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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.

Michael


> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> 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