickness before you culture your cells. Then at the
microscope you just adjust the collar accordingly.

II
At the microscope you first try to find a really small detail(s) in your
sample and then adjust the collar for best image quality (image z-stacks
or something like that).

III
Image some cell adhering beads(?) what you can just add to your sample.
Unfortunately I dont know if such beads even exists.

IV
Buy some other dishes with more accurate glass thickness. Is there any?

Any suggestions and tips are warmly welcome.



Best Regards,
Teemu Ihalainen
-------------------------------------------------
M.Sc., Nanoscience
University of Jyväskylä
Department of Biological and Environmental science
Molecular Biology, room B 212.2
Survontie 9 B2, 40500 Jyväskylä, Finland
Tel. [phone]
Mobile [phone]
-------------------------------------------------

 

George McNamara, Ph.D.
University of Miami, Miller School of Medicine
Image Core
Miami, FL 33010
[hidden email]
[hidden email]
305-243-8436 office
http://home.earthlink.net/~pubspectra/
http://home.earthlink.net/~geomcnamara/
http://www.sylvester.org/health_pro/shared_resources/index.asp (see Analytical Imaging Core Facility)

[Guy Cox]

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

But ... but .....

 

(a) this will be nowhere near accurate enough

(b) setting the ring manually once you know what to

look for takes only a minute or two

 

If you are paying more than $8 per minute for confocal

time you are being seriously overcharged!

 

                                                              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)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
______________________________________________
Phone [phone]     Fax [phone]
Mobile [phone]
______________________________________________
     http://www.guycox.net

 

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of George McNamara
Sent: Sunday, 9 December 2007 6:08 AM
To: [hidden email]
Subject: Re: Coverslip thickness and correction collar ... option Ib

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

option Ib. Measure of the glass thickness before you culture your cells. Then, for this lens, use dishes whose coverglasses are 170 um (or whatever thickness works best for the lens at its current "optimal" setting).

If one in five are the right thickness, than the cost of that dish, for this experiment, has gone from about $2 to $10. Should take less than a minute to measure a dish using a 20x, decent NA, dry lens. Compared to the cost of the confocal time (or of an LSM510), or of your time to do manual image analysis, or cost of the analysis computer and image analysis software, the extra cost is trivial. Plus, you can sort the "rejects" and use them with lenses each thickness is optimized for.



At 08:25 AM 12/6/2007, you wrote:

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

Dear list,

I have a small question considering coverslip thickness correction when
using water immersion objectives and I was hoping that you could help me
out.

We have bought a water immersion objective from Olympus (UPLSAPO 60x, 1.2
NA) with a correction collar. The objective works nicely in our LSM510,
when imaging living cells it gives good images, the PSF is better compared
to oil immersion objectives, etc. So, everything goes according to the
theory.

There is only one problem. The objecive is very "picky" about the
coverslip thickness (again, like the theory predicts...). We are using
glass bottom dishes from MatTek which have the thickness in range of
160-190um. Is there some simple way to adjust the correction collar when
imaging your sample?

The adjustment is easy to do when you are imaging for example ps-specks
but when you have your real sample, it is trickier. I have thought/found a
couple of ways to do it (below) but Im not sure which is the best and are
there other ways to do it.

I
Measure of the glass thickness before you culture your cells. Then at the
microscope you just adjust the collar accordingly.

II
At the microscope you first try to find a really small detail(s) in your
sample and then adjust the collar for best image quality (image z-stacks
or something like that).

III
Image some cell adhering beads(?) what you can just add to your sample.
Unfortunately I dont know if such beads even exists.

IV
Buy some other dishes with more accurate glass thickness. Is there any?

Any suggestions and tips are warmly welcome.



Best Regards,
Teemu Ihalainen
-------------------------------------------------
M.Sc., Nanoscience
University of Jyväskylä
Department of Biological and Environmental science
Molecular Biology, room B 212.2
Survontie 9 B2, 40500 Jyväskylä, Finland
Tel. [phone]
Mobile [phone]
-------------------------------------------------

 

George McNamara, Ph.D.
University of Miami, Miller School of Medicine
Image Core
Miami, FL 33010
[hidden email]
[hidden email]
305-243-8436 office
http://home.earthlink.net/~pubspectra/
http://home.earthlink.net/~geomcnamara/
http://www.sylvester.org/health_pro/shared_resources/index.asp (see Analytical Imaging Core Facility)

No virus found in this incoming message.
Checked by AVG Free Edition.
Version: 7.5.503 / Virus Database: 269.16.17/1177 - Release Date: 7/12/2007 1:11 PM

No virus found in this outgoing message.
Checked by AVG Free Edition.
Version: 7.5.503 / Virus Database: 269.16.17/1178 - Release Date: 8/12/2007 11:59 AM

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

INTERNATIONAL SCHOOL OF BIOPHYSICS «ANTONIO BORSELLINO» 36th Course: MULTIDIMENSIONAL OPTICAL FLUORESCENCE MICROSCOPY TOWARDS NANOSCOPY
ERICE–SICILY: 19 – 29 APRIL 2008
Sponsored by the: • Italian Ministry of Education, University and Scientific Research • Sicilian Regional Government, Companies.

LECTURERS
Fluorescence Spectroscopy, GFP Photophysics
• R. BIZZARRI, NEST-INFM, SNS, Pisa, IT;  Optics, Confocal Microscopy, THG • F. BRAKENHOFF, University of Amsterdam, NL;  FRAP, Single particle tracking • K. BRAECKMANS, Ghent University, BE; Single molecule force spectroscopy • J. BRUJIC, New York University, USA;  Fluctuation Microscopies for biological tissues GIUSEPPE CHIRICO, University of Milan-Bicocca, IT; Micro-particle manipulation • D. COJOC, TASC, INFM, Trieste, IT;  SHG, CARS, 2PE • C. COMBS, NIH, Bethesda, USA;  2PE, 3D imaging • A. DIASPRO, University of Genoa, IT;  Micro/Nano Optical Manipulation • E. Di FABRIZIO, Unversity of Catanzaro Magna Graecia, IT;  Raster Image Correlation Spectroscopy, Photon Counting • M. DIGMAN, UC Irvine, USA; High-content screening • M. FARETTA, IFOM-IEO, Milan, IT;  Correlative Microscopy • U. FASCIO, University of Milan, IT;  Fluorescence Lifetime, FRET • H.C. GERRITSEN, Utrecht University, NL; FCS, Global Data Analysis • E. GRATTON, UC Irvine, USA;  Photonic crystals, nanophoton
ics • M. GU, Swinburne Univ. of Technology, Victoria, AU;  Time lapse imaging • S. GUIDO, Universit of Naples, IT;  Fluorescence Optical Nanoscopy • S. HELL, MPI, Goettingen, DE;  Scanning Microscopy, Optical aberrations • M. MARTINEZ CORRAL, Univ. of Valencia, ES;  Optical systems, Scanning Microscopy • F. QUERCIOLI, CNR-ISC, Florence, IT; 2PE, Fast scanning methods • P. SAGGAU, Baylor College of Med. Hou
ston, Texas, USA;  Correlative Microscopy at cryo-Temperatures • A. SARTORI, Institut Pasteur, Paris, FR;  Light Scattering, FCS applications • P.L. SAN BIAGIO, CNR-IBF, Palermo, IT;  Molecular landscapes by means of AFM • G. SCOLES, Princeton University, USA;  Linear and Non linear Optical Microscopy • C. SHEPPARD, Ntl Univ. of Singapore, Singapore;  Optical Microscopy, 3D imaging, Photonic Forces • E. STELZER, EMBL, Heidelberg, DE;  Laser scissors and tweezers in cell biology • I. TOLIC-NORRELYKKE, MPI, Dresden, DE; Fluorescence imaging in Neuroscience • V. TORRE, SISSA, Trieste, IT; Quantitative colocalization • C. USAI, CNR-IBF, Genoa, IT; Confocal Microscopy, Structured light methods • T. WILSON, University of Oxford, UK;  Photoswitch-activatable fluorescent proteins, Lifetime • F. WOUTERS, Univ. of Goettingen, DE.
--------
----------------------------------------------------
"Follow knowledge wherever it leads us." (reading Galileo Galilei)
-----------------------------------------------------
Alberto Diaspro, EBSA President-Elect, MicroScoBIO LAMBS-IFOM, Department of Physics, University of Genoa, Via Dodecaneso 33, 16146 Genoa, Italy - fax [phone] - tel [phone]; URLs: www.lambs.it;
EBSA is Biophysics in Europe - European Biophysical Societies' Association www.ebsa.org
----------------------------------------------

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Search the CONFOCAL archive at
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----- Original Message -----
From: "[hidden email]" <[hidden email]>
To: <[hidden email]>
Sent: Sunday, December 09, 2007 1:38 PM
Subject: Alby - after FOM 2008 in Japan --- Erice 2008, Sicily

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> INTERNATIONAL SCHOOL OF BIOPHYSICS «ANTONIO BORSELLINO» 36th Course:
> MULTIDIMENSIONAL OPTICAL FLUORESCENCE MICROSCOPY TOWARDS NANOSCOPY
> ERICE-SICILY: 19 - 29 APRIL 2008
> Sponsored by the: . Italian Ministry of Education, University and
> Scientific Research . Sicilian Regional Government, Companies.
>
> LECTURERS
> Fluorescence Spectroscopy, GFP Photophysics
> . R. BIZZARRI, NEST-INFM, SNS, Pisa, IT;  Optics, Confocal Microscopy, THG
> . F. BRAKENHOFF, University of Amsterdam, NL;  FRAP, Single particle
> tracking . K. BRAECKMANS, Ghent University, BE; Single molecule force
> spectroscopy . J. BRUJIC, New York University, USA;  Fluctuation
> Microscopies for biological tissues GIUSEPPE CHIRICO, University of
> Milan-Bicocca, IT; Micro-particle manipulation . D. COJOC, TASC, INFM,
> Trieste, IT;  SHG, CARS, 2PE . C. COMBS, NIH, Bethesda, USA;  2PE, 3D
> imaging . A. DIASPRO, University of Genoa, IT;  Micro/Nano Optical
> Manipulation . E. Di FABRIZIO, Unversity of Catanzaro Magna Graecia, IT;
> Raster Image Correlation Spectroscopy, Photon Counting . M. DIGMAN, UC
> Irvine, USA; High-content screening . M. FARETTA, IFOM-IEO, Milan, IT;
> Correlative Microscopy . U. FASCIO, University of Milan, IT;  Fluorescence
> Lifetime, FRET . H.C. GERRITSEN, Utrecht University, NL; FCS, Global Data
> Analysis . E. GRATTON, UC Irvine, USA;  Photonic crystals, nanophoton
>
> ics . M. GU, Swinburne Univ. of Technology, Victoria, AU;  Time lapse
> imaging . S. GUIDO, Universit of Naples, IT;  Fluorescence Optical
> Nanoscopy . S. HELL, MPI, Goettingen, DE;  Scanning Microscopy, Optical
> aberrations . M. MARTINEZ CORRAL, Univ. of Valencia, ES;  Optical systems,
> Scanning Microscopy . F. QUERCIOLI, CNR-ISC, Florence, IT; 2PE, Fast
> scanning methods . P. SAGGAU, Baylor College of Med. Hou
> ston, Texas, USA;  Correlative Microscopy at cryo-Temperatures . A.
> SARTORI, Institut Pasteur, Paris, FR;  Light Scattering, FCS applications
> . P.L. SAN BIAGIO, CNR-IBF, Palermo, IT;  Molecular landscapes by means of
> AFM . G. SCOLES, Princeton University, USA;  Linear and Non linear Optical
> Microscopy . C. SHEPPARD, Ntl Univ. of Singapore, Singapore;  Optical
> Microscopy, 3D imaging, Photonic Forces . E. STELZER, EMBL, Heidelberg,
> DE;  Laser scissors and tweezers in cell biology . I. TOLIC-NORRELYKKE,
> MPI, Dresden, DE; Fluorescence imaging in Neuroscience . V. TORRE, SISSA,
> Trieste, IT; Quantitative colocalization . C. USAI, CNR-IBF, Genoa, IT;
> Confocal Microscopy, Structured light methods . T. WILSON, University of
> Oxford, UK;  Photoswitch-activatable fluorescent proteins, Lifetime . F.
> WOUTERS, Univ. of Goettingen, DE.
> --------
> ----------------------------------------------------
> "Follow knowledge wherever it leads us." (reading Galileo Galilei)
> -----------------------------------------------------
> Alberto Diaspro, EBSA President-Elect, MicroScoBIO LAMBS-IFOM, Department
> of Physics, University of Genoa, Via Dodecaneso 33, 16146 Genoa, Italy -
> fax [phone] - tel [phone]; URLs: www.lambs.it;
> EBSA is Biophysics in Europe - European Biophysical Societies' Association
> www.ebsa.org
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[S. Pagakis (IIBEAA)]

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

>
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of George McNamara
> Sent: Sunday, 9 December 2007 6:08 AM
> To: [hidden email]
> Subject: Re: Coverslip thickness and correction collar ... option Ib
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> option Ib. Measure of the glass thickness before you culture your
> cells. Then, for this lens, use dishes whose coverglasses are 170 um
> (or whatever thickness works best for the lens at its current
> "optimal" setting).
>
> If one in five are the right thickness, than the cost of that dish,
> for this experiment, has gone from about $2 to $10. Should take less
> than a minute to measure a dish using a 20x, decent NA, dry lens.

Dear George

On a recent test I did, I discovered something that I had not expected.
A dry objective would significantly underestimate the thickness of a
glass coverslip.

For a #1 coverslip, a 20x dry objective would measure it as 98microns
thick, where as a 63oil objective as 138microns thick.
For a #1.5 coverslip the values were 113microns and 160microns
respectively.

As I said, I wasn't expecting it because the 20 lens was coverslip
corrected.

So you have to be very careful when using dry objectives to measure
anything in Z.

regards

*********************************
Stamatis Pagakis Ph.D.
Biological Imaging Unit
Biomedical Research Foundation, Academy of Athens
[hidden email]




> Compared to the cost of the confocal time (or of an LSM510), or of
> your time to do manual image analysis, or cost of the analysis
> computer and image analysis software, the extra cost is trivial. Plus,
> you can sort the "rejects" and use them with lenses each thickness is
> optimized for.

> George McNamara, Ph.D.
> University of Miami, Miller School of Medicine
> Image Core
> Miami, FL 33010
> [hidden email]
> [hidden email]
> 305-243-8436 office
> http://home.earthlink.net/~pubspectra/
> http://home.earthlink.net/~geomcnamara/
> http://www.sylvester.org/health_pro/shared_resources/index.asp (see
> Analytical Imaging Core Facility)
>
>
>
>
> No virus found in this incoming message.
> Checked by AVG Free Edition.
> Version: 7.5.503 / Virus Database: 269.16.17/1177 - Release Date:
> 7/12/2007 1:11 PM
>
>
> No virus found in this outgoing message.
>  Checked by AVG Free Edition.
>  Version: 7.5.503 / Virus Database: 269.16.17/1178 - Release Date:
> 8/12/2007 11:59 AM

[Zoltan]

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

The apparent thickness of a layer with a real depth of  'x' is 'x/n', where n is the RI (viewing from vacuum; otherwise n1/n2).  I guess the coverslip correction doesn't correct for that; that's why your were surprised, I guess.

 

Zoltan 

On Dec 10, 2007 1:52 PM, S. Pagakis (IIBEAA) <[hidden email]> wrote:

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

>

> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of George McNamara
> Sent: Sunday, 9 December 2007 6:08 AM
> To: [hidden email]
> Subject: Re: Coverslip thickness and correction collar ... option Ib
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> option Ib. Measure of the glass thickness before you culture your
> cells. Then, for this lens, use dishes whose coverglasses are 170 um
> (or whatever thickness works best for the lens at its current
> "optimal" setting).
>
> If one in five are the right thickness, than the cost of that dish,
> for this experiment, has gone from about $2 to $10. Should take less
> than a minute to measure a dish using a 20x, decent NA, dry lens.

Dear George

On a recent test I did, I discovered something that I had not expected.
A dry objective would significantly underestimate the thickness of a
glass coverslip.

For a #1 coverslip, a 20x dry objective would measure it as 98microns
thick, where as a 63oil objective as 138microns thick.
For a #1.5 coverslip the values were 113microns and 160microns
respectively.

As I said, I wasn't expecting it because the 20 lens was coverslip
corrected.

So you have to be very careful when using dry objectives to measure
anything in Z.

regards

*********************************
Stamatis Pagakis Ph.D.
Biological Imaging Unit
Biomedical Research Foundation, Academy of Athens
[hidden email]

> Compared to the cost of the confocal time (or of an LSM510), or of
> your time to do manual image analysis, or cost of the analysis
> computer and image analysis software, the extra cost is trivial. Plus,
> you can sort the "rejects" and use them with lenses each thickness is
> optimized for.

> George McNamara, Ph.D.

> University of Miami, Miller School of Medicine
> Image Core
> Miami, FL 33010
> [hidden email]
> [hidden email]
> 305-243-8436 office
> http://home.earthlink.net/~pubspectra/
> http://home.earthlink.net/~geomcnamara/
> http://www.sylvester.org/health_pro/shared_resources/index.asp (see
> Analytical Imaging Core Facility)
>
>
>
>
> No virus found in this incoming message.
> Checked by AVG Free Edition.
> Version: 7.5.503 / Virus Database: 269.16.17 /1177 - Release Date:
> 7/12/2007 1:11 PM
>
>
> No virus found in this outgoing message.
>  Checked by AVG Free Edition.
>  Version: 7.5.503 / Virus Database: 269.16.17/1178 - Release Date:
> 8/12/2007 11:59 AM

--
--
Zoltan Cseresnyes
Facility manager, Imaging Suite
Dept. of Zoology University of Cambridge
Downing Street, Cambridge
CB2 3EJ    UK

Tel.: (++44) (0)1223 769282
Fax.: (++44) (0)1223 336676

[James Pawley]

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

>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>>
>>From: Confocal Microscopy List
>>[mailto:[hidden email]] On Behalf Of George McNamara
>>Sent: Sunday, 9 December 2007 6:08 AM
>>To: [hidden email]
>>Subject: Re: Coverslip thickness and correction collar ... option Ib
>>
>>Search the CONFOCAL archive at
>>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>>option Ib. Measure of the glass thickness before you culture your
>>cells. Then, for this lens, use dishes whose coverglasses are 170
>>um (or whatever thickness works best for the lens at its current
>>"optimal" setting).
>>
>>If one in five are the right thickness, than the cost of that dish,
>>for this experiment, has gone from about $2 to $10. Should take
>>less than a minute to measure a dish using a 20x, decent NA, dry
>>lens.
>
>
>Dear George
>
>On a recent test I did, I discovered something that I had not
>expected. A dry objective would significantly underestimate the
>thickness of a glass coverslip.
>
>For a #1 coverslip, a 20x dry objective would measure it as
>98microns thick, where as a 63oil objective as 138microns thick.
>For a #1.5 coverslip the values were 113microns and 160microns respectively.
>
>As I said, I wasn't expecting it because the 20 lens was coverslip corrected.
>
>So you have to be very careful when using dry objectives to measure
>anything in Z.
>
>regards
>
>*********************************
>Stamatis Pagakis Ph.D.
>Biological Imaging Unit
>Biomedical Research Foundation, Academy of Athens
>[hidden email]

Hi all.

The dry, coverslip lens is ONLY corrected for imaging immediately
next to the far side of a coverslip of the specified thickness. The
image of the near side will be poorly corrected, and as you noted, in
the wrong place. How much "the wrong place" depends on how well you
fill the BFP because this determines the effective NA on the
illumination  side..

Dry lenses are even more fussy that oil lenses of the same NA about
the thickness of the coverslip.

Dry lenses are even more fussy that oil lenses of the same NA about
the thickness of the coverslip.

Dry lenses are even more fussy that oil lenses of the same NA about
the thickness of the coverslip.

This sad news brought to you with best wishes for the Holiday
Season(translation: the time when we get to correct all the papers)

Cheers,

Jim P.

>
>
>
>>Compared to the cost of the confocal time (or of an LSM510), or of
>>your time to do manual image analysis, or cost of the analysis
>>computer and image analysis software, the extra cost is trivial.
>>Plus, you can sort the "rejects" and use them with lenses each
>>thickness is optimized for.
>
>>George McNamara, Ph.D.
>>University of Miami, Miller School of Medicine
>>Image Core
>>Miami, FL 33010
>>[hidden email]
>>[hidden email]
>>305-243-8436 office
>>http://home.earthlink.net/~pubspectra/
>>http://home.earthlink.net/~geomcnamara/
>>http://www.sylvester.org/health_pro/shared_resources/index.asp (see
>>Analytical Imaging Core Facility)
>>
>>
>>
>>
>>No virus found in this incoming message.
>>Checked by AVG Free Edition.
>>Version: 7.5.503 / Virus Database: 269.16.17/1177 - Release Date:
>>7/12/2007 1:11 PM
>>
>>
>>No virus found in this outgoing message.
>>  Checked by AVG Free Edition.
>>  Version: 7.5.503 / Virus Database: 269.16.17/1178 - Release Date:
>>8/12/2007 11:59 AM

--
               ****************************************
Prof. James B. Pawley,                 Ph.  608-263-3147
Room 223, Zoology Research Building,                         FAX  [phone]
250 N. Mills St., Madison, WI, 53706  [hidden email]
"A scientist is not one who can answer questions but one who can
question answers."  Theodore Schick Jr., Skeptical Enquirer, 21-2:39

[Craig Brideau]

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

Spherical aberration caused by the index mismatch (air to glass) will
alter the apparent depth when making these sorts of measurements.
Unless you know the exact index of refraction of the glass you cannot
correct for this error when measuring thickness using a dry lens.
Even a wet or oil lens can have some error if the index of the glass
is not matched, but for dry lenses it is the worst as the index
difference is highest.

Craig

> Dear George
>
> On a recent test I did, I discovered something that I had not expected.
> A dry objective would significantly underestimate the thickness of a
> glass coverslip.
>
> For a #1 coverslip, a 20x dry objective would measure it as 98microns
> thick, where as a 63oil objective as 138microns thick.
> For a #1.5 coverslip the values were 113microns and 160microns
> respectively.
>
> As I said, I wasn't expecting it because the 20 lens was coverslip
> corrected.
>
> So you have to be very careful when using dry objectives to measure
> anything in Z.
>
> regards
>
> *********************************
> Stamatis Pagakis Ph.D.
> Biological Imaging Unit
> Biomedical Research Foundation, Academy of Athens
> [hidden email]
>
>
>
>
> > Compared to the cost of the confocal time (or of an LSM510), or of
> > your time to do manual image analysis, or cost of the analysis
> > computer and image analysis software, the extra cost is trivial. Plus,
> > you can sort the "rejects" and use them with lenses each thickness is
> > optimized for.
>
> > George McNamara, Ph.D.
> > University of Miami, Miller School of Medicine
> > Image Core
> > Miami, FL 33010
> > [hidden email]
> > [hidden email]
> > 305-243-8436 office
> > http://home.earthlink.net/~pubspectra/
> > http://home.earthlink.net/~geomcnamara/
> > http://www.sylvester.org/health_pro/shared_resources/index.asp (see
> > Analytical Imaging Core Facility)
> >
> >
> >
> >
> > No virus found in this incoming message.
> > Checked by AVG Free Edition.
> > Version: 7.5.503 / Virus Database: 269.16.17/1177 - Release Date:
> > 7/12/2007 1:11 PM
> >
> >
> > No virus found in this outgoing message.
> >  Checked by AVG Free Edition.
> >  Version: 7.5.503 / Virus Database: 269.16.17/1178 - Release Date:
> > 8/12/2007 11:59 AM
>

[George McNamara]

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

The only way to know which - if either - was the correct answer is to
stand the coverglass on edge, and measure that. Did you do this?

I did not mention this with respect to the dishes, because that
measurement would require breaking or unglueing the glass.

Jim P - thanks, thanks, and thanks again, for your point about where
the lenses are corrected for.



At 08:52 AM 12/10/2007, you wrote:

>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>>
>>From: Confocal Microscopy List
>>[mailto:[hidden email]] On Behalf Of George McNamara
>>Sent: Sunday, 9 December 2007 6:08 AM
>>To: [hidden email]
>>Subject: Re: Coverslip thickness and correction collar ... option Ib
>>
>>Search the CONFOCAL archive at
>>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>>option Ib. Measure of the glass thickness before you culture your
>>cells. Then, for this lens, use dishes whose coverglasses are 170
>>um (or whatever thickness works best for the lens at its current
>>"optimal" setting).
>>
>>If one in five are the right thickness, than the cost of that dish,
>>for this experiment, has gone from about $2 to $10. Should take
>>less than a minute to measure a dish using a 20x, decent NA, dry lens.
>
>
>Dear George
>
>On a recent test I did, I discovered something that I had not
>expected. A dry objective would significantly underestimate the
>thickness of a glass coverslip.
>
>For a #1 coverslip, a 20x dry objective would measure it as
>98microns thick, where as a 63oil objective as 138microns thick.
>For a #1.5 coverslip the values were 113microns and 160microns respectively.
>
>As I said, I wasn't expecting it because the 20 lens was coverslip corrected.
>
>So you have to be very careful when using dry objectives to measure
>anything in Z.
>
>regards
>
>*********************************
>Stamatis Pagakis Ph.D.
>Biological Imaging Unit
>Biomedical Research Foundation, Academy of Athens
>[hidden email]
>
>
>
>
>>Compared to the cost of the confocal time (or of an LSM510), or of
>>your time to do manual image analysis, or cost of the analysis
>>computer and image analysis software, the extra cost is trivial.
>>Plus, you can sort the "rejects" and use them with lenses each
>>thickness is optimized for.
>
>>George McNamara, Ph.D.
>>University of Miami, Miller School of Medicine
>>Image Core
>>Miami, FL 33010
>>[hidden email]
>>[hidden email]
>>305-243-8436 office
>>http://home.earthlink.net/~pubspectra/
>>http://home.earthlink.net/~geomcnamara/
>>http://www.sylvester.org/health_pro/shared_resources/index.asp (see
>>Analytical Imaging Core Facility)
>>
>>
>>
>>
>>No virus found in this incoming message.
>>Checked by AVG Free Edition.
>>Version: 7.5.503 / Virus Database: 269.16.17/1177 - Release Date:
>>7/12/2007 1:11 PM
>>
>>
>>No virus found in this outgoing message.
>>  Checked by AVG Free Edition.
>>  Version: 7.5.503 / Virus Database: 269.16.17/1178 - Release Date:
>> 8/12/2007 11:59 AM
>
>
>
>
>
>
>George McNamara, Ph.D.
>University of Miami, Miller School of Medicine
>Image Core
>Miami, FL 33010
>[hidden email]
>[hidden email]
>305-243-8436 office
>http://home.earthlink.net/~pubspectra/
>http://home.earthlink.net/~geomcnamara/
>http://www.sylvester.org/health_pro/shared_resources/index.asp (see
>Analytical Imaging Core Facility)
>
>

[Jan Peychl]

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

Dear all,

as  posted here  already some time ago by Jens Rietdorf,
there is a company which sells coverslips of selected thickness.
I hope that this might be of  help ( no commercial interest from my side).:

http://www.hecht-assistent.de/e_microscopic/cover_glasses.html
search for :
Assistent" Cover glasses, selected thickness, CE
1014 thickness 0,17 ± 0,01 mm
1015 thickness 0,17 ± 0,02 mm

Hope that this might help.

Jan


George McNamara wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> The only way to know which - if either - was the correct answer is to
> stand the coverglass on edge, and measure that. Did you do this?
>
> I did not mention this with respect to the dishes, because that
> measurement would require breaking or unglueing the glass.
>
> Jim P - thanks, thanks, and thanks again, for your point about where
> the lenses are corrected for.
>
>
>
> At 08:52 AM 12/10/2007, you wrote:
>
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>>>
>>> From: Confocal Microscopy List
>>> [mailto:[hidden email]] On Behalf Of George McNamara
>>> Sent: Sunday, 9 December 2007 6:08 AM
>>> To: [hidden email]
>>> Subject: Re: Coverslip thickness and correction collar ... option Ib
>>>
>>> Search the CONFOCAL archive at
>>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>>
>>> option Ib. Measure of the glass thickness before you culture your
>>> cells. Then, for this lens, use dishes whose coverglasses are 170 um
>>> (or whatever thickness works best for the lens at its current
>>> "optimal" setting).
>>>
>>> If one in five are the right thickness, than the cost of that dish,
>>> for this experiment, has gone from about $2 to $10. Should take less
>>> than a minute to measure a dish using a 20x, decent NA, dry lens.
>>
>>
>>
>> Dear George
>>
>> On a recent test I did, I discovered something that I had not
>> expected. A dry objective would significantly underestimate the
>> thickness of a glass coverslip.
>>
>> For a #1 coverslip, a 20x dry objective would measure it as 98microns
>> thick, where as a 63oil objective as 138microns thick.
>> For a #1.5 coverslip the values were 113microns and 160microns
>> respectively.
>>
>> As I said, I wasn't expecting it because the 20 lens was coverslip
>> corrected.
>>
>> So you have to be very careful when using dry objectives to measure
>> anything in Z.
>>
>> regards
>>
>> *********************************
>> Stamatis Pagakis Ph.D.
>> Biological Imaging Unit
>> Biomedical Research Foundation, Academy of Athens
>> [hidden email]
>>
>>
>>
>>
>>> Compared to the cost of the confocal time (or of an LSM510), or of
>>> your time to do manual image analysis, or cost of the analysis
>>> computer and image analysis software, the extra cost is trivial.
>>> Plus, you can sort the "rejects" and use them with lenses each
>>> thickness is optimized for.
>>
>>
>>> George McNamara, Ph.D.
>>> University of Miami, Miller School of Medicine
>>> Image Core
>>> Miami, FL 33010
>>> [hidden email]
>>> [hidden email]
>>> 305-243-8436 office
>>> http://home.earthlink.net/~pubspectra/
>>> http://home.earthlink.net/~geomcnamara/
>>> http://www.sylvester.org/health_pro/shared_resources/index.asp (see
>>> Analytical Imaging Core Facility)
>>>
>>>
>>>
>>>
>>> No virus found in this incoming message.
>>> Checked by AVG Free Edition.
>>> Version: 7.5.503 / Virus Database: 269.16.17/1177 - Release Date:
>>> 7/12/2007 1:11 PM
>>>
>>>
>>> No virus found in this outgoing message.
>>>  Checked by AVG Free Edition.
>>>  Version: 7.5.503 / Virus Database: 269.16.17/1178 - Release Date:
>>> 8/12/2007 11:59 AM
>>
>>
>>
>>
>>
>>
>>
>> George McNamara, Ph.D.
>> University of Miami, Miller School of Medicine
>> Image Core
>> Miami, FL 33010
>> [hidden email]
>> [hidden email]
>> 305-243-8436 office
>> http://home.earthlink.net/~pubspectra/
>> http://home.earthlink.net/~geomcnamara/
>> http://www.sylvester.org/health_pro/shared_resources/index.asp (see
>> Analytical Imaging Core Facility)
>>
>>