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This was already answered by Arne Seitz in the very first reply to this query!

The spot on the sample is a diffraction-limited spot - an Airy disk.  This is a function of the NA of the lens and has nothing to do with the pixel size you select on the scope.  The radius of the Airy disk is the resolution of that objective.

If you underfill the BFP (pupil) of the objective you are effectively reducing the NA and the spot will therefore get larger, making your resolution worse.  This is obviously a bad thing to do.  The Leica has a beam-expander so this should not happen (unless you start fiddling).

If you overfill the BFP you will lose some light, that's all.  Resolution and spot-size is unchanged, and you can always dial in more laser.  

In multiphoton you get a resolution improvement of 1.414 (sqrt 2) which compensates for the longer wavelength.  (800nm in MP is equivalent to 565nm in confocal or widefield)  This explains why you see much the same resolution in both MP and confocal.   (In principle you can get the same benefit in confocal, but only with a very tiny pinhole, which is impracticable.  Normal confocal fluorescence has the same resolution as widefield fluorescence).  

                                                                                 Guy



-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Pascal Weber
Sent: Tuesday, 28 August 2012 4:07 PM
To: [log in to unmask]
Subject: Re: focused laser beam size on stage

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We have one Leica TCS SP5 MP confocal system and I would like to find out how the size of the laser beam on the stage is controlled?

It is possible to controlled the size of the point in 2P. It's mentionned in one answer you have to inderfill, but never over. To do that you have to add a lense in the laser beam.

 When we change the pixel size to meet Nyquist criteria, does the laser focal point on the specimen change its size accordingly?

It's only change with the NA.

 Will the focused beam has same size to the pixels? What is the smallest focus size of the laser beam on the stage we can achieve?

I always obtain the same résolution with a 2P than a normal confocal but in deep (over 1mm in tthe brain of over weeks mouse)


 Does two photon excitation has the same scenarios? 

No because the pulse power is over a confocal and you can excite many fluo 
at the same time. You have less bleaching due to the small excitaion point. 
Infact may be the 'confocal' system if you use multi wave length 2P laser.
Thank you very much in advance for your kind advice.