For specific "spacers" our 15 micron fluorescent beads were designed for CLSM based Z (depth) calibration.  We also have 6 micron ones if you prefer.  

These also show the effect of color and RI mismatch on Z depth accuracy.  Simple protocol included.  

FocalCheck™ microspheres, 15 µm, fluorescent green ring stain⁄dark red throughout
Cat. No. F-7238
Or
FocalCheck™ microspheres, 15 µm, fluorescent dark-red ring stain⁄green throughout
Cat. No. F-7239

Kind Regards,

Mike Ignatius
Molecular Probes/Life Technologies

-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Patrick Van Oostveldt
Sent: Tuesday, May 11, 2010 10:24 AM
To: [log in to unmask]
Subject: Re: Optical Section Thickness-Leica SP5

If you really want to determine the thickness of a section you should  
take account of the fact that the refractive index of the medium can  
change the estimated thickness. Cfr. Paper of Visser, Oud and  
Brakenhoff in the early days of CSLM. Just google to "visser  
refractive index" and you will find the pdf print.

My experience is that fixation can also affect effective thickness of  
the section due to shrinking or swelling.

A test is possible by using a fluorescent dye mounted under a  
coverglass with specific spacers and measure the number of steps  
necessary to image the whole object.

A simple test is the use of some fluorescentive index and axial  
...Quoting "Linda Barthel" <[log in to unmask]>: textile fiber of PE  
which is round and where you can directly check if XY diameter  
corresponds to XZ distances.

Bye

Patrick Van Oostveldt




> Seems to be a simple enough question-but having trouble getting a simple
> answer.  Operating a Leica SP5 Confocal microscope, software: LAS-AF ver.
> 2.0.0 software.  How do we determine the optical sections thickness of our
> images?  This must be a simple oversight on our part.
> Thanks,
> Linda Barthel
> Gregg Sobocinski
> University of Michigan
> Dept of MCDB
> Ann Arbor
>



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