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I agree with this statement but this applies only to widefield fluorescence, absolutely NOT to confocal.  See another post (which I am about to write).

                                             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:[log in to unmask]] On Behalf Of Daniel White
Sent: Tuesday, 4 February 2014 3:42 AM
To: [log in to unmask]
Subject: why does high NA excitation illumination give better resolution in fluorescence microscopy?

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Hi again all,

So from the informative answers so far...
it seems that the idea of filling the back focal plane of the objective (acting as the condenser lens) in epi fluorescence, both widefield and confocal, is more about getting lots of light into the sample in order to get enough signal to then be able to see the resolution that is there, since effective resolution is limited by signal to nose (sorry, noise)

In other words, is there enough light going in (and consequently coming back out) that we can see the Rayleigh criterion dip between the two peaks of the object images,  despite any noise that's present?

Is this following statement then true?:

In the case of infinite signal to noise, where contrast is optimal and not limited by noise, where illumination power is close to saturation of the fluorescence excited state, exposure time is long enough, etc....
the effective NA of illumination (back aperture filling) has no effect on achievable lateral or axial resolution - only signal:noise does.

Are there cases where this is false? Anisotropy?

cheers

Dan