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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 ([log in to unmask])
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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