Matt,

Fred's reply is particularly relevant. I would make a suggestion, 
though, which is that because of R.I. mismatch and spherical 
aberration the actual z-axis resolutions is almost always worse than 
the theoretical by a significant amount. For example, a 1.4 NA oil 
objective at 10 um into a sample should have a z- res about 0.45 um 
(FWHM) using 488 nm. In actual practise, it is usually closer to 
0.6-0.8 um and it gets worse (much worse) the further into a sample 
you go. In any event, the closer you stay near the coverslip boundary 
the more justified it is to use a high sampling number. In the case 
of a layer of cells 20 um thick, a 128 sections gives ~0.16 um 
spacing. Much more than you need to satisfy Nyquist even if 
everything is perfect for 488 nm excitation. Realistically, 64 
sections should do the job in a real sample. Fewer than that starts 
to become risky for a 1.4 NA lens.

Depending on what you are doing, 64 frames should be enough.

Good luck
Mario

>Hi all,
>
>I have a question concerning Z sectioning.  We have a Leica SP2 and 
>when set the top and bottom range for a Z stack, the software 
>automatically calculates the optimised number of sections, which is 
>nearly always over 100!  This is however not practical as the stacks 
>take too long to acquire.  Can anyone shed some light on how the 
>software decides how many slices are necessary and if I haven't got 
>time to take 100, how can I work out what will be a sufficient 
>number.
>
>Thanks for your help in advance.
>
>Matt Pearson.


-- 
________________________________________________________________________________
Mario M. Moronne, Ph.D.

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