Perkin Elmer OPAL 7 kit experiences

[daj1u06]

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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.

[George McNamara]

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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-fp1488001kt

Spectral 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.

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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
-----------------------------------------------------------------
Max Planck Florida Institute for Neuroscience
1 Max Planck Way
Jupiter, FL 33458
-----------------------------------------------------------------

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Dave Johnston
Sent: Mittwoch, 12. Dezember 2018 02:56
To: [hidden email]
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.

[Giang, William]

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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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Subject: Re: Perkin Elmer OPAL 7 kit experiences

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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
-----------------------------------------------------------------
Max Planck Florida Institute for Neuroscience
1 Max Planck Way
Jupiter, FL 33458
-----------------------------------------------------------------

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Dave Johnston
Sent: Mittwoch, 12. Dezember 2018 02:56
To: [hidden email]
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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[Steffen Dietzel]

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oh, wow, now we have a vendor that is using some value in the
neighborhood of the actual peak of the *emission* to name dyes, just to
add to the general confusion.

Is there any reason to prefer the Opal fluors over those of vendors that
freely publish the spectra of their dyes? Cost, maybe? From the maxima
of the emission/excitation (not easily found at
http://www.perkinelmer.de/lab-products-and-services/application-support-knowledgebase/opal/opal-multiplex-ihc-assay.html),
it does not seem to be something that would contain properties not
available elsewhere.

For those of you who have seen the spectra, do they look comparable to
other dyes, concerning e.g. spectral width, or is there something that
the vendor wants to hide?

Steffen


Am 12.12.2018 um 18:01 schrieb [hidden email]:

> *****
> 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 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
> -----------------------------------------------------------------
> Max Planck Florida Institute for Neuroscience
> 1 Max Planck Way
> Jupiter, FL 33458
> -----------------------------------------------------------------
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On Behalf Of Dave Johnston
> Sent: Mittwoch, 12. Dezember 2018 02:56
> To: [hidden email]
> Subject: Perkin Elmer OPAL 7 kit experiences
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&amp;data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7Ccbf93e7c3f7644d8e56508d660075471%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636801981952636848&amp;sdata=yaXvYNL%2BkzF6clei2aoCXxoL5w%2FAM88sga7JzckIoYw%3D&amp;reserved=0
> Post images on https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&amp;data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7Ccbf93e7c3f7644d8e56508d660075471%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636801981952636848&amp;sdata=c5jzg%2FUG9jwv8neteVtSWzu4IvOc731U1qbi0aFExhM%3D&amp;reserved=0 and include the link in your posting.
> *****
>
> 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.

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Biomedical Center (BMC)
Head of the Core Facility Bioimaging

Großhaderner Straße 9
D-82152 Planegg-Martinsried
Germany

http://www.bioimaging.bmc.med.uni-muenchen.de

[Douglas Richardson]

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Hi Steffen,

I had some chats with Perkin when they were developing these years ago and
they said they were doing extensive optimization to minimize FRET and
quenching between the dyes.  That said, we routinely do 6-8plex with
standard alexa and atto dyes and I've never noticed these effects to impact
the imaging to a great degree. The bigger struggle is getting somewhat
equivalent emission with limited laser lines.

-Doug

On Thu, Dec 13, 2018 at 11:25 AM Steffen Dietzel <[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.
> *****
>
> oh, wow, now we have a vendor that is using some value in the
> neighborhood of the actual peak of the *emission* to name dyes, just to
> add to the general confusion.
>
> Is there any reason to prefer the Opal fluors over those of vendors that
> freely publish the spectra of their dyes? Cost, maybe? From the maxima
> of the emission/excitation (not easily found at
>
> http://www.perkinelmer.de/lab-products-and-services/application-support-knowledgebase/opal/opal-multiplex-ihc-assay.html),
>
> it does not seem to be something that would contain properties not
> available elsewhere.
>
> For those of you who have seen the spectra, do they look comparable to
> other dyes, concerning e.g. spectral width, or is there something that
> the vendor wants to hide?
>
> Steffen
>
>
> Am 12.12.2018 um 18:01 schrieb [hidden email]:
> > *****
> > 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 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
> > -----------------------------------------------------------------
> > Max Planck Florida Institute for Neuroscience
> > 1 Max Planck Way
> > Jupiter, FL 33458
> > -----------------------------------------------------------------
> >
> > -----Original Message-----
> > From: Confocal Microscopy List <[hidden email]> On
> Behalf Of Dave Johnston
> > Sent: Mittwoch, 12. Dezember 2018 02:56
> > To: [hidden email]
> > Subject: Perkin Elmer OPAL 7 kit experiences
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> >
> https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&amp;data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7Ccbf93e7c3f7644d8e56508d660075471%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636801981952636848&amp;sdata=yaXvYNL%2BkzF6clei2aoCXxoL5w%2FAM88sga7JzckIoYw%3D&amp;reserved=0
> > Post images on
> https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&amp;data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7Ccbf93e7c3f7644d8e56508d660075471%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636801981952636848&amp;sdata=c5jzg%2FUG9jwv8neteVtSWzu4IvOc731U1qbi0aFExhM%3D&amp;reserved=0
> and include the link in your posting.
> > *****
> >
> > 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.
>
> --
> ------------------------------------------------------------
> Steffen Dietzel, PD Dr. rer. nat
> Ludwig-Maximilians-Universität München
> Biomedical Center (BMC)
> Head of the Core Facility Bioimaging
>
> Großhaderner Straße 9
> D-82152 Planegg-Martinsried
> Germany
>
> http://www.bioimaging.bmc.med.uni-muenchen.de
>

[daj1u06]

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*****

Thanks to everyone who has responded. The reason that the user wants to go with OPAL dyes rather than "home brew"is because this is for a large clinical trial, with tissues to be stained on an automated stainer and so they were wanting to use a validated, self contained option designed for the machine. If anyone is interested in the partial curves we have calcumated from spectral scanning opal520/570/650/690, let me know and I can send over the leica spectral files. There is quite a variation in curve width and shoulder size from the 4 we have looked at.

DAJ.