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Arne Seitz on
URL: http://confocal-microscopy-list.275.s1.nabble.com/why-does-high-NA-excitation-illumination-give-better-resolution-in-fluorescence-microscopy-tp7581531p7581533.html
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Hi Dan,
I directly move on to your "one last thing":
In a previous post Jim Pawley pointed out the zero frequency support along the z-axis would only be true in a aperture free system. I'm too lazy to find the post in the archives but I remember the publication which demonstrates this fact:
Biophys J. 1990 Feb;57(2):325-33.
Determination of three-dimensional imaging properties of a light microscope system. Partial confocal behavior in epifluorescence microscopy.
Hiraoka Y, Sedat JW, Agard DA.
Nevertheless there is still the problem why zero fold zero is not zero.....
If I remember it correctly you have to do a convolution of the emission and excitation OTF. Thus it is not a multiplication. You can come to the resulting OTF of a confocal microscope if draw the excitation OTF and you draw the emission OTF on a different piece of paper an cut it out. Now you place the center of the cut out OTF on every point of the first OTF and you combine the outlines of the overlaid OTFs. This results in the OFT of the confocal microscope.
I found a nice illustration of what I tried to explain in the following lecture from Kai Wicker (page 15):
http://www.iap.uni-jena.de/iapmedia/Lecture/Advanced+Optical+Microscopy1365976800/AOM_Slides+AOM+121203.pdfThis is clearly not a math-free explanation but at least something that can be derived graphically.
Hope that helps.
Cheers
Arne
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Subject: why does high NA excitation illumination give better resolution in fluorescence microscopy?
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Thanks Arne, Guy, Retro et al. for all the great answers and comments.
Now we get to the CRUNCH:
We agree that the NA of illumination has no effect on epi wide field fluorescence resolution.
But for confocal, we hear very strong claims about NA of illumination strongly affecting resolution, both lateral and axial.
Here is where my understanding needs some support....
Can we say this statement is true?:
As a form of structured illumination microscopy, scanning point confocal can benefit from a high illumination NA and proper back aperture beam filling, because the resolution is determined by the product of the excitation and detection point spread functions.
In other words,
Because in confocal, the illumination pattern is itself high spatial frequency, contrasty, focused and small, rather than featureless and large as in widefield, this illumination contrast over space multiplies with the detection point spread function to increase resolution, and is also the source of the optical sectioning power of a confocal.
If that's the case, then it begins to make more sense, to a stupid biochemist like myself with a very tenuous grasp on maths.
I understand at least that the square of a Gaussian function is a bit sharper than a Gaussian. But not that much.
One last thing:
If the axial response of a confocal has real detectable power, where does that come from?
The product of nothing and nothing is nothing. Widefield detection psf has a nothing response axially, right? Does the illumination psf in confocal somehow have some non zero power axially, in z direction? How is the confocal really able to fill in the missing wedge in the axial direction in Fourier space... when zero times zero is zero? Or are my zeros an over simplification?
Just looking for plain natural language explanations instead of maths and equations that pretty image fixated cell biologists and dummies like me are unlikely to comprehend.
Cheers
Dan