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Basic question

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Christian Elowsky

Basic question

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I have a basic question, how does one calculate the physical plane from a
confocal image? Meaning without under or oversaturation, what is the plane
thickness calculation if the pinhole is set to 1 A.U.?

Basically, I have a 4x image z-series from a Nikon A1 and need to explain
what physically is represented in each plane. The pinhole was set to 1 A.U.

Unless of course the calculations are in the image data and I've had no luck
finding it yet. I'm also happy to figure it out, if someone wants to point
me at the right textbook.

Thanks.

Julio Vazquez-2

Re: Basic question

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There is a formula on page 11 of a publication by Zeiss titled "Confocal Laser Scanning Microscopy Principles" for calculating the thickness of confocal optical sections:

http://zeiss-campus.magnet.fsu.edu/referencelibrary/pdfs/ZeissConfocalPrinciples.pdf

best,

Julio Vazquez
Fred Hutchinson Cancer Research Center

----- Original Message -----
From: "Christian Elowsky" <[hidden email]>
To: "Confocal Microscopy List" <[hidden email]>
Sent: Monday, January 4, 2016 12:36:40 PM
Subject: Basic question

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To join, leave or search the confocal microscopy listserv, go to:
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Post images on http://www.imgur.com and include the link in your posting.
*****

I have a basic question, how does one calculate the physical plane from a
confocal image? Meaning without under or oversaturation, what is the plane
thickness calculation if the pinhole is set to 1 A.U.?

Basically, I have a 4x image z-series from a Nikon A1 and need to explain
what physically is represented in each plane. The pinhole was set to 1 A.U.

Unless of course the calculations are in the image data and I've had no luck
finding it yet. I'm also happy to figure it out, if someone wants to point
me at the right textbook.

Thanks.
--
Julio Vazquez, PhD
Director of Scientific Imaging
Office: 206.667.1215
Lab: 206.667.4205
[hidden email]

Fred Hutchinson Cancer Research Center
1100 Fairview Ave. N., Mail Stop DE-512
Seattle, WA 98109
fredhutch.org

Tim Feinstein

Re: Basic question

In reply to this post by Christian Elowsky

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Hi Christian,

The question is a bit tricky to answer simply, but I will try. The
effective thickness of a confocal section depends on the wavelength of
light (in crude terms, blue focuses more tightly than red or far red), the
numerical aperture (NA) of the objective, the pinhole size and whether
you're imaging through air, water or oil. You can never focus light down
to a perfect point and you never get back an image of a single fluorophore
as a perfect little point*, instead fluorophores appear appear on the
detector as a fuzzy area centered on their actual position of the
fluorescing molecule. This is known as the point spread function (PSF).
The size of the PSF determines how wide or tight your Z spacing has to be
- any Z spacing significantly smaller than the Z dimension of the PSF will
sample the same fluorophore repeatedly. A 4x objective for example has a
low NA and so each point of light results in a PSF that measures several
microns in the Z dimension. If we say a PSF is three microns tall, Z
spacing of half a micron is grossly excessive whereas ten micron Z steps
will have 'gaps' where many fluorophores are not imaged. Optimal Z
spacing is known as the Nyquist criterion and represents a best effort to
capture all fluorophores in a 3D sample without sampling any single
fluorophore in more than one or two sequential images.

Among reference books that I have used, Optical Imaging Techniques in Cell
Biology from Guy Cox was the most approachable (some chapters cover
general imaging theory) and the Handbook of Biological Confocal Microscopy
by Jim Pawley is definitely the most thorough.

Best,

Tim

Timothy Feinstein, Ph.D.
Research Scientist
University of Pittsburgh Department of Developmental Biology

(*) People are working on that. Metamaterials suggest some interesting
possibilities.

On 1/4/16, 3:36 PM, "Confocal Microscopy List on behalf of Christian
Elowsky" <[hidden email] on behalf of [hidden email]>
wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>Post images on http://www.imgur.com and include the link in your posting.
>*****
>
>I have a basic question, how does one calculate the physical plane from a
>confocal image? Meaning without under or oversaturation, what is the
>plane
>thickness calculation if the pinhole is set to 1 A.U.?
>
>Basically, I have a 4x image z-series from a Nikon A1 and need to explain
>what physically is represented in each plane. The pinhole was set to 1
>A.U.
>
>Unless of course the calculations are in the image data and I've had no
>luck
>finding it yet. I'm also happy to figure it out, if someone wants to
>point
>me at the right textbook.
>
>Thanks.
>
>

jerie

Re: Basic question

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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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*****

Hi Christian,
also make sure the lens was correctly entered into the microscope software.
The software calculates the 1AU projected diameter of the pinhole from the
data on magnification and aperture and maybe wavelength. What exactly is
regarded to be 1AU has been a matter of interpretation. Is it the fwhm of
the airy disk or is it the 1st zero crossing of the Bessel function? So it
may make sense to ask the manufacturer, how the 1AU setting was calculated
instead of using the definition of one manufacturer and the section
thickness calculated of another ;) Because most manufacturers nowadays use
a single pinhole for all detectors/wavelength, I don't know in how far
absolute wavelength goes into their calculation or if they just use a mean
wavelength.
Needless to say that misalignments, mounting and immersion media also will
affect the number.
A simple way to derive a theoretical number may be to use the sampling
calculator of svi.nl, because they care about the different manufacturers;
a more practical approach may be to use large spheres, and image under the
same conditions like in your experiment to measure the effect of the
pinhole.

So depending on who is reviewing your work it may be a basic question to
answer indeed ;)

My 2 cents, cheers, Jens

Christian Elowsky

Re: Basic question

On the Nikon A1, the pinhole is calculated from the longest wavelength of emission (well the center of the range). I'm aware this means there will be some differences in the "DAPI" and "CY5" channels, something I have never needed to ponder before. A fact which is a bit ridiculous. Thank you for the input, it really is great that people take the time to answer these questions.

Thank you Jens, Tim and Julio.

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of jens rietdorf
Sent: Monday, January 04, 2016 4:43 PM
To: [hidden email]
Subject: Re: Basic question

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Hi Christian,
also make sure the lens was correctly entered into the microscope software.
The software calculates the 1AU projected diameter of the pinhole from the data on magnification and aperture and maybe wavelength. What exactly is regarded to be 1AU has been a matter of interpretation. Is it the fwhm of the airy disk or is it the 1st zero crossing of the Bessel function? So it may make sense to ask the manufacturer, how the 1AU setting was calculated instead of using the definition of one manufacturer and the section thickness calculated of another ;) Because most manufacturers nowadays use a single pinhole for all detectors/wavelength, I don't know in how far absolute wavelength goes into their calculation or if they just use a mean wavelength.
Needless to say that misalignments, mounting and immersion media also will affect the number.
A simple way to derive a theoretical number may be to use the sampling calculator of svi.nl, because they care about the different manufacturers; a more practical approach may be to use large spheres, and image under the same conditions like in your experiment to measure the effect of the pinhole.

So depending on who is reviewing your work it may be a basic question to answer indeed ;)

My 2 cents, cheers, Jens

Joshua Zachary Rappoport

Re: Basic question

Actually you can select in NIS Elements which excitation wavelength you want used for the pinhole calculation
More specifically you pick a pinhole size in microns, which is fixed for all laser lines, and then you can see what A.U. number that corresponds to for each

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christian Elowsky
Sent: Tuesday, January 05, 2016 9:07 AM
To: [hidden email]
Subject: Re: Basic question

On the Nikon A1, the pinhole is calculated from the longest wavelength of emission (well the center of the range). I'm aware this means there will be some differences in the "DAPI" and "CY5" channels, something I have never needed to ponder before. A fact which is a bit ridiculous. Thank you for the input, it really is great that people take the time to answer these questions.

Thank you Jens, Tim and Julio.

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of jens rietdorf
Sent: Monday, January 04, 2016 4:43 PM
To: [hidden email]
Subject: Re: Basic question

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Hi Christian,
also make sure the lens was correctly entered into the microscope software.
The software calculates the 1AU projected diameter of the pinhole from the data on magnification and aperture and maybe wavelength. What exactly is regarded to be 1AU has been a matter of interpretation. Is it the fwhm of the airy disk or is it the 1st zero crossing of the Bessel function? So it may make sense to ask the manufacturer, how the 1AU setting was calculated instead of using the definition of one manufacturer and the section thickness calculated of another ;) Because most manufacturers nowadays use a single pinhole for all detectors/wavelength, I don't know in how far absolute wavelength goes into their calculation or if they just use a mean wavelength.
Needless to say that misalignments, mounting and immersion media also will affect the number.
A simple way to derive a theoretical number may be to use the sampling calculator of svi.nl, because they care about the different manufacturers; a more practical approach may be to use large spheres, and image under the same conditions like in your experiment to measure the effect of the pinhole.

So depending on who is reviewing your work it may be a basic question to answer indeed ;)

My 2 cents, cheers, Jens

Guy Cox-2

Re: Basic question

Bio-Rad used to make a big point of this since they had a separate pinhole for each channel on the old MRC-series machines. But it's hard to see quite what experiment this will make a difference to. After all your XY resolution will be worse in the CY5 channel anyway.

You could reset your pinhole between each collection if it really matters. Other, simpler suggestions would be to detect the DAPI at longer than optimal wavelength - it is very bright and has a very wide spectrum, or else to close the pinhole below 1 AU. But unless you are very scrupulous about imaging conditions your actual PSF will be enlarged by spherical aberration anyway so the 1 AU pinhole becomes a bit idealistic.

Guy

Guy Cox, Honorary Associate Professor
School of Medical Sciences

Australian Centre for Microscopy and Microanalysis,
Madsen, F09, University of Sydney, NSW 2006

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Joshua Zachary Rappoport
Sent: Wednesday, 6 January 2016 2:25 AM
To: [hidden email]
Subject: Re: Basic question

Actually you can select in NIS Elements which excitation wavelength you want used for the pinhole calculation More specifically you pick a pinhole size in microns, which is fixed for all laser lines, and then you can see what A.U. number that corresponds to for each

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christian Elowsky
Sent: Tuesday, January 05, 2016 9:07 AM
To: [hidden email]
Subject: Re: Basic question

On the Nikon A1, the pinhole is calculated from the longest wavelength of emission (well the center of the range). I'm aware this means there will be some differences in the "DAPI" and "CY5" channels, something I have never needed to ponder before. A fact which is a bit ridiculous. Thank you for the input, it really is great that people take the time to answer these questions.

Thank you Jens, Tim and Julio.

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of jens rietdorf
Sent: Monday, January 04, 2016 4:43 PM
To: [hidden email]
Subject: Re: Basic question

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Hi Christian,
also make sure the lens was correctly entered into the microscope software.
The software calculates the 1AU projected diameter of the pinhole from the data on magnification and aperture and maybe wavelength. What exactly is regarded to be 1AU has been a matter of interpretation. Is it the fwhm of the airy disk or is it the 1st zero crossing of the Bessel function? So it may make sense to ask the manufacturer, how the 1AU setting was calculated instead of using the definition of one manufacturer and the section thickness calculated of another ;) Because most manufacturers nowadays use a single pinhole for all detectors/wavelength, I don't know in how far absolute wavelength goes into their calculation or if they just use a mean wavelength.
Needless to say that misalignments, mounting and immersion media also will affect the number.
A simple way to derive a theoretical number may be to use the sampling calculator of svi.nl, because they care about the different manufacturers; a more practical approach may be to use large spheres, and image under the same conditions like in your experiment to measure the effect of the pinhole.

So depending on who is reviewing your work it may be a basic question to answer indeed ;)

My 2 cents, cheers, Jens