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Listers,

I strongly recommend to look back 10 years, there was a threat on the
confocal listserver mentioning this article:

Contrast, resolution, pixelation, dynamic range and signal-to-noise :
fundamental limits to resolution in fluorescence light microscopy -
E.H.K. Stelzer - J. Microscopy (1998) 189 : 15-24.

Basically, to meet the Rayleigh criterion under even moderate noise
conditions you need at least 8 samples to achieve the required contrast
to distinguish the peaks of 2 neighbouring Airy patterns. Translated for
the objective and lambda mentioned (1.3NA, 532nm ex.) this results in a
pixelsize of about 58nm(!). The Nyquist calculator from SVI even returns
51nm as the required pixel size.

Overall, in my opinion, the pinhole does not improve lateral resolution
at all under the conditions encountered in imaging of fluorescently
labeled biomaterials.

Cheers, jens

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-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On
Behalf Of Ian Dobbie
Sent: Mittwoch, 2. April 2008 11:49
To: [log in to unmask]
Subject: Re: lateral resolution

Search the CONFOCAL archive at
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Michael Weber <[log in to unmask]> writes:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> The correct pixel size is just correct by chance, since the Zeiss 
> software doesn't consider the pinhole diameter in the calculation of
XY scaling.

There is some justification for this. Using the Rayleigh criterion, the
resolution is defined by the distance to the first minimum of the Ariry
disk. A confocal pinhole does not change this at all, although it does
reduce the FWHM of the peak (ie the shape changes but the peak still has
the same total width). Where a confocal really wins is in Z resolution.

Ian