Posted by
Joshua Zachary Rappoport on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Basic-question-tp7584586p7584596.html
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
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From: Confocal Microscopy List [mailto:
[hidden email]] On Behalf Of Christian Elowsky
Sent: Tuesday, January 05, 2016 9:07 AM
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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.
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From: Confocal Microscopy List [mailto:
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Sent: Monday, January 04, 2016 4:43 PM
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Subject: Re: Basic question
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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