Dear Confocal Tutors,

        I apologize for bringing up a subject that was dealt some time ago, but I
keep finding trouble with it. As you can infer for the subject heading, I
am one of the sufferers of having only one laser in our confocal microcope
(Meridian) and I am ambitious enough to want to do multiple labeling
experiments. Currently, I usually work with the Oregon green conjugates
(and propidium iodide-type dyes for DNA visualization). But I would also
like to label two different cellular components besides the nuclei. I need
some dye with intermediate spectral characteristics between OrGreen and PI.
I tried tetramethylrhodamine (TMR)antibody conjugates with no success. My
selection of it was based on the fact that, as stated in the Molecular
Probes Handbook (page 30, Fig 1.9), protein conjugation with TMR originates
an additional absorption peak at about 520nm. I supposed that I could
efficiently excite it using the Ar 514nm line. I could not, even at the
highest laser power. The images were faint and blurry (it was not the prep,
since the label with OrGreen worked beautifully on the same prep).
        The spectral characteristics of carboxyrhodamine could sound suitable, but
from what I have read, they don't yield good protein conjugates (I need an
antibody or an avidin compound) and in fact these are not in the Mol Probes
product list.

        Do you have any suggestions? (not buying another laser, of course! unless
you want to donate it!  :)

        Thank you very much


                                        Susi