Re: pinhole-thickness
Posted by
Julio Vazquez on
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Valeria,
There is a Zeiss publication where they give the formula that calculates the thickness of the optical sections as a function of pinhole diameter, wavelength, refractive index, and numerical aperture. As Michael said, it is a rather complex formula. The more you open the pinhole, the more out of focus contribution you will get from farther away from the focal plane, so it's a somewhat arbitrary definition... it all depends where you set the threshold for what you "see" and what you don't "see" (typically, expressed in units of FWMH or full-width at max height). In any event, if you send me an email, I can reply with an attachment of the Zeiss paper (I did not get your original posting, so I don't have your personal address).
As a side note, the Zeiss LSM 510 acquisition software will tell you what your optical thickness is for any combination of objective, wavelength and pinhole aperture. This is used to for instance make sure you are collecting images of similar "thickness" in different channels by adjusting the pinhole aperture, for instance for colocalization studies.
The publication is titled: Microscopy from Carl Zeiss. Principles. Confocal laser scanning microscopy. You may be able to Google it too...
--
Julio Vazquez
Fred Hutchinson Cancer Research Center
1100 Fairview Ave. N., mailstop DE-512
Seattle, WA 98109-1024
= On Jul 8, 2008, at 7:02 AM, MODEL, MICHAEL wrote:
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Hi Valeria
I don't think there's a simple formula, but in the 2nd edition of
Confocal Handbook there is a chapter by Wilson that talks about the
effect of pinhole on resolution.
Mike
Michael Model, Ph.D.
Confocal Microscopy Core
Dpt. Biological Sciences
Kent State University
Kent, OH 44242
tel. 330-672-2874
-----Original Message-----
Behalf Of Valeria Berno
Sent: Tuesday, July 08, 2008 7:47 AM
Subject: pinhole-thickness
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Dear all,
I know this is a very basic question but I am having hard time to find
the
answer.
How can I calculate the thickness of the optical section I am aquiring
with different pinhole size?
So waht is its relation with objective resolution?
Thanks in advance
Valeria