Dear Florian,

the refractive index of  1% Agarose is close to water, difference seems
neglectable. We did recordings of 1 Micrometer Tetraspeck beads with  a
LEICA 63x 1.2 N.A. water immersion lens, and the performance at 140
micrometers depth was almost the same as with beads located directly at the
cover slip.

Much more important factors in this context:

If you scan with a high aperture water immersion lens (e.g. 63x 1.2 N.A. )
calculated for use with a cover slip 0.17 mm: correct for cover slip
thickness with the correction collar (if there is one): Do a xz scan of the
coverslip reflection signal, adjust collar for max. peak in combination
with minimum width of the reflection signal.

If there is no correction collar, be sure to use high quality 0.17 mm cover
slips (check thickness with a micrometer measuring device available at your
mechanical workshop, since, according to my experience, you cannot trust
manufacturers: several batches of expensive, low-tolerance cover slips were
far beyond specifications regarding thickness).



Hope this helps,

Guenter


At 11:32 10.09.2001 +0800, you wrote:
>--
>Dear all,
>
>does anyone of you know the refractive index of 1% low melting
>agarose in water? My second question is about mounting medium: I want
>to do antibody staining on zebrafish embryos; I use a FITC-Alexa
>secondary antibody. I used 1% low-melting agarose, but maybe this s
>not the optimal medium... so, if anybody has experience with this, I
>would be happy about suggestions.
>
>Thank you very much,
>
>Florian Ulrich
>Heisenberg Group
>MPI-CBG, Dresden
>Pfotenhauerstrasse 108
>01099 Dresden
>Germany
>0049 - 351 - 2102689

------------------------------------------
Dr. Guenter Giese
Light Microscopy Facility
Dept. of Biomedical Optics, MPI fuer Medizinische Forschung
Jahnstr. 29, D-69120 Heidelberg, Germany
Phone (+49) 6221-486-360 (Fax: -325)
e-mail: [log in to unmask]
http://sun0.mpimf-heidelberg.mpg.de/~ggiese/lightmicro/index.html