From:   BODKIN::8803630P     "PATRICK CORLEY BIO DEPT" 14-SEP-1995 23:20:59.87
To:     ME
CC:
Subj:
 
 
 
Dear colleagues,
 
                Thank you for your informative letters/comments regarding dual
labelling of samples the use of mowiol, and subsequent visualisation of same
by confocal microscopy on a Leica TCS4d.
 
                We have our machine about four months now and even in that time
I have come to agree that due to detector bleed through of FITC into the red it
is best to acquire both data sets sequentially. The labels that I have been
using are FITC and Texas Red. We are to have one final return visit by our
'local'  Leica representative in October, and I feel that in order not to waste
this opportunity I shall be well prepared with questions and queries.
 
        The filter set that I use for the above are as follows
 
        LASER line 488 nm
 
        DD 488/568
 
        BP-FITC
 
        Detector one:    ( for detection of FITC )
 
        ______________
        LASER line 568 nm
 
        DD 488/568
 
        LP-590
 
        Detector one    ( for detection of Texas Red )
 
 
 
        Our group is interested in co-localisation of fluorophores within
intercellular compartments. The question which has entered our
thoughts is with what confidence may one say that both fluorophores are truly
co-localised? That is to say , the two fluorophores are within X nm
of each other, given that the axial (x,y) resolution @ 488nm is say 200nm and
the z resolution is say 300 nm ( limited by mechanical considerations ? ).
this would then influence whether or not one may  say two antigens were in the
same compartment, given the size of secretory or endosomal compartments.
 
        I believe that there is a type of software package called  a
co-localisation package, and our Leica rep tells me that the multi colour
analysis package is used for this purpose, I was wondering if anyone has  used
this package? It is not currently installed on our microscope so I haven't been
able to investigate if in fact it would be useful for determining
co-localisation.
 
        Regarding the mowiol I have  taken on board the point about using the
matched Leica immersion oil possibly being enough, my cells are bound to a
coverslip by poly-l-lysine so effectively they are at the glass interface.
I find that  sometimes my mowiol sets nicely and sometimes not. Is there a general
preference for any particular mounting media?
 
 
        Your comments are much  appreciated.
 
        with special thanks to : Dr. Matthew Hannah, Heidelberg,
                                 Dr. Victor Fridrich, New York,
                                 Douglas Cromey, Arizona
                                 and Abdel-Salam Al-Drouby, Wales,
 
yours sincerely         Patrick Corley
                        [log in to unmask]
                        Dept. of Biochemistry
                        University College. Galway Ireland