Dear All,

In our Lab we have got the CLSM Olympus FV500 with three laser light source
(Ar 488nm, HeNe-G 543nm and HeNe-R 633nm emission) via three integrated
laser ports.
When a fluorescence dye is chosen the laser and light path setting are done
automatically.
In our experiment we are using seconds antibodies conjugated with Cy.5,
TRICT and FITC. DNA in nucleus we label with PI. When we use one channel
(one dye i.e. Cy.5 or PI) scan is O.K. But when two channels are using (Cy.5
and PI) on the scan with Cy.5 the very intense and shine "shadows" of
nucleus appear. When we use second antibodies conjugate with FITC this
problem does not exist.
Maybe somebody from the list could help me to resolve this problem...
Thanks in advance