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pinhole-thickness

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Valeria Berno Valeria Berno
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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
mmodel mmodel
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Re: pinhole-thickness

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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-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Valeria Berno
Sent: Tuesday, July 08, 2008 7:47 AM
To: [hidden email]
Subject: pinhole-thickness

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

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
Eric Scarfone Eric Scarfone
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Re: pinhole-thickness

In reply to this post by Valeria Berno
Search the CONFOCAL archive at
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Valeria
I would say that the size of your optical section is entirely
dependant on the NA of your objective. What happens when you open your
pinhole is that you let photons coming from "outside" contribute to
image formation. Of course, you also get back difracted light coming
from your focal plane that you otherwise you would have rejected.
The end effect is more a blurring than a "thickening".
the simplest solution is to get a infra red pulsed laser! No need of
pinhole with multiphoton!
Cheers
Eric

Eric Scarfone, PhD, CNRS,
Center for Hearing and communication Research
Department of Clinical Neuroscience
Karolinska Institutet

Postal Address:
CFH, M1:02
Karolinska Hospital,
SE-171 76 Stockholm, Sweden

Work:  +46 (0)8-517 79343,
Cell:  +46 (0)70 888 2352
Fax:   +46 (0)8-301876

email:  [hidden email]
http://www.ki.se/cfh/


----- Original Message -----
From: Valeria Berno <[hidden email]>
Date: Tuesday, July 8, 2008 1:47 pm
Subject: pinhole-thickness
To: [hidden email]

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> 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
>
Julio Vazquez Julio Vazquez
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Re: pinhole-thickness

In reply to this post by mmodel
Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal -
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:

Search the CONFOCAL archive at

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-----
From: Confocal Microscopy List [[hidden email]] On
Behalf Of Valeria Berno
Sent: Tuesday, July 08, 2008 7:47 AM
Subject: pinhole-thickness

Search the CONFOCAL archive at

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
Guillermo Palchik Guillermo Palchik
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Re: pinhole-thickness

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal The publication link is:

On Jul 8, 2008, at 1:57 PM, Julio Vazquez wrote:

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
-
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:

Search the CONFOCAL archive at

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-----
From: Confocal Microscopy List [[hidden email]] On
Behalf Of Valeria Berno
Sent: Tuesday, July 08, 2008 7:47 AM
Subject: pinhole-thickness

Search the CONFOCAL archive at

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
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