Posted by
Guy Cox-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/why-does-high-NA-excitation-illumination-give-better-resolution-in-fluorescence-microscopy-tp7581519p7581525.html
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy*****
Arne,
This is not quite true. In terms of confocal imaging with a pinhole of one Airy diameter or larger, the lateral resolution is determined only by the excitation NA, not the detection NA. They are or course the same, but you must expand the illumination beam to fill the pupil of the objective or you will lose resolution.
In confocal we are also concerned with axial (Z) resolution, and here both NAs are important, very important since the axial resolution depends on the square of the NA.
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:
[hidden email]] On Behalf Of Seitz Arne
Sent: Tuesday, 4 February 2014 4:25 AM
To:
[hidden email]
Subject: Re: why does high NA excitation illumination give better resolution in fluorescence microscopy?
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy*****
Hi Dan,
the discussions so far are strictly true for wide field microscopy. There the NA of the condenser should not play a role (given enough SNR). And to my understanding light-sheet microscopy is just a prominent example of such a scenario.
For confocal microscopy things are to my understanding a bit more complicated. The gain in lateral resolution with an infinitesimal small pinhole is due to the fact that the excitation point-spread function is convolved with the emission point spread function. Thus in that case the NA of the condensor should play a role. But as the lateral resolution improvement is hardly seen in practice this is just a theoretical limitation.
Cheers
Arne
> 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