Posted by
jerie on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Basic-question-tp7584586p7584590.html
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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