Ulrich Seydel asked about visualizing DiI and DiA on the MRC-1000.
 
        We have been double labeling pathways in mammalian brain with DiI and
DiA for 3 years.  These labels can be imaged with epifluorescence using narrow
band blue and green cubes, and in the case of persistent breakthrough the use
of a short pass 540 filter will almost completely eliminate the breakthrough.
On the MRC-1000 you cannot use simultaneous imaging of red/green.  We have
consulted Molecular Probes for information on the emission curves for DiI and
DiA.  We have gotten almost no help.  They tell us what the emission of DiA is
in methanol, but several of their newsletters have indicated that this
radically shifts when in tissue.  In fact, 3 of their publications listed
different amounts of shifts!  Based on our experimental use, we think that DiA
may have an even wider emission curve than DiI.  We have used the following
procedures to produce a double label image on the MRC-1000.
 
        Try collecting separate fields sequentially using the Membrane Label
settings for DiI and DiA.  That eliminates 95% of the problem.  If you are not
dealing with a super-bright object, it can be 100% effective.  If not, go into
the Custom mode and reduce the problem signal (in our hands it is always the
DiA).  This usually works.  As an absolute last resort, if you are completely
satisfied that the object is really breaking through, rather than representing
a double-labeled object, you can play with the brightness and contrast setting
to make that problem object visually unnoticeable.  The end result is that we
get beautiful images of the two separate labels within a single visual field.
Let me know if you have any further problems.
        ANDREA ELBERGER
        Dept. of Anatomy and Neurobiology
        The University of Tennessee, Memphis
        tel. (901) 448-4101
        email: [log in to unmask]
                                             Nka.   asitem lesbhATsu, oe