The theory for Z axis elongation says that the focal point in the sample
moves less (or more if the index of the sample is higher than the immersion
media) than the focus is moved by a factor that is the ratio of the indices
of refraction.  For water this is 1.3/1.5 or about 85% NOT 2.5 times as was
stated here earlier.  This effect is described by Maxwell's elongation
formula which is covered in Born and Wolf, Principles of Optics p.166 of
the 2nd edition.  An excellent original reference to all of this theory is
Rayleigh, Phil. Mag. (5) 21:466 (1886).  We have measured the elongation
quite accurately and find agreement with the theory for the first few
microns inside the cover slip.  However, after that the spherical
aberrations begin to dominate and the apparent elongation becomes greater.
We have found that 1.3 and 1.4 NA lenses are limited to less than 10
microns for quantitative imaging in living cells, but we have no problem
with the 1.0 and lower NAs.  Of course it is still preferable to use a
water immersion lens with no cover slip in an upright scope if deep
sectioning is needed.
 
                                Dave Piston
                                Vanderbilt University