Re: Coverslip thickness and correction collar ... option Ib

Posted by Craig Brideau on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Coverslip-thickness-and-correction-collar-tp591122p591128.html

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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)
> >
> >
> >
> >
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