FRET with YPET and mKO?

[Adrian Smith-6]

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Hi all,

We have a user who has setup a FRET pair using YPet and mOrange.

We are having some trouble getting definitive results with it with the
fitler sets we have available. Looking at the spectra it seems like this is
far from an ideal pair (eg lots of overlap in donor and acceptor excitation
spectra so it is hard to excite donor with also directly exciting acceptor)?

Does anyone have experience with this pair and could point us in the right
direction?

Regards,

Adrian Smith
Centenary Institute, Sydney AU

[Pamela Young]

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Hey Adrian!

I¹m really interested in talking with you about this.  I have a user with
similar crosstalk issues.  We are using 2P excitation and doing FRET-FLIM.
 No matter how hard we try to block out any acceptor fluorescence, we can
clearly see a three component decay that very nicely picks out the
lifetime of the donor, the FRETed donor, and the acceptor (though only
about 1% contribution).

Thanks,
Pam

Dr Pamela A. Young
 | Light and Optical Microscopist
Australian Centre for Microscopy & Microanalysis

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On 28/03/2014 2:23 pm, "Adrian Smith" <[hidden email]>
wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>Post images on http://www.imgur.com and include the link in your posting.
>*****
>
>Hi all,
>
>We have a user who has setup a FRET pair using YPet and mOrange.
>
>We are having some trouble getting definitive results with it with the
>fitler sets we have available. Looking at the spectra it seems like this
>is
>far from an ideal pair (eg lots of overlap in donor and acceptor
>excitation
>spectra so it is hard to excite donor with also directly exciting
>acceptor)?
>
>Does anyone have experience with this pair and could point us in the right
>direction?
>
>Regards,
>
>Adrian Smith
>Centenary Institute, Sydney AU