Dear Dr. Corley,
 
        I am using the old Leica confocal (E5 machine) and would be happy
to correspond with others using Leica systems.  I use the same filter
arrangement you specified in your message and I haven't seen any
bleed-through from Fl to TxRed.  This used to be a considerable problem for
me when I used the BioRad system, but since switching to Leica the problem
has gone away.  I believe the suggestions made by Erik Manders to the list
may help you.  It turns out that my Kr/Ar laser is tuned so that the 568
line is considerably stronger than the 488 one.  As the laser dies out, the
longer wavelengths are lost first.  By tuning it so that the 568 line has
more power initially, you get a bit more life out of the laser. I believe
this contributes to my lack of bleed through.  With the new Leica PMT
(present in all new machines) which is more sensitive in the FL emission
range, you should see no reduction in image quality even with less Fl laser
excitation. To mimic this situation you could try attenuating the FL light
as Erik Manders suggests.
 
        I run a confocal facility and many people here are interested in
co-localization too.  I am also looking for insight into this phenomenon.
 
        I used to use Mowiol with the addition of PPD as an anti-fade
agent.  I then discovered Vectashield, from Vector Co. and like it much
better.  We've done side by side tests with Vectashield and other
commercial mounting mediums as well as our home made mowiol/PPD and the
Vectashield prevented fading much longer.  It is also equally good at
preventing fading of both FL and Tx-Red.  It is a glycerol based medium,
but the down side is that it doesn't harden.  You have to nail polish
around the edges of the coverslip.  We've decided this is a small price to
pay for being able to look at our specimens over and over again with
virtually no fading.  I've kept specimens in Vectashield at -20 for over a
year and see no degradation.
 
        In terms of immersion oil:  The word I have is to use Zeiss oil.
Even the Leica reps here have told me that.  As I understand it there's not
a significant difference with respect to refractive index matching among
the major oils. What does seem to be important is viscosity.  The Zeiss oil
produces less "drag" on the specimen and this is important for doing stable
z-series.  Several confocal reps have told me this, so I stick with Zeiss
oil.
 
        I hope any of this is useful.  Happy confocalling!
 
Judy Drazba
 
 
 
Judy Drazba, Ph.D.  ([log in to unmask])
(Director, Confocal Microscopy Facility)
Department of Neurosciences, NC-3
The Cleveland Clinic Foundation
9500 Euclid Avenue
Cleveland, OH 44195-5001
Office (216)445-3760
Lab    (216)444-8712
FAX    (216)444-7927