Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear all,

I have a question regarding spectral unmixing using the LSM 510 Meta.  I
have these cells that I've stained for eGFP, CM-DiI, PI and Alexa-Fluor
647.   AF647  localized to the membrane, PI the nucleus, eGFP would be the
whole cell, and CM-DiI the cytoplasm.

 I am able to obtain individual emission fingerprint for each of the dyes.
however, I wasn't able to obtain the image for the four colors combined.The
image that I've obtained looks exactly the same in all four channel, ie.
AF647 can be detected in the whole cell, which isn't correct.

Interestingly, if the emission fingerprint was obtained from the
combination slide (eGFP, DiI, PI and AF 647), it seems to work.  What's
happening here?

Regards,
Ivy