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The resolution, assuming the BFP of the objective is 
filled (not always the case) will be given in fluorescence
by the Rayleigh formula r = 0.5lambda/NA.  This applies 
to widefield or scanned microscopy.  With a very small 
pinhole there is an additional 'confocal' resolution 
improvement of 1.414 (root 2) (r confocal = r wf / 1.414).
The full improvement will only be given by an infinitely
small pinhole ... For some examples of finite sized 
pinholes see Guy Cox & Colin Sheppard, 2004.  Practical 
limits of resolution in confocal and non-linear microscopy.  
Microscopy Research & Technique, 63, 18-22	.

In practice you can get some lateral resolution improvement
in reflection mode but you won't get much, if any, in
fluorescence.  So when you open your pinhole you are not
really losing lateral resolution.  You are losing depth
resolution quite substantially.  There really shouldn't be
any reason to open the pinhole above 1 Airy unit since (unless
the system is misaligned) that will let through all the in 
focus light.  After that you're just letting in out of 
focus light.

                                              Guy



Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
    http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
______________________________________________
Phone +61 2 9351 3176     Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
     http://www.guycox.net
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Jan Grawé
Sent: Tuesday, 1 April 2008 6:01 PM
To: [log in to unmask]
Subject: lateral resolution

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Dear all,

I find that there is someting I have not understood regarding lateral resolution in our confocal (Zeiss 510 meta).
As I understand it, lateral resolution should be dependent on excitation wavelength and NA solely. 
But what is the case if we open the pinhole a bit above 1 AU? For some (admittedly special) samples, we use up to about 4AU.
As far as I understand it, on a 510, using a 40x
1.3 objective, 532ex/575em, zoom 1, the pinhole is 300um, and the object-side projected pinhole diameter is then about 2,2um. As the PMT detector itself is not position-sensitive any light within this 2.2um circle in the focal plane would be detected. The practical resolution would then be determined by the laser spot size (?).
Using the zeiss "optimal frame size" calculator for nyqvist sampling on the above setting gives me 2048*2048 pixels, which is the max available, maybe it would calculate an even higher nunber of pixels if the option was available. 2048*2048 gives me a pixel size of 0.11*0.11 um , which to me seems small in relation to the spot size of the laser.
Is this reasoning correct, or where am I wrong?

Thanks in advance,

Jan Grawé
Cell Analysis Core Facility
Rudbecklaboratoriet/C5
Dag hammarskjölds väg 20
SE-75185 Uppsala
SWEDEN

Phone: +(0)18-4714657
Cell:   +(0)70-2577874
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www.rudbeck.uu.se/cellanalys

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