Hi again,

In my yesterday's procedure to measure coverslip thickness I forgot to
mention refractive correction.  Sorry about that!  The correct procedure as
described in the Zeiss manual should be as follows:

Choose reflection/fluorescence mode on your confocal microscope with a
small pinhole diameter and no emission filters (or an emission filter that
passes the exciter laser line - for example an LP510 for the 543nm line of
the HeNe-laser).

Scan a series of z-sections through your coverslip with about 500nm step-width.

Determine the distance between the two reflection intensity peaks and
adjust the correction collar on the objective accordingly.

On the Zeiss confocal software the refractive correction can be set in the
z-section menu.  You should choose the value RC=n2/n1, where  n2 is the
refraction index of the coverslip (1.525 +/- 0.0015) and  n1 is the
refraction index of the media in between the lense and the coverslip.  n1=1
for air,  n1=1.455 for glycerol, and n1=1.33 for water.  Therefor, the
correct value for water immersion objectives should be about 1.146.  The
Zeiss software will make the correction automatically if you choose the
correct value.  Otherwise, if your software does not offer this option, you
should multiply the measured value  with the correction factor to obtain
the excact coverslip thickness.

We investigated some rather thick and intensly fluorescent specimen.  Using
a Zeiss C-Apochromat 40x/1.2W Korr we collected images as deep as
0.15-0.2mm inside of the specimen with a surprisingly good resolution.  In
my experience, the correct settings on the objective collar were essential
for the image quality.

Regards,
Jacques

---------------------------------------
Dr. Jacques Paysan
Universitaet Tuebingen
Institut fuer Physiologie, Abt. II
Ob dem Himmelreich 7
D-72074 Tuebingen
Germany

Telefon: (07071) 29 771 73
Telefax: (07071) 87 815

Website:  http://bongo.physiologie.medizin.uni-tuebingen.de/paysan/