Re: reflected light profilometry trrough plastic - mystery bands

[mcammer]

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A few weeks ago there was a discussion here about measuring depth by reflectance.

We had a similar issue.  Yes there are "ghost" reflections as we moved away from the glass surfaces, but we expected these because of the 3D nature of diffraction patterns.

The bigger problem was that the answers we were getting for depth measurements were unexpectedly small.

A user came in to image a home built chamber with a glass face.  The results we were getting for depth seemed too small.  Therefore, we made a chamber with cut glass slides to check this.  A description with pictures etc is at http://microscopynotes.com/710/10X_cal_issue/index.html , but here is the short version:

When imaged through the glass slide to the coverslip we got the same results as imaging through the coverslip to the slide.

When imaging through air or dilute water based dye, we got the same results.

My conclusion is that there is a calibration constant off somewhere in the confocal system; it's probably just a software problem.

Next call:  Zeiss service.

Regards,
Michael

=========================================================================
Michael Cammer, Microscopy Core & Skirball Institute, NYU Langone Medical Center
                      Cell:  914-309-3270     ** Office: Skirball 2nd Floor main office, back right **
          http://ocs.med.nyu.edu/microscopy & http://microscopynotes.com/


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[Moulding, Dale]

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Hi Michael,
Is it just needing correction for refractive index? 660um imaged through the glass will be 660 x 1.51 to give the expected 1mm (air to glass RI mismatch) and a 1.31 air to water correction in the other set up?
Cheers
Dale




On 5 Apr 2016, at 21:15, Cammer, Michael <[hidden email]<mailto:[hidden email]>> wrote:

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A few weeks ago there was a discussion here about measuring depth by reflectance.

We had a similar issue.  Yes there are "ghost" reflections as we moved away from the glass surfaces, but we expected these because of the 3D nature of diffraction patterns.

The bigger problem was that the answers we were getting for depth measurements were unexpectedly small.

A user came in to image a home built chamber with a glass face.  The results we were getting for depth seemed too small.  Therefore, we made a chamber with cut glass slides to check this.  A description with pictures etc is at http://microscopynotes.com/710/10X_cal_issue/index.html , but here is the short version:

When imaged through the glass slide to the coverslip we got the same results as imaging through the coverslip to the slide.

When imaging through air or dilute water based dye, we got the same results.

My conclusion is that there is a calibration constant off somewhere in the confocal system; it's probably just a software problem.

Next call:  Zeiss service.

Regards,
Michael

=========================================================================
Michael Cammer, Microscopy Core & Skirball Institute, NYU Langone Medical Center
                     Cell:  914-309-3270     ** Office: Skirball 2nd Floor main office, back right **
         http://ocs.med.nyu.edu/microscopy & http://microscopynotes.com/


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[mcammer]

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Dale Moulding pointed out that I skipped a step of essential math.  Multiplying the glass distances by 1.5 and the water distances by 1.33 corrects the problem.
Thank you Dale and sorry to waste time of anyone else who read my initial post.
Regards,
Michael
=========================================================================
Michael Cammer, Microscopy Core & Skirball Institute, NYU Langone Medical Center
                      Cell:  914-309-3270     ** Office: Skirball 2nd Floor main office, back right **
          http://ocs.med.nyu.edu/microscopy & http://microscopynotes.com/

From: Cammer, Michael
Sent: Tuesday, April 05, 2016 3:33 PM
To: Confocal Microscopy List ([hidden email])
Subject: Re: reflected light profilometry trrough plastic - mystery bands

A few weeks ago there was a discussion here about measuring depth by reflectance.

We had a similar issue.  Yes there are "ghost" reflections as we moved away from the glass surfaces, but we expected these because of the 3D nature of diffraction patterns.

The bigger problem was that the answers we were getting for depth measurements were unexpectedly small.

A user came in to image a home built chamber with a glass face.  The results we were getting for depth seemed too small.  Therefore, we made a chamber with cut glass slides to check this.  A description with pictures etc is at http://microscopynotes.com/710/10X_cal_issue/index.html , but here is the short version:

When imaged through the glass slide to the coverslip we got the same results as imaging through the coverslip to the slide.

When imaging through air or dilute water based dye, we got the same results.

My conclusion is that there is a calibration constant off somewhere in the confocal system; it's probably just a software problem.

Next call:  Zeiss service.

Regards,
Michael

=========================================================================
Michael Cammer, Microscopy Core & Skirball Institute, NYU Langone Medical Center
                      Cell:  914-309-3270     ** Office: Skirball 2nd Floor main office, back right **
          http://ocs.med.nyu.edu/microscopy & http://microscopynotes.com/


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[Arvydas Matiukas]

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Michael,
 
 In provided pictures the thickness is measured correctly when  measured in xy plane
but is "compressed" when measured in z-direction. I see that your z-measurements could be
corrected close to true value by multiplying by refractive index of the medium in which
the (reflected) beam propagates: i.e. ~1.5 for glass and 1.33 for water.
 
I noticed this "z-miscalibration" when imaging large depths (2-5 mm) in cleared tissue
using 10x dry objective on LSM 510. Confocal software (LSM and Zen) in  within z-stack
has a setting for refractive index correction. The default value is 1.00 , and setting it to
the medium specific value restores correct  z and thickness values inside the imaged sample.
 
I  saw a paper that explains this z- compression phenomenon but do not have the reference at hand.
 
Best,
Arvydas
 
 

 
Arvydas Matiukas, Ph.D.
Director of Confocal&Two-Photon Core
SUNY Upstate Medical University
Email: [hidden email]
>>> "Cammer, Michael" <[hidden email]> 4/5/2016 3:33 PM >>>
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A few weeks ago there was a discussion here about measuring depth by reflectance.

We had a similar issue.  Yes there are "ghost" reflections as we moved away from the glass surfaces, but we expected these because of the 3D nature of diffraction patterns.

The bigger problem was that the answers we were getting for depth measurements were unexpectedly small.

A user came in to image a home built chamber with a glass face.  The results we were getting for depth seemed too small.  Therefore, we made a chamber with cut glass slides to check this.  A description with pictures etc is at http://microscopynotes.com/710/10X_cal_issue/index.html , but here is the short version:

When imaged through the glass slide to the coverslip we got the same results as imaging through the coverslip to the slide.

When imaging through air or dilute water based dye, we got the same results.

My conclusion is that there is a calibration constant off somewhere in the confocal system; it's probably just a software problem.

Next call:  Zeiss service.

Regards,
Michael

=========================================================================
Michael Cammer, Microscopy Core & Skirball Institute, NYU Langone Medical Center
                      Cell:  914-309-3270     ** Office: Skirball 2nd Floor main office, back right **
          http://ocs.med.nyu.edu/microscopy & http://microscopynotes.com/


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[Stanislav Vitha-2]

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Yes, the correction factor n2/n1 is sufficient for most cases.
(n2 = RI of the sample; n1 = RI of the objective's immersion medium)
There is some residual error in this calculation. If you wanted to be really
precise, look at this paper: Kuypers, L.C., W.F. Decraemer, J.J. Dirckx, and
J.P. Timmermans, A procedure to determine the correct thickness of an
object with confocal microscopy in case of refractive index mismatch. J
Microsc, 2005. 218(Pt 1): p. 68-78.

I have not used Zeiss confocals much in last 20 years, but remember that
when I worked with Zeiss LSM 10 (yes, before LSM 310 or 210) it actually
had this correction implemented when you did Z-scans and depth profiling.  

Stan Vitha
Microscopy and Imaging Center
Texas A&M University


On Tue, 5 Apr 2016 20:44:51 +0000, Moulding, Dale
<[hidden email]> wrote:

>Hi Michael,
>Is it just needing correction for refractive index? 660um imaged through
the glass will be 660 x 1.51 to give the expected 1mm (air to glass RI
mismatch) and a 1.31 air to water correction in the other set up?
>Cheers
>Dale
>
>
>On 5 Apr 2016, at 21:15, Cammer, Michael
<[hidden email]<mailto:[hidden email]>
> wrote:

>
>A few weeks ago there was a discussion here about measuring depth by
reflectance.
>
>We had a similar issue.  Yes there are "ghost" reflections as we moved
away from the glass surfaces, but we expected these because of the 3D
nature of diffraction patterns.
>
>The bigger problem was that the answers we were getting for depth
measurements were unexpectedly small.
>
>A user came in to image a home built chamber with a glass face.  The
results we were getting for depth seemed too small.  Therefore, we made a
chamber with cut glass slides to check this.  A description with pictures etc
is at http://microscopynotes.com/710/10X_cal_issue/index.html , but here
is the short version:
>
>When imaged through the glass slide to the coverslip we got the same
results as imaging through the coverslip to the slide.
>
>When imaging through air or dilute water based dye, we got the same
results.
>
>My conclusion is that there is a calibration constant off somewhere in the
confocal system; it's probably just a software problem.
>
>Next call:  Zeiss service.
>
>Regards,
>Michael
>