Posted by
Daniel White-2 on
Feb 05, 2014; 7:53pm
URL: http://confocal-microscopy-list.275.s1.nabble.com/why-does-high-NA-excitation-illumination-give-better-resolution-in-fluorescence-microscopy-tp7581531.html
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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