On Thu, 14 Sep 1995 23:22:57 +0000, PATRICK CORLEY BIO DEPT wrote:
 
>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
 
HI,
I have also a Leica TCS 4d and I'm using the Multi-Color analysis package.
I think it's very useful for co-localization analysis; it makes a binary
combined image of the two single images (green and red) and a
cytofluorogram that represent the % of colocalization. I think that this
software is useful and easy to see the colocalization areas and to correct
the optical crosstalk. You can mark the colocalization areas (or others)
and whatch them in a special look-up table. I have only some problems to
understand the quantification of marked areas. I think that quantification
analysis is not very fine but Leica told me that they're working in that.
 
 
Susanna Castel
 
 
UNITAT DE MICROSCOPIA CONFOCAL
SERVEIS CIENTIFICO-TECNICS.UNIVERSITAT DE BARCELONA
LLUIS SOLE I SABARIS, 1-3 08028 BARCELONA, SPAIN
Tel. 34 3 402 13 52  Fax. 34 3 402 13 98
E-mail: [log in to unmask]