On May 15, 10:32am, immunogenetics wrote:
> Subject: None
> To all confocal users
>
> Our institute has recently purchased an Optiscan Personal Confocal System
> model F900e.  The laser is a Argon Ion Laser with two laser lines 488 nm and
> 514 nm (It is also capable of running 50/50).  Two channels can be run
> simultaneously with a splitter which allows 510-550 nm to channel 1 and above
> 550 nm to channel 2.  Channel 1 can be filtered with two long-pass filters at
> 515 or 530 nm and Channel 2 can be filtered with two long-pass filters at 550
> or 590 nm.
>
> I would like to do two colour fluorescence looking at the co-localisation of
> two proteins.  I was wondering if anyone has a similar confocal setup and
what
> fluorescent dye combinations are appropriate for the above filter sets (with
> minimal bleed through).  The fluorescent dyes need to be conjugated to
> antibodies.
>
You should try Texas Red (sulforhodamine 101) and FITC. The emission peak of
TR is about 615nm . FITC has lots of signal between 550 and 600nm. So, if you
only have two channels, try splitting the channels at about 600nm.

On my web site I have a descriprion of a filter set I installed in a Biorad
MRC1024. It has three channels, but the filters I use for TR should work for
you. Can you replace your filters or add filters?

Also, if you avoid saturation, you can compensate for FITC bleed through in
your current setup by determining the bleedthrough with a control sample and
then subtracting the weighted FITC signal from the combined signal. In the FITC
channel, use a bandpass filter with sharp skirts so that you dont get rhodamine
bleedthrough in the FITC channel.

Finally, if your FITC signal is really strong, you can use 514nm to excite your
sample. That increases the rhodamine excitation, and decreases the FITC
excitation.

--aryeh

--
Aryeh Weiss                          | email: [log in to unmask]
Department of Electronics            | URL:   http://optics.jct.ac.il/~aryeh
Jerusalem College of Technology      | phone: 972-2-6751146
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