Posted by
George McNamara on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Perkin-Elmer-OPAL-7-kit-experiences-tp7588991p7588993.html
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Hi Dave,
If you have 488 and 514 nm laser lines you can better discriminate
between Opal 520 and 540. Chris Baumann published in 1998 (J Histochem
Cytochem 46: 1073-1076, DOI: 10.1177/002215549804600911) with a Leica
SP1 separation of EGFP and EYFP, similar separation to your Opal's.
The Leica SP series is usually best operated in 'emission band' mode,
with sequential scan tracks, rather than 'routinely' spectral scanning.
Sequential bands imaging is a lot faster than scanning spectra. If you
have HyD(s), I strongly recommend using them in photon counting mode (I
usually recommend users start with 10 line accumulation on our SP8 with
two 2nd gen HyD's ... acquiring both together can be useful; acquiring
'autofluorescence dominant' data, i.e. 440nm excitation -> 450-490nm,
can also help). For those with Leica SP8's see also p.s..
http://www.perkinelmer.com/product/opal-570-reagent-pack-fp1488001ktSpectral DAPI
494/525 nm Opal 520
523/536 nm Opal 540
550/570 nm Opal 570
588/616 nm Opal 620
627/650 nm Opal 650
676/694 nm Opal 690
I encourage careful choice of fluorophores -> targets, so that your
specimens do not overlap hard-to-separate.
Acquiring and posting Leica SP confocal spectral emission scanning of
each of the Opal's (single plex) may be of broad interest to the
community - especially if graphed along with the older tyramide reagents
on PerkinElmer's web page, ex. fluorescein, Cy3, Cy3.5, Cy5.
With respect to PerkinElmer -- Opal (and Vectra product lines) are now
at Akoya Biosciences -- I recommend comparing their "strip the
antibodies&HRP after each cycle" to using PeroxAbolish (Biocare Medical)
to kill the HRP. The latter approach is likely to leave a lot more
tyramide on the specimen. See Takahashi et al 2012 Cell Transplant 21:
113-125, PMID 21929847, doi: 10.3727/096368911X586747 (some data
acquired on the Zeiss LSM510 (non META) I managed at U Miami).\
Molecular Probes has had a license for tyramide for a long time and lots
of Alexa Fluor tyramide conjugates. You could substitute any Opal with
some Alexa Fluor tyramide (near or very different spectrum, depending on
your experiment needs and instrumentation).
sincerely,
George
p.s. Leica HyD detectors ... could someone with both 2nd gen HyD(s)
(original HyD on SP8) and 3rd gen SMD HyD(s) (introduced earlier in
2018) please send me a comparison of photon counting rates for the two
detector types. Yes, I hosted a Leica SP8 FALCON demo a couple of months
ago, but timing worked out that I was unable to do the comparison here.
thanks.
On 12/12/2018 2:56 AM, Dave Johnston wrote:
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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.