Posted by
Mario-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Z-sections-tp1142113p1142696.html
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.
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Mario M. Moronne, Ph.D.
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