Lutz,

>
> Steffen, you still have to watch the coverglass thickness since your
> embedding media and sample do have likely a refractive index that is smaller
> than 1.515 (glass, oil). Therefore most objectives produce the least
> spherical aberrations when the object lies directly beneath the coverglass.
> Taken into consideration that for the condition that the sample refracts by
> 1.33 (Water) with the objective corrected in such way, a different coverslip
> thickness would shift the sample-to-coverslip distance that is necessary for
> aberration corrected imaging. In the older days with the 160mm tubus people
> could correct for that by changing the tubus length, but now with ICS we
> have to be a bit careful.
>
> Due to that the PSF is variant in the axial dimension, which may be a burden
> for deconvolution if the PSF is not measured and applied at more then one
> axial position. However, the closer the refractive index of the sample
> matches that of the immersion, the less variant the PSF will become.
>
> Lutz

all this about the spherical aberration and varying PSF I accept, but
still, what is the difference between
a) having an oil objective with a working distance of, say, 90um, which
is 90um above a 170um coverslip thus focussing directly beneath the
coverslip, and
b) having the same objective 160 um above a 100um coverslip

In the second case the 70um of glass are just replaced by immersion oil,
which shouldn't make a difference, and in both cases the objective
should focus directly beneath the coverslip.

Joachim


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Joachim Walter, Dipl. Phys.
Institut für Anthropologie und Humangenetik der LMU München
AG Cremer
Richard-Wagner-Straße 10/I
D-80333 München               Tel. +49 - 89-2180-6713
Germany                       Fax. +49 - 89-2180-6719
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