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