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Giang, William on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Perkin-Elmer-OPAL-7-kit-experiences-tp7588991p7588996.html
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Hi Nicolai,
I've been using a web tool (
https://automeris.io/WebPlotDigitizer/ ) to extract spectral data. I hope you find it as useful as I have.
Thanks,
Will
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Hi Dave,
one of our confocal users here has been tinkering around with Perkin Elmer's OPAL kits, testing various multicolor labeling schemes. At our request, Perkin Elmer sent us spectral data on the OPAL 7 dyes, albeit only as a png image file. For our purposes this was good enough, although proper data would have been a lot more useful. I'll send you the image of the spectra, if that would be of use to you (just shoot me an email).
In general, you will need to do quite some spectral unmixing to get reasonable data when using some of the 'spectrally close' dyes. Any pre-separation of the dyes with excitation and detection multiplexing (such as was suggested with the 488nm & 514nm excitation for the 520 & 540 dyes, respectively) will go a long way towards acquiring useful data without incurring horrendous spectral uncertainty. But if you don't need all 7 channels, best avoid the close ones entirely, if you have that option.
Hope it helps!
Nicolai Urban
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Sent: Mittwoch, 12. Dezember 2018 02:56
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Subject: Perkin Elmer OPAL 7 kit experiences
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Hi All, I was wondering if anyone has had experience of using the Perkin Elmer & probe OPAL kit with confocal, especially with Leica spectral detection systems. With a fair amount of workup we have users using the OPAL 4 kit reliably but the OPAL 7 seems to provide significant extra challenges, particularly the very close overlap of the first 2 probes ("520" and "540" (actually 525 and 536 peaks) and the fact that no spectra seem to be available - the PE imaging systems would appear to rely on spectral unmixing using propriatory data. We have generated partial emision spectra for the 4 colour kit by lambda scanning but those likely wouldn't be good enough for unmixing purposes, which we would rather not have to rely on, and don't have a means of generating excitation spectra. Our best suggestion to date is to use the 4 colour kit on 2 consecutive serial sections. Any other suggestions and thoughts gratefully received.
Dave Johnston, Biomedical Imaging Unit, Southampton.
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