Hello everyone!
 
        I am wondering if somebody can help me out with the
following situation.  We are labeling specific bacteria that
are located inside of dentine tubules of human carious teeth.
We have tryed different ways of labeling and mounting trying to
improve the resolution of the images; however, the images still
are not as good as we would like to, because the main goal
of our experiments is to be able of counting individual
bacterium in non-disturbed dentin.  We use 100-um-thick samples
that we label with specific antibodies and post-label with
different dyes (FITC, TRITC, TxRed, and Bodipy).  After labeling,
samples are analyzed with a Noran Odyssey CLSM with the 480nm
and 523nm excitation wavelentgh, taking 20 sequential optical slices
(starting from around 4um of the top) ), 0.5um apart.  Now,
what we are doing, that we think has improved the images, is to
dry and immerse the specimens in immersion oil for at least half
hour before they are analyzed with a 60X (N.A. 1.4) oil immersion
Nikon objective without coverslip.  The use of cover slips is not
easy in this kind of samples and we have not found improvement with
their use.
        My knowledge in optical physics is limited (maybe too limited),
but it makes sense to me that in this situation, where the specimen
tubules are filled up with oil, the light will travel in a more adequate
refractive index than glycerol or other mounting media.
Can anybody share some ideas on how to improve this technique?.
Also, Is it possible to put antifading agents in the immersion oil? if so,
which one would you recommend?.
 
        I appreciate any help.
 
        Thank you,   Carlos
 
P.S. By the way, does anybody around there know what Na Salt Fluorescein
labels when we are labeling bacteria with this dye.  Does it locate in the
cell membrane? or does it penetrate and label esterases?.
 
Dr. Carlos Gonzalez Cabezas,    [log in to unmask]
Confocal and Electron Microscopes Facility
Indiana University, School of Dentistry