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Richard - I believe that your 'HOWEVER' is a bit more complicated than you suggest and also not quite as bad as you say in that you do not loose as much working distance. I did a simple ray tracing based upon Snell's Law for the rays going through the cover slip to find that for large NA the "lost" working distance in your example is not 20um but 20/n = 20/1.523 = 13.1um. This is the case for NA < 0.3 and assumes that objective to coverslip is air. For larger NA, WD lost is even less than 20/n and goes to zero for the largest NA = 1 in air.
Here are the details of the formula if you are interested. If x is the coverslip mismatch thickness (20um in your example) and d is the actual lost WD then
d = x * sqrt { [(1/NA)^2 - 1] / [(n/NA)^2 - 1] }
You can see that when NA is very small, the 1/NA terms dominate and the -1 is negligible. We have sqrt{1/n^2} = 1/n.
In the limit as NA goes to 1, the numerator goes to 1-1=0 such that d goes toward 0 -- almost no loss of WD when NA is large.
If you plot d/x, you get a quadrant of an ellipse with a low NA asymptote of 1/n = 0.657 and a high NA asymptote of 0.
Lauren M. Peterson, PhD Lead Physicist - Photonics
Michigan R&D Center (formerly ERIM - Veridian)
General Dynamics - Advanced Information Systems
1200 Joe Hall Dr., P.O. Box 990 Ypsilanti, MI 48197
734-480-5303 -480-5252 FAX
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-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Richard E. Edelmann
Sent: Monday, October 01, 2007 3:34 PM
To: [log in to unmask]
Subject: Re: Working distance
Stéphane:
To my understanding the cover slip is considered an optical element
of the lens itself - which is why coverslip thickness is so critical,
and the working distance is the distance "beyond" the optical
elements of the lens. So if your lens calls for a 0.17mm coverslip
and has a WD or 300um, you get 300um beyond the 170um coverslip.
HOWEVER, if your coverslip is really 190um ( 0.160-0.190mm) you only
get 280um. If you are using a correction collar then you can correct
the optics to include the thicker 190um coverslip, and thereby
recover your "lost" 20um.
On 30 Sep 2007 at 12:50, Stéphane Pagès wrote:
>
> Dear List,
> I wonder about the exact definition of working distance. As I
> understood, in the case were the objective is not corrected for
> coverslip, this distance is the between the front of the objective and
> the specimen to be imaged.
> In the case were I use a a coverslip corrected objective, what does it
> mean?. For example I use a 63X water objective from Olympus with a
> working distance of 300 um. The coverslip thickness is 150 um. What is
> the effective distance between the front of the objective and the
> specimen ?? 300 um ??? Here
> www.microscopyu.com/articles/ formulas/formulasworkingparfocal.htm
> I saw that the distance is between the objective and the upper side of
> the coverslip ??
> I don t know if I misunderstood something or not
>
> Thanks, Stephane
> --
> Stéphane Pages PhD
> Neurobiologie cellulaire
> Centre de Recherche Université Laval Robert-Giffard (CRULRG)
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Richard E. Edelmann, Ph.D.
Electron Microscopy Facility Director
364 Pearson Hall
Miami University, Oxford, OH 45056
Ph: 513.529.5712 Fax: 513.529.4243
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