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Dear Jan,

As you have indicated, when opening up the pinhole the resolution is
determined by the laser beam only. Laterally, your focused laser beam will
have a resolution of approx. 250nm. Following Nyquist, the pixel size should
indeed be about 100 to 110 nm. So, everything seems to be fine.

Best regards,

Kevin


> -----Oorspronkelijk bericht-----
> Van: Confocal Microscopy List [mailto:[log in to unmask]]
> Namens Jan Grawé
> Verzonden: dinsdag 1 april 2008 10:01
> Aan: [log in to unmask]
> Onderwerp: lateral resolution
> 
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> 
> 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