Re: [EXTERNAL EMAIL] CONFOCALMICROSCOPY Digest - 11 Dec 2018 to 12 Dec 2018 (#2018-273)

[Jared K. Buks]

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I use Opal all the time. Look for the new kit which swaps 540 and 650 for 480 and 780. Plus know that the group was sold to Akoya Biosciences.

If you want more info let me know.

Jared Burks
MDACC

Sent from my iPhone

> On Dec 13, 2018, at 12:02 AM, CONFOCALMICROSCOPY automatic digest system <[hidden email]> wrote:
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> Topics of the day:
>
>  1. Perkin Elmer OPAL 7 kit experiences (4)
>  2. ABRF 2019, San Antonio, TX. March 23-26 2019
>  3. Fwd: ECBO 2019 - Advances in Microscopic Imaging - 23-27 June 2019 Munich
>     - call for papers - deadline 16 January 2019
>
> ----------------------------------------------------------------------
>
> Date:    Wed, 12 Dec 2018 01:56:08 -0600
> From:    Dave Johnston <[hidden email]>
> Subject: Perkin Elmer OPAL 7 kit experiences
>
> *****
> 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, 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.
>
> ------------------------------
>
> Date:    Wed, 12 Dec 2018 12:01:40 +0000
> From:    "Cole, Richard W (HEALTH)" <[hidden email]>
> Subject: ABRF 2019, San Antonio, TX. March 23-26 2019
>
> *****
> 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.
> *****
>
> ABRF 2019- Imaging Track
>
> Three days of world-renowned speakers and hot topics in light microscopy in sunny San Antonio, TX. March 23-26 2019.  Take advantage of the collegial environment to network and share ideas about instrumentation, reagents, methods and the challenges of running a shared resource. Bring your sunglasses and come see the bright future of imaging.
> Sessions include:  Open-Mic (submit your abstract for a chance to give a talk and get a FREE REGISTRATION)
>
> Additional sessions:
> Super-resolution image analysis
> Super-resolution- alternatives
> Imaging flow cytometry
> Spectroscopy
> Particle tracking
> Light microscopy careers- academic & industry
> Light microscopy research group (LMRG)
>
> More info, please see: https://conf.abrf.org/
>
>
> Richard Cole
> Research Scientist V
> Director: Advanced Light Microscopy & Image Analysis Core
> Wadsworth Center
>  
> Research Assistant Professor
> Dept. of Biomedical Sciences
> School of Public Health State University of New York
>
> 120 New Scotland Avenue, Albany N.Y. 12208
> 518-474-7048 Phone
> 518-408-1730 Fax
>
> ------------------------------
>
> Date:    Wed, 12 Dec 2018 07:40:52 -0500
> From:    George McNamara <[hidden email]>
> Subject: Re: Perkin Elmer OPAL 7 kit experiences
>
> *****
> 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,
>
> 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:
>> *****
>> 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, 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.
>
> ------------------------------
>
> Date:    Wed, 12 Dec 2018 14:13:09 +0100
> From:    "Debora Olivier (Keller)" <[hidden email]>
> Subject: Fwd: ECBO 2019 - Advances in Microscopic Imaging - 23-27 June 2019 Munich - call for papers - deadline 16 January 2019
>
> *****
> 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.
> *****
>
> FYI:
>
> *(please share the information with potentially interested colleagues or
> students)*
>
>
>
> Call for Papers
>
> SPIE / OSA EUROPEAN CONFERENCES ON BIOMEDICAL OPTICS
>
> 23–27 June 2019, ICM—International Congress Center, Munich, Germany
>
> http://spie.org/conferences-and-exhibitions/european-conferences-on-biomedical-optics
>
> Submit abstracts by 16 January 2019
>
>
>
> Advances in Microscopic Imaging (EB103)
>
> *Conference Chairs: *Emmanuel Beaurepaire, Ecole Polytechnique
> (France); Francesco
> Saverio Pavone, LENS - Lab. Europeo di Spettroscopie Non-Lineari (Italy)
>
> *Program Committee: *Paul J. Campagnola, Univ. of Wisconsin-Madison
> (USA); Laurent
> Cognet, Institut d’Optique, Université de Bordeaux (France); Vincent R.
> Daria, The Australian National Univ. (Australia); Valentina Emiliani, Lab.
> de neurophysiologie et nouvelles microscopies (France); Paul M. W.
> French, Imperial
> College London (United Kingdom); Irene Georgakoudi, Tufts Univ. (USA); Rainer
> Heintzmann, Institut für Photonische Technologien e.V. (Germany); Jan
> Huisken, Max-Planck-Institut für molekulare Zellbiologie und Genetik
> (Germany); U. Valentin Nägerl, Univ. de Bordeaux (France); Jerome Mertz, Boston
> Univ. (USA); Nozomi Nishimura, Cornell Univ. (USA); Dan Oron, Weizmann
> Institute of Science (Israel); Shy Shoham, Technion-Israel Institute of
> Technology (Israel); Peter T. C. So, Massachusetts Institute of Technology
> (USA); Vinod Subramaniam, Univ. Twente (Netherlands); Ivo Vanzetta,
> Aix-Marseille
> Univ. (France); Alipasha Vaziri, Rockefeller Univ. (USA)
>
>
>
> This conference will explore the rapidly developing field of microscopic
> imaging and applications, with approaches including multidimensional
> microscopy, light-sheet-based approaches, super-resolution microscopies,
> multiphoton imaging, and photomanipulation. Consideration will be given to
> the characteristics of the overall system design, as well as to contrast,
> image formation, image recording, and digital methods of producing and
> displaying the resulting reconstruction. Recent innovations in
> multi-dimensional microscopy have an important impact on the biological and
> medical fields ranging from cellular and developmental biology to
> neurosciences. We hope that the broad range of relevant topics presented at
> this conference will encourage the interaction among physicists, optical
> engineers, computer image analysts, and biologists.
>
> Papers are invited on all areas of development and application of novel
> optical microscopies including, but not limited to, the following and
> related areas:
>
> • super-resolved optical imaging (e.g. PALM/STORM, SIM, STED)
>
> • fast volumetric imaging approaches (e.g. SPIM, DSLM, ultramicroscopy)
>
> • multiphoton microscopy, SHG, THG, CARS, SRS, FWM imaging
>
> • adaptive optics, spatial and temporal control of the excitation
>
> • single molecule microscopy and microanalysis
>
> • phase-, holographic-, absorption-, polarization-based microscopy
>
> • spectroscopic analysis in microscopy
>
> • image contrast enhancement approaches such as near field surface effects
>
> • FRET, FLIM, fluorescence correlation spectroscopy
>
> • applications to cell biology, developmental biology, animal models
>
> • in-vivo tissue microscopy
>
> • optogenetics; instrumentation, reagents and applications
>
> • new contrast agents and reporters of tissue structure and function
>
> • ultra-microscopy / light sheet imaging of optically cleared brain
>
> • fast volumetric imaging approaches for neuro-microscopy
>
> • hybrid and multimodality approaches to neuroimaging
>
> • functional microscopy.
>
>
>
> *This conference will include a workshop on *Quantitative Phase
> Imaging, Workshop
> Chair: Gabriel Popescu, Univ. of Illinois at Urbana-Champaign, USA and
> Pietro Ferraro, CNR-ISASI, Italy
>
> ------------------------------
>
> Date:    Wed, 12 Dec 2018 17:01:48 +0000
> From:    "[hidden email]" <[hidden email]>
> Subject: Re: Perkin Elmer OPAL 7 kit experiences
>
> *****
> 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.
>
> ------------------------------
>
> Date:    Wed, 12 Dec 2018 17:15:43 +0000
> From:    "Giang, William" <[hidden email]>
> Subject: Re: Perkin Elmer OPAL 7 kit experiences
>
> *****
> 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 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
>
> ________________________________
> From: Confocal Microscopy List <[hidden email]> on behalf of [hidden email] <[hidden email]>
> Sent: Wednesday, December 12, 2018 12:01 PM
> To: [hidden email]
> Subject: Re: Perkin Elmer OPAL 7 kit experiences
>
> External Sender. Use caution with links and attachments.
> For more information:  http://it.emory.edu/external
>
>
>
> *****
> 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.
>
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> End of CONFOCALMICROSCOPY Digest - 11 Dec 2018 to 12 Dec 2018 (#2018-273)
> *************************************************************************

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