CONFOCALMICROSCOPY Archives

September 1993

CONFOCALMICROSCOPY@LISTS.UMN.EDU

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Subject:
From:
"David K. Hanzel" <[log in to unmask]>
Reply To:
Confocal Microscopy List <[log in to unmask]>
Date:
Thu, 23 Sep 1993 16:12:23 -0700
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Just to add my two cents:
        Martin Wessendorf is quite right that "Quantum Red" is
NOT the anwser to our dreams.
        1. the conjugated phycoerythrin seems only slightly less
sensitive to bleaching than phycoerythrin alone.
        2. the phycoerythrin emission is right on top of your green
dye. [remember Quantum Red was developed for FACS]
 
        I have to differ on the concept that "there is no way to do
SPECIFIC two-color confocal with a single laser line", there are several
ways:
        1.  Two dyes with similar excitation and vastly different emission
e.g. FITC and Propidium Iodide, excite with 488 collect FITC between 515-545nm
and collect PI >620nm
        2.  Despite comments to the contrary FITC and TexasRed can be a very
rewarding combination to work with;
excite with either 488+514 or if the FITC is sufficiently bright 514 alone,
collect FITC between 525-555 [note longer than above to reject the 514 line]
collect TexasRed >610nm or >625nm [less than 2% of the FITC crosses over]
Certainly this is not a very sensitive mode for either dye but it provided
sufficient rejection of cross-talk to allow for the most critical applications.
happy scanning
david hanzel

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