Multiplex

20210512W (start date) note: Multiplex Microscopy

Some alternative terms: fluorescence image cytometry, spatialomics, spatial genomics (usually single molecule RNA, can be DNA), spatial proteomics.

Goal: maximize the amount of useful information, in as few stain-image-destain cycles as possible (ideally N=1), from each cell or 2D tissue section (most common(, or 3D tissue or organoid or whole-mount (organ, embryo, larva, adult animal [or plant]). This page mentions a few publications (by others) I think are useful (ex: Ce3D and IBEX from ron Germain's lab). 

I have a long standing interest in multiplex -- aka multi-Probe -- fluorescence microscopy, having started "MPMicro" for a lecture at the Nikon AQLM course at MBL in 5/1995 (26 years ago on starting this page 5/2021). MPMicro is available online at https://works.bepress.com/gmcnamara/2 (please do not hit the print button).

Below is a summary I posted on linkedin and add some more text here -- the linkedin page has some graphics of spectra (we have hit a limit on our web hosting site * budget wrt graphics here), see also https://searchlight.semrock.com and other spectra search web sites, or if you prefer Microsoft Excel data, my (2006) PubSpectra data set is at https://works.bepress.com/gmcnamara/9

GM Suggestion = Hypothesis: Could Multiplex different brands

GM Suggestion = Hypothesis: Could Multiplex different brands using the same excitation and emission -- especially if you have the right microscope, such as a spectral fluorescence lifetime imager (you could buy the image core a fully loaded Leica STELLARIS 8 with 5 HyD S SiPMs inside, and 4 more on X1 port, for us to figure out - other brands&models possible), and right software. Easiest if the different markers are in different locations, such as different cells.

For example, may be able to multiplex ALL the BD Brilliant Ultraviolets (BUV's) with Bio-Rad's StarBright UltraViolets (SBUVs) and add in fluorescent quantum dots (QDots). Repear with Brilliant Biolets, SBVs, etc. 

https://www.linkedin.com/pulse/superbright-tandems-outed-thermofisher-now-has-plus-series-mcnamara/
Brilliant Ultraviolets, Violets, Blues, Yellow-Green(s) ... BD Biosciences (some BVs available BioLegend, Jackson Immunores). ... See below for possible Alexa Fluor acceptors of BV's. 
Super Bright   ... ThermoFisher
Super Nova    ... Beckman Coulter
StarBright      ... Bio-Rad ... see below ... StarBright Ultraviolet (340nm excitation max, should excite at 368nm wrt FISHscope], StarBright Violet, StarBright Blue
Above are, or likely, "polymer dyes", based on Nobel Prize in Chemistry 2000, http://sirigen.com/sirigen_technology.html (Sirigen was acquired by BD Biosciences - more on this below).

How similar are BV's and SuperNova's? ... patent case(s) may provide answer https://www.maxval.com/blog/becton-dickinson-filed-a-case-against-beckman-coulter-over-alleged-patent-infringement/  ... not that this will affect customers too much, though some patent lawyers and law firm(s) likely to be the big financial winners. 

Brilliants were invented by Sirigen (acquired by BD Biosciences), which was cofounded by 2000 Chemistry Nobel laureates to commercialize their discovery of electron carrying polymers. 
--
some recent fluorophore families based on other technologies:

Nova Fluor (NovaFluor) ... Phitonex, acquired by ThermoFisher ... based on what I refer to as DNA origami (Duke U. spin out). More on NovaFluor's below (19plex 202111 - 17plex spectra, plus 2 deliberately dimmer variants).

CPN                   ... StreamBio UK ... conjugated polymer nanoparticles https://www.streambio.co.uk   (may need to wait for LinkBright product line to get into Sigma-Aldrich web site to avoid crazy shipping charges from U.K.). More on CPNs below. 

StabiLux Biosciences - https://stabiluxbiosciences.com/ - NovoLux ... lacks details on the technology.

==> I am happy to add other academics and vendors weblinks here ... please contact me on linkedin and/or my JHMI email (see "About Us"). Note: I do not have any commercial interest in any company involved in fluorescence. 

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more options ... Alphathera (4/2022) announced new product(s) for their oYo-Link lableing system with the ability to install any of 10 peptide epitope tags onto IgG antibody heavy chain, see 

https://alphathera.com/news/alphathera-launches-the-oyo-link-epitope-tag-for-enhanced-multiplexing-capability 

https://alphathera.com/epitope-tag/p/epitope-tag 

Specifications table (molecular weight in parentheses, kD):

His12 Tag (8.7)

DYKDDDDK Tag (12.2)

V5 Tag (13.4)

S Tag (14.4)

VSVg Tag (13.2)

NWS Tag (12.7)

S1 Tag (12.5)

AU1 Tag (11.7)

AU5 Tag (11.6)

HSV1 Tag (13.1)

See https://alphathera.com/all-products  for all their products - all make covalent bond, typically ProteinG-IgG heavy chain (UV activated site specific crosslink). 

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GM 20211130 - 

Philosophies to Dye for ...

I came across Alexa Fluor 610-X as being the brightest small molecule fluorescent dye of ~40 tested by Maillard et al 2020 Universal quenching of common fluorescent probes by water and alcohols (yes, you read that correctly: water quenches fluorophores, D2O does not). Chem Sci 12: 1352. https://pubs.rsc.org/en/content/articlelanding/2021/sc/d0sc05431c (also discussed below). 

The Brilliants, SuperBrights, etc, summarized above, andfurther discussed below, are probably less susceptible to the H2O quenching phenomenon.

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20220210U: Brief note - many ways to improve fluorophore behavior. One, that fits nicely with recombinant proteins (genes) with 6His tag added, is to use Ni+ : NTA, at optimal distance, to quench fluorophore triplet state (dark state) while permittign singlet excited state to emit photon (fluorescence). Example: Alexa Fluor 647 without protection 40,000 photons, trisNTA-AF647 ~700,000 photons (almost 20x more photons - see fig2F below). Nice paper:

Glembockyte V, ... Costa, Tampe 2018 Tris-N-Nitrilotriacetic Acid Fluorophore as a Self-Healing Dye for Single-Molecule Fluorescence Imaging. J Am Chem Soc 140: 11006−11012.  DOI: 10.1021/jacs.8b04681. NTA-linker-fluorophore

Glembockyte 2018 JACS fig2F - tris-NTA-fluorophore

 

20211029 update: "Best and Brightest" note

I am big on BV421 and other modern fluorophores because some of these greatly improve on fluorescence Brightness, where Brightness = E.c. * QY / 1000 (Extinction coefficient, Quantum yield),  ex.BV421 B=1500, fluorescein = 81. this is like getting paid $1500/hr vs $81/hr (to complete the analogy, if you get paid $100/hr as a round number and work 2,080 hours per year, this would be $208,000 annual salary, $81*2250 = $168,480 vs $1500*2250 = $3,120,000 ..which would you prefer?). Yet, many biologists still use fluorescent (ex. "FITC conjugated" secondary antibody, or Alexa Fluor 488 Brightness = 67 (pH insensitive and more photostable than fluorescein, B=67 from  https://www.geomcnamara.com/fluorophore-table ).

I came across Maillard et al 2020, who compared 42 fluorophores. I was amazeed to see that Alexa Fluor 610-X ("AF610X") a fluorophore I had never paid attention to, was 2.4x brighter than Alexa Fluor 594 (which spectrally, and maybe chemically, is similar to Texas Red, one of the first fluorophores the founders of Molecular Probes developed ... they eventually sold to Invitrogen, which was acquired by ThermoFisher). Again: would you like to get paid 2.4x more than you do now?   ... FYI (I believe) ThermoFisher/Molecular Probes "custom conjugation" group can put AF610X on any antibody in their catalog, and sell to you at same price as "catalog" antibodies. I also note that some Dyomics ("DY") fluorophores had high Brightness values. I do not Brightness value is not everything: performance at various degrees of labeling (DoL) on antibody, photostability, lack of going into triplet excited state and/or other dark states (which are good for PALM/STORM precision localization microscopy), reactive oxygen species (often consequence of going into triplet states) play a  role in success. 

Maillard J et al 2021 Universal quenching of common fluorescent probes by water and alcohols. Chem. Sci. 12: 1352. https://pubs.rsc.org/en/content/articlelanding/2021/sc/d0sc05431c

Alexa Fluor 610-X highest Brightness B=121 in H2O (highest Brightness of the 42 dyes), over 2x that of Alexa Fluor 594, B=54 in H2O. ... Alexa Fluor 610-X in D2O is B=140, 

Maillard 2020 ChemSci - graph featuring Alexa Fluor 610-X AF610-X

see also   https://www.linkedin.com/pulse/maillard2020-chemsci-42-fluorophores-paper-values-george-mcnamara/

Another look at Maillard 2020 ChemSci data: two 'classes" of fluorescence lifetimes, long, 3.5-5.0 ns, and short, 1.0-2.0 ns. (all 42 fluorophores graphed, on brightest tabulated, since you should avoid "unbright" fluorophores - Excel sheet available from me). These are "in D2O" values -- the "in H2O" proportionally dimmer and shorter lifetimes (not shown, available in my Excel sheet based on data from their supplemental PDF). I note that X-axis of their figure panel "g" above and "my" Brightness vs Tau below are the same (Brigrness). ... Yes, the graph below does not have spectral info - so what? You cvan get the Excel file from me or figue it out for yourself. I suggest a kick-butt imaging system would be a Leica STELLARIS 8+++ (or how about ST9 !) ... STELLARIS FALCON with 8 HyD S (SiPMs) and one HyD R (NIR, for wavelengths range that "R" outperforms "S"), wgite light lase wit NKT Photonics UV-Extend (down to 350nm excitation), ALL the best ~4ns and best ~1ns fluorophores, plus the Brilliant Ultraviolets (BUV, ~350nm excitation hence UV-EXTEND), Brilliant Violets (~395 excitation, also UV-EXTEND), Brilliant Blues (only ~5), and best dyes below (tyramide signal amplification [TSA] or methyl-luminol (may be 10x better than tyramide) ... potentially 8plex or more chlorins (see NIRVana Sciences content on this page). --> say 40plex in one acquisition. I note Leica STELLARIS could be operated in "push-broom" ode, of line scan one axis, move stage in other axis, as big as specimen (ex. adult human brain slice or liver slice). 

Maillard 2020 Brightness vs Tau

20220504W same data as above (plus some Abberior dyes) with new graph using emission wavelength maxima - then data table below.

Fluorescent Dyes Performance (20220504W)


Maillard 2020 Dye      wavelength em max (nm)     Tau_s1 (H2O) (ns)     B(H2O)/40      Brightness (H2O)     [you can fix formatting by copying to Excel etc]
AF488 519 4.0 1.6 65
STAR GREEN 519 4.1 1.6 63
ATTO488 520 4.1 1.8 72
STAR 488 524 3.9 1.4 58
DY505 530 4.1 1.7 67
STAR 512 530 4.1 1.7 70
ATTO520 538 3.7 1.0 42
AF514 542 4.0 1.6 66
AF532 554 3.0 0.9 37
5CR6G 557 3.9 2.0 79
DY530 561 3.9 2.1 83
Cy3B 572 1.4 1.0 39
AF546 573 3.8 2.4 95
ATTO565 590 3.9 1.7 67
DY590 599 3.0 2.3 91
AF568 603 3.4 1.3 51
 STAR 580 607 3.5 1.9 77
STAR ORANGE 616 4.5 1.3 52
AF594 617 3.8 1.3 53
AF610X 623 3.7 3.0 121 Alexa Fluor 610-X
STAR 600 623 4.2 2.0 81
ATTO594 626 3.9 2.4 97
AF633 647 3.2 1.6 65
STAR 635P 651 3.3 2.7 108
ATTO633 651 3.3 1.8 72
STAR 635 655 2.8 2.4 97
STAR RED (KK114) 655 3.4 1.7 66
FLUX 640 (Abberior) 660 0.8 1.3 53
ATTO647N 664 3.7 2.4 96
Cy5 665 1.0 1.8 73
AF647 665 1.0 2.0 81
FLUX 647 666 1.1 1.8 70
DY649P1 676 1.1 2.3 93
ATTO655 679 1.9 0.9 35
FLUX 660 (Abberior) 680 0.9 1.0 40
Cy5.5 694 0.9 1.5 60
FLUX 680 (Abberior) 695 0.9 1.1 45
ATTO680 698 1.9 0.9 38
AF680 702 1.2 1.7 66
AF700 714 0.9 1.2 50
 

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Some deuterated pricing (April 20, 2022) 

https://www.sigmaaldrich.com/US/en/product/aldrich/151882

151882-10G ...      $28.50 ... 10 grams is ~10 mL

151882-100G ...  $152.00

151882-1KG ...  $1,320.00

for comparison (more viscous, a'la VectaShield)

https://www.sigmaaldrich.com/US/en/product/aldrich/447498 

Glycerol-d8 (fully deuterated)

447498-1G ... $159.00

gylcerol-d8

I suspect (but I'm no chemist) based on my understanding of Maillard et al 2020 ChemSci that only the OH's need to be replaced with OD's for deuterated glycerol to be effective -- that is to minimize quenching of fluorophores. Glycerol is traditionally used in fluorescence microscopy for both refractive index increase compared to water and to increase viscosity. The latter limits diffusion of O2, so usually best practice to avoid vortexing glycerol solutions (plus, vortexing, shaking, stirring would get air bubbles into the media, which would mess up image quality). 

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ThermoFisher has a brief table of some of the basic parameters of some of their fluorophores - but not AF610-X (Maillard 2020 supplemental pdf has fluorescent lifetimes in H2O and D2O for all 42 tested -- I generated an Excel table from their data and calculated Brightness values - on my linkedin page).

https://www.thermofisher.com/us/en/home/references/molecular-probes-the-handbook/tables/fluorescence-quantum-yields-and-lifetimes-for-alexa-fluor-dyes.html 

fluorescence lifetimes of same dye on different conjugates (example)

20220118U: please see publication and especially supplemental table for mre details on this.

Fluorophore (fluorescent dye) free in solution vs conjugates can have different fluorescence lifetimes ("Tau") and quantum yields (Q.Y.) -- and different linker lengths and "local environment" (such as very nearby guanine on oligonucleotide, or tryptophan adjacent to dye on proteins) - as nicely shown in the example below. GM notes that higher Tau and QY usually "go together".

Niehorster ... Sauer 2016 Multi-target spectrally resolved fluorescence lifetime imaging microscopy. Nat Meth 13: 257-262. https://www.nature.com/articles/nmeth.3740   doi:10.1038/nmeth.3740
ATTO488 
4.16ns free (dye in solution)
3.98ns EdU/DNA (in chromatin)
3.43ns Goat-anti-Rabbit antibodies ("GaR") (polyclonal) ("whole IgG", Sigma-Aldrich, 36098)
3.00ns Phalloidin (not explicit if bound to F-actin in cells - probably)
1.90ns anti-giantin (rabbit IgG, abcam ab24586)

GM note: ideally would use site specific conjugation, such as C-terminal amino acid of nanobody (12 kDa VHH only), or single site on a monoclonal antibody (mAb ~155 kDa, could be "cut down" with enzyme to ~50 kDa Fab), instead of "random lysines" on each IgG antibody molecule of a population of polyclonal antibodies (~155 kDa, lots of different amino acid sequences in population, and random lysines, such as degree of labeling DoL ~3 out of ~20 surface lysines on average).

???Missed opportunity by these authors???: FRET or its quenching variant (QRET) can be used to "tune" donor lifetime (moreFRET results in on average shorter donor lifetime) AND acceptor lifetime has interesting behavior (over statistics of many events counted) in that there is a delayed rise in acceptor lifetime when FRETting. That is FLIM-FRET of donor-acceptor, especially with defined site labeling (ex. COOH of nanobody) and linker length between conjugate and dye, and of linker (spacer) between donor and acceptor. Potential additional degree of freedom: conjugate-linker-donor-spacer-acceptor and conjugate-linker-acceptor-spacer-donor may behave differently even if linker and spacer are the same in each.

As noted above (Alexa Fluor 488 B=67) Brightness of many fluorophores are tabulated at https://www.geomcnamara.com/fluorophore-table and in our 2017 CPHG (open access) 

https://currentprotocols.onlinelibrary.wiley.com/doi/10.1002/cphg.42

Back to Dyomics (several DY dyes had high Brightness in Maillard 2020) ... their products https://dyomics.com/en/products  include several families of MetaStokes dyes. Many vendors do not include extinction coefficient (Ec) or quantum yield (QY) and/or "MegaStokes" aka "Long Stokes Shift" (LSS) dyes have odest or low quantum yields. If you want to use MegaStokes dyes to get the ability to excite more fluorophores with the same LED or laser line, either "just do it" (order the stuff and try it), or look at Brilliants, Nova Fluors, StreamBio CPNs and/or other similar modern fluorophores. 

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20220524Tue: Additional Long Stokes Shift dyes ("LSS", most are modestly longer Stokes shifts)

--> LSS facilitates multiplexing by using the same excitation wavelength for multiple fluorophores with different Stokes shifts [Stokes shift = emission maximum wavelength minus excitation maximum wavelength). This web page summarizes several other ways to get multiple fluorophores of different emission maxima -- and spectra -- from each excitation wavelength. These include (not limited to): Brilliants and similar fluorescent polymers (polymer with conventional flurophore attached), Stream.Bio CPNs, ThermoFisher Nova Fluors (acquired Phitonex, using DNA origami to position donor and acceptor fluorophores at precise distance and Kappa squared orientation for FRET).

--> Jiang et al compared azetidine rings ("square rings" from Luke Lavis lab) and other fluorophore modifications and (not surprisingly) found their new modification best.

Jiang G ... Yan L 2022 A synergistic strategy to develop photostable and bright dyes with long Stokes shift for nanoscopy. 
Nat. Communs 13:2264  https://doi.org/10.1038/s41467-022-29547-3
Name  λab(nm)      λem(nm)  Stokes shift/nm            φ          ε(M−1 cm−1)       ε × φ/1000    Δ(ε × φ) [YL vs parent]      Scaffold
YL578    578             634                  56                     0.74           89,000                66.4                    2                                     Rhodamine
RhB        553             580                  27                     0.31         105,000               32.4

11          548a            612a                64                    0.62a         51,000a               31.6                  2.5
R-2        518              546                  29                    0.21            60,000                12.6

12          577a            623a               46                    0.71a          83,300a               59.1                  3.2
R-3         552             581                 24                    0.18           103,000                18.5

13           406b          513b              117                   0.55b           32,400b               17.8                  5.2
R-4         372b          470b                98                   0.19b           18,000b                 3.4

14           464b          556b                92                   0.67b           26,200b               17.5                   8.1                                  Coumarin
R-5         430b          484b                54                   0.06b          32,200b                  2.2

15           439b          575b              136                   0.40b          25,100b                10.0                  8.0                                   Boranil
R-6         401b          462b                 61                   0.04b          31,300b                 1.3

aMeasured in PBS (25 mM, pH 7.4).
bMeasured in PBS (25 mM, pH 7.4) containing 20% EtOH. Δ(ɛ × φ) represents the ratio of the increased brightness.

 

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Brilliants were developed by Sirigen (acquired by BD Biosciences for $55M) using technology (and patents) developed out of work that won the Nobel Prize in Chemistry in 2000, see

http://sirigen.com/sirigen_technology.html

http://sirigen.com/microscopic.html 

see also BioLegend (acquired by PerkinElmer ... BioLegend licensed BV's from Sirigen, but do not have access to BUVs, BBs, BYGs) Brilliant Violets web pages

https://www.biolegend.com/en-us/brilliant-violet   web pages [About, Technology] stating BV421 has extinction coefficient 2,500,000 M-1cm-1, quantum yield 0.65, so Brightness (Ec*QY/1000) ~1500 vs fluorescein B=81

 

20211011Mon - GM table of POSSIBLE BV421 donor --> Alexa Fluor acceptors (I had previously identified BV570 as BV421 -> AF546, more candidates today - 480 and 510 are different polymers than BV421 (have different spectra ... which comes from different chemistry).

20220119W found nice history of Sirigen and Brilliants, and alternative possible fluorophores (some of which do not make spectral sense to me - and no idea where their Stokes shift math comes from, gven 421 - 407 = 14)

    L. Della Ciana, 2019 New Trends in Fluorescent Reporters in Biology in Springer Series of Fluorescence 19. Author affiliation: Cyanagen srl, Bologna, Italy (see for some products, https://www.cyanagen.com/search-for-applications/ ).  (page 335) Table 5 Optical properties of Brilliant Violet polymer dyes

DY682 and other Dy dyes are from Dyomics, ex https://dyomics.com/en/products/far-red-excitation/dy-682  (unfortunately not in Semrock SpectraViewer) 

BV donor to acceptor (candidates)
Brilliant Violet Alexa Fluor acceptor - GM hypothesis Della Ciana table 5 ex em Stokes Shift (Della Ciana)
BV421 base polymer (of all tandems below)   407 421 106
BV480 base polymer   - -  
BV510 base polymer   405 510 133
BV570 Alexa Fluor 546 Cy3 407 570 168
BV605 Alexa Fluor 568 Cy3.5 407 605 175
BV650 Alexa Fluor 633 Dy610 407 650 261
BV711 Alexa Fluor 700 Dy682 407 711 304
BV750 -   407 750  
BV785 Alexa Fluor 750 Dy752 407 786 379

Table note: I used https://searchlight.semrock.com    to match fluorophorer spectra - not exact matches: the emission peaks are very close, some of the emission tails have "bumps", some do not. 

If I am correct, one could use 405nm to excite ALL the Brilliant Violets, and "appropriate laser line(s)" to excite stand alone Alexa Fluor's as antibody conjugates (or tyramide signal amplification products), single molecule RNA FISH, DNA-PAINT, etc. Yes, the BV-->acceptor mighthave some signal on direct acceptor excitation, butcould be corrected by "spectral unmixing" (multichannel unmixing) vs the BV-acceptor.

If anyone finds better candidate(s) for ecceptors, please email me suggestions to update here.

I use Semrock Searchlight for spectral comparisons,   https://searchlight.semrock.com/#    (login required for full benefit of the spectral web site).

BioLegend fluorophore families

BioLegend fluorophore families

BioLegend 20211012U (plus updates) https://www.biolegend.com/en-us/fluorophore-families 
(congratulations - I hope - to BioLegend for being acquired by PerkinElmer for ~$5.25B, August 2021)

https://www.biolegend.com/en-us/spark-dyes 

Spark Violet     423          ex 405nm   ("423" added early 2022)
Spark Violet     538        ex 405nm
Spark Blue      550        ex 488nm, optional 405nm
Spark Blue      574
Spark YG        570        ex 555nm max
Spark YG        581        ex 561nm
Spark YG        593        ex 561nm
Spark NIR      685        ex 633nm (or 638nm, 640nm)

KIRAVIA Blue 520    ex 488nm ("distinct class of multimers with distinct chemistry" ... 8 kDa)

APC/Fire      750       ex 650nm (or 638nm, 640nm)
APC/Fire      810       ex 650nm (or 638nm, 640nm)
PE/Fire         640       ex 488nm or 565nm (modest to high 488-561nm)
PE/Fire         700       ex 488nm or 565nm (modest to high 488-561nm)
PE/Fire         810       ex 488nm or 565nm (modest to high 488-561nm)
GM note: APC and PE fluoorphores are typically used in flow cytometry, not microscopy, because "tend to photobleach quickly". Can get an image if very careful with settings and workflow, such as focus using transmitted light. Usually commercial PE is "R-PE" not B-PE. 

BioLegend also offers Brilliant Violets (BV###) - see above-- licensed from Sirigen before Sirigen was acquired by BD Biosciences (BD also offers Brilliant Ultraviolets BUV###, Brilliant Blues BB###, Brilliant Yellow-Green BYG###).

 

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11/2021   ThermoFisher NovaFluors ... ThermoFisher acquired Phitonex 12/2020.

Product info from https://www.thermofisher.com/us/en/home/life-science/cell-analysis/flow-cytometry/novafluor-dyes.html  - see web page for excitation and emission maxima.     I note there is potential to use fast FLIM (fast fluorescence lifetime imaging microscopy, examples: Leica STELLARIS 8, Becker&Hickl, PicoQuant, ISS) to distinguish the 610/30S from 610/70S and the 660/120S from 660/40S pairs -- that is, the brighter product (high #S) should have shorter donor lifetime, and dimmer product (lower #S) longer donor lifetime -- each shorter than the donor alone product NovaFluor Blue 510. . 

  1 NovaFluor Blue 510
  2 NovaFluor Blue 530
  3 NovaFluor Blue 555
  4 NovaFluor Blue 585
          NovaFluor Blue 610/30S (dimmer acceptor)
  6 NovaFluor Blue 610/70S      (brighter acceptor)
  7 NovaFluor Blue 660/120S    (dimmer acceptor)
          NovaFluor Blue 660/40S  (brighter acceptor)
  9 NovaFluor Red 660
10 NovaFluor Red 685
11 NovaFluor Red 700
12 NovaFluor Red 710
13 NovaFluor Yellow 570
14 NovaFluor Yellow 590
15 NovaFluor Yellow 610
16 NovaFluor Yellow 660
17 NovaFluor Yellow 690
18 NovaFluor Yellow 700
19 NovaFluor Yellow 730

(for clarity, TM trademarks removed from NovaFluor names). 

Spectra below from FluoroFinder (17plex, ignoring 2 dim) added 20220126W -- First Nova Color [Blue, Yellow, Red] are the "base dye", rest of color are tandem FRET donor (base dye) --> acceptor(s). The "phiton" technology is a spinout of a Duke University lab, using clever DNA structure (DNA origami) that looks like a "+" (four arms) with dye(s) positioned inside to protect from environment (re: O2 and other quenchers) and to space and orient dyes to each other to optimize tandems (ex: position dyes end to end or in parallel, with specific spacing, for dipoles to be oriented for optimal FRET (fluorescence resonance energy transfer). 100% FRET efficiency is an ideal way to make efficient use of dyes and spectra: 3 excitation wavelengths enabled 17 fluorophores shown below -- various molecules excite different wavelengths (due to donors) and emit at same wavelengths (enabling no moving parts in front of multiplex detectors). Also looks like the "base" Nova Yellow and Nova Red and tandemized to the base Nova Blue for 2 FRET molecules, and base Nova Yellow is tandemized with the base Nova Red for another tandem. 

FluoroFinder - Nova Fluors (NovaFluors) Phitonex ThermoFisher

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20211117W - more on StreamBio UK CPNs = conjugated nanoparticles ... potentially bright enough to see SINGLE molecules ... iron core to enable being pulled down by magnet and outer bright fluorescent shell.

https://www.streambio.co.uk

https://www.streambio.co.uk/wp-content/uploads/2020/07/CPN_Product-list_ver1.0_jun2020_v2.1_Updated_V2.pdf 

Product                         ex max / em max        GM notes - FV3000RS confocal and FISHscope have all necessary ex wavelengths
CPN 420 (Violet)                390 / 420            405nm laser or ~395nm LED
CPN 435 (Indigo)               390 / 435            405nm laser or ~395nm LED    
CPN 475 (Blue)                  390 / 475            405nm laser or ~395nm LED    
CPN 510 (Green)               450 / 510            ~440nm light source (FV3000RS laser usually off - user can turn on then off)
CPN 550 (Yellow)              470 / 550            ~488nm light source (laser or LED).
CPN 610 (Orange)            480 / 610            ~488nm light source (laser or LED).
CPN 680 (Red)                 400 / 680              405nm laser or ~395nm LED ... Long Stokes Shift (LSS)    

two NIR CPNs NOT likely to be useful on our fluorescence microscopes:
CPN 900 (IR-I)                  650 / 900            Emission "way out" in NIR - not suitable for our microscopes.
CPN 1130 (IR-II)               750 /1130           Emission "way out" in NIR - not suitable for our microscopes.    

Thought experiment: the seven usable-on-our-microscopes CPNs could enable "dedensify by multiplexing". for example, if the same primary antibody or nanobosy (or FISH probe set) were labeled with EACH of the seven CPNs, and applied at same density to a specimen, with saturaton binding (and assuming equal affinity and kinetics), the density for each would be 1/7th os a single plex. This could facilitate single molecule separation -- maybe not perfect but a lot faster (7 exposures) than a usual STORM single molecule localization experiment. With the right microscope (ex 405 & 440 & 488 nm excitation, 8 cameras of optimzed emission wavelenths [enable some other fluorophore to be imaged]) could acquire one exposure (and then do Z-series, tile scan, etc). Similar logic would apply to Brilliant Violets, Brilliant Ultraviolets, ThermoFisher/Phitonix NovaFluors etc. 

***

Bio-Rad 20211118Thur ... 3 excitation wavelengths, potentially 8+9+7 = 24plex (ignoring potential to spectrally -- or multichannels -- resolve with Brilliants, SuperBrights, Nova Fluors, conventional fluorophores).

StarBright Ultraviolet (SBUV) 8plex ... max excitation ~340nm (so may work on FISHscope, 368nm excitation; not on confocals 405nm excitation). Shpuld bne excitable by 2-photon (~700nm) and/or 3-photon (~1000nm) light.

https://www.bio-rad-antibodies.com/flow-cytometry-starbright-ultraviolet-dyes.html
StarBright Dye                                Max Ex, nm         Max Em, nm       Brightness (1–5)        Comparison Dye
StarBright UltraViolet 400 Dye            335                        394                        3                        BUV395  = Brilliant Ultraviolet 395
StarBright UltraViolet 445 Dye            347                        440                        3                        Alexa Fluor 350
StarBright UltraViolet 510 Dye            340                        513                        3                        BUV496
StarBright UltraViolet 575 Dye            340                        569                        4                        BUV563
StarBright UltraViolet 605 Dye            340                        609                        4                        BUV615
StarBright UltraViolet 665 Dye            340                        669                        4                        BUV661
StarBright UltraViolet 740 Dye            344                        743                        4                        BUV737
StarBright UltraViolet 795 Dye            340                        792                        2                        BUV805

Bio-Rad SBUV availableity - 20220330Wed table

Excitable by the 355 nm laser {GM: FISHscope 368nm LED should work}, StarBright UltraViolet (SBUV) Dyes
https://www.bio-rad-antibodies.com/flow-cytometry-starbright-ultraviolet-dyes.html
Fluorophore                                  ... Availability       ... Comparison 
                                                          (20220330Wed)     Fluorophore
StarBright UltraViolet 400 Dye ... now                        BUV395
StarBright UltraViolet 445 Dye ... soon                       Alexa Fluor 350
StarBright UltraViolet 510 Dye ... now                        BUV496
StarBright UltraViolet 575 Dye ... soon                       BUV563
StarBright UltraViolet 605 Dye ... soon                       BUV615
StarBright UltraViolet 665 Dye ... now                        BUV661
StarBright UltraViolet 740 Dye ... soon                       BUV737
StarBright UltraViolet 795 Dye ... now                        BUV805

 

StarBright Violet (SBV) 9plex

https://www.bio-rad-antibodies.com/flow-cytometry-starbrightviolet.html
SBV Dye Range
StarBright Dye                         Max Ex, nm              Max Em, nm       Brightness (1–5)          Comparison Dye
StarBright Violet 440 Dye             383                          436                          5                          Super Bright (SB) 436 , Pacific Blue, eFluor 450
StarBright Violet 475 Dye             405                          479                          4                          Brilliant Violet (BV) 480
StarBright Violet 515 Dye             402                          516                          5                          BV510, Amethyst Orange
StarBright Violet 570 Dye             402                          570                          4                          BV570
StarBright Violet 610 Dye             402                          607                          5                          BV605, SB600
StarBright Violet 670 Dye             401                          667                          5                          BV650, SB645
StarBright Violet 710 Dye             402                          713                          5                          BV711, SB702
StarBright Violet 760 Dye             403                          760                          5                          BV750
StarBright Violet 790 Dye             402                          782                          4                          BV785, SB780

StarBright Blue (SBB) 7plex
https://www.bio-rad-antibodies.com/flow-cytometry-starbrightblue.html
SBB Dye Range
StarBright Dye                           Max Ex, nm Max Em, nm                Brightness (1-5)              Comparison Dye
StarBright Blue 520 Dye              TBD                         TBD                      TBD                          FITC, A488, BB515
StarBright Blue 580 Dye              475                          580                        4                              -
StarBright Blue 615 Dye              475                          611                        3                              -
StarBright Blue 675 Dye              474                          675                        5                             PerCP
StarBright Blue 700 Dye              470                          705                        5                             PerCP-Cy5.5, BB700, PerCP-eFluor710
StarBright Blue 765 Dye              474                          763                        4                             -
StarBright Blue 810 Dye              475                          808                        3                             -

Bio-Rad 202202 SuperBright fluorophores emission spectra 488, 405, 355nm excitation laser lines (368nm on FISHscope should excite SBUV's)  (gm light edits from their web site)

Bio-Rad 202202 SuperBright emission spectra

 

BioRad posted online potentially useful pdf:

Antigen Density for Human and Murine Surface Markers

Antigen Density for Human and Murine Surface Markers
https://www.bio-rad-antibodies.com/flow-antigen-density.html
https://www.bio-rad-antibodies.com/static/2018/antigen-density/antigen-density-common-murine-markers.pdf
* GM note: Log10 graphs, simplistic, few markers for mouse and human. One example of simplistic is CD25 (IL-2Ralpha), which is low on most human lymphocytes, with exceptiion of CD4 Treg's (very high) and activated CD8 T-cells (aka CTL cytolytic T-cells). Their graph does not reflect this.


Good feature: Page 3 is on "Antigen Density for Human and Murine Surface Markers" -- see the PDF for details!!! Summary:
1. titrate your antibody.
2. Stain your "ABC" (antibody binding capacity) beads (intended for flow cytometry and uses PE - could be adapted for fluorescence microscopy by using bright, photostable fluorophore - GM suggests BV421).
3. Create a standard curve (adapt for microscope!).
4. Measure the antigen density (adapt for microscope!).


GM note: Flow cytometer PMT is reporting entire cell fluorescence - depending on what microscope you use, what magnification and resolution (mag, NA, pixel and voxel size, type of detector [PMT, HyD, CCD, sCMOS, EMCCD - and their settings and exposure time, ixel dwell time], optical sectioning ... also any autofluorescence) you may want to (a) measure entire bead or cell as "one dot" (ex. low mag), or probably better under your standard imaging conditions (i.e. high resolution Z-series) measure bead and cell surface (pixels, voxels) intensities, then compute total (surface) intensity level. 

 

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Academic and commercial multiplex publications

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10/2021 multiplex paper - ChipCytometry ... Christian Hennig, MD, is cofounder of ChipCytometry, aka Zellkraftwerk ("cell power plant" in German), acquired by Canopy Biosciences, acquired by Bruker. GM first met Christian after he gave a keynote talk at ISAC 2012 (or 2011?) (Baltimore Convention Center). The company has shown on their web site up to 90plex and could go higher. Walter Schubert (Nature Biotechnol ~2006) published 100plex (50 cycles of 2plex), minimal methods and probably one firld of view of one specimen (aka postage stamp microscopy). This ChipCytometry paper features FFPE tissue sections -- earlier work focused on single cell suspensions attached to imaging chamber. GM has past pricing estimates for each of ChipCytometry and Akoya Biosciences CODEX2 (in 2021 they just call it CODEX): each expensive per run (i.e. 30plex 10x10 mm) but may be worth it (expensive relative to 3plex + DAPI).. 

Jarosch 2021 CellRepMeth - Multiplexed imaging automated signal quantification FFPE tissues ChipCytometry Henning Busch - Canopy Bruker

https://doi.org/10.1016/j.crmeth.2021.100104
https://www.sciencedirect.com/science/article/pii/S2667237521001636#mmc2
Multiplexed imaging and automated signal quantification in formalin-fixed paraffin-embedded tissues by ChipCytometry
Sebastian Jarosch, Jan Köhlen, Rim S.J.Sarker, Katja Steiger, Klaus-Peter Janssen, Arne Christians, ChristianHennig, Ernst Holler, Elvira D'Ippolito, Dirk H.Busch

Highlights
• ChipCytometry is an optical imaging method based on staining/bleaching cycles

• ChipCytometry allows multiplexed imaging and is now applicable to FFPE sections

• A fine-tuned analysis pipeline leads to accurate automatic signal quantifications

• High data quality enables deep investigation of spatial and phenotypic parameters


Motivation
Simultaneous in situ analyses of a multitude of cells and markers is necessary for gaining insights into tissue biology and pathology. Here we describe the application of ChipCytometry, a user-friendly optical imaging-based technology for multiplexed staining, to widespread used formalin-fixed paraffin-embedded tissues. The multiplexing of up to 30 markers in combination with a developed open-source workflow for signal quantification facilitated high-dimensional tissue analyses.


***
12/2020 article from Ron Germain's lab, 65plex (max 11plex per imaging cycle ... under-utilizing capability of their Leica SP8 ... I suggest the new Leica STELLARIS 8 with 5 internal S detectors and 8 external S detectors on X1 port [XS8] would enable much more simultaneous plex,
https://www.pnas.org/content/117/52/33455.long 

12./2021 review (failed to cite McNamara ... Levenson 2020 !!!) by many of the multiplex players, including Ron Germain and Garry Nolan:

Spatial mapping of protein composition and tissue organization: a primer for multiplexed antibody-based imaging.
Hickey JW, Neumann EK, Radtke AJ, Camarillo JM, Beuschel RT, Albanese A, McDonough E, Hatler J, Wiblin AE, Fisher J, Croteau J, Small EC, Sood A, Caprioli RM, Angelo RM, Nolan GP, Chung K, Hewitt SM, Germain RN, Spraggins JM, Lundberg E, Snyder MP, Kelleher NL, Saka SK.
Nat Methods. 2021 Nov 22. doi: 10.1038/s41592-021-01316-y. Online ahead of print.
PMID: 34811556 
https://www.nature.com/articles/s41592-021-01316-y

McNamara ... Levenson 2020 = 

New Technologies to Image Tumors.
McNamara G, Lucas J, Beeler JF, Basavanhally A, Lee G, Hedvat CV, Baxi VA, Locke D, Borowsky A, Levenson R.
Cancer Treat Res. 2020;180:51-94. doi: 10.1007/978-3-030-38862-1_2.
PMID: 32215866 
https://link.springer.com/chapter/10.1007%2F978-3-030-38862-1_2 (book chp and access to the eBook - most academics will have online access)
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8370749                      (PubMed Central's ugly format, only virtue is it is free).

Note: neither web page has my supplemental Excel file - anyone interested can contact me by email for the Excel file. Lists many 'players' that did not get mentioned by name in the book chp.

2/2022 research paper on automating IBEX:

Radtke AJ ... Germain RN 2022 IBEX: an iterative immunolabeling and chemical bleaching method for high-content imaging of diverse tissues. Nature Protocols 17: 378–401. 
https://www.nature.com/articles/s41596-021-00644-9
Adds automation, lower concentration LiBH4 with perfusion.  

***

multiplex advice links:

A Spatial Biology Startup Guide – Part 1 -- The top 20 questions for ensuring preanalytical quality control
Michael Surace (AstraZeneca), Houssein A. Sater, Carlos Andrea, Jeffrey Chun Tatt Lim, Carmen Ballesteros-Merino, Jaime Rodriguez Canales, Joe Yeong | 02/09/2022
https://thetranslationalscientist.com/tools-techniques/a-spatial-biology-startup-guide-part-1

The Society for Immunotherapy of Cancer statement on best practices for multiplex immunohistochemistry (IHC) and immunofluorescence (IF) staining and validation.
Taube JM, Akturk G, Angelo M, Engle EL, Gnjatic S, Greenbaum S, Greenwald NF, Hedvat CV, Hollmann TJ, Juco J, Parra ER, Rebelatto MC, Rimm DL, Rodriguez-Canales J, Schalper KA, Stack EC, Ferreira CS, Korski K, Lako A, Rodig SJ, Schenck E, Steele KE, Surace MJ, Tetzlaff MT, von Loga K, Wistuba II, Bifulco CB; Society for Immunotherapy of Cancer (SITC) Pathology Task Force. 
J Immunother Cancer. 2020;8(1):e000155. doi: 10.1136/jitc-2019-000155. PMID: 32414858 

 

***

11/2021 preprint from Joe Beechem et al at NanoString

High-Plex Multiomic Analysis in FFPE Tissue at Single-Cellular and Subcellular Resolution by Spatial Molecular Imaging

Shanshan He, ... Joseph M. Beechem
doi: https://doi.org/10.1101/2021.11.03.467020

https://www.biorxiv.org/content/10.1101/2021.11.03.467020v1

NanoString SMI 960 RNAs & 80 proteins (ssDNA oligo antibodies; could go plexier)

Spatial Molecular Imager (SMI) is an automated microscope imaging system with microfluidic reagent cycling, for high-plex, spatial in-situ detection of multiomic targets (RNA and protein) on FFPE and other intact samples with subcellular resolution. The key attributes of the CosMxTM SMI platform (NanoString®, Seattle, WA) include: 1) high-plex and high-sensitivity imaging chemistry that works for both RNA and protein detection, 2) three-dimensional subcellular-resolution image analysis with a target localization accuracy of ∼50 nm in the XY plane, 3) large Hamming-distance encoding sch

Equipment

pc Computer Workstation #1

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Keyence BZ-X700 (open access)

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MetaMorph Key 1 (#34135)

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zzzz Retired Perkin-Elmer EnVision Plate Reader

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zzzZeiss Axio Imager Upright Microscope, optionally...

6/2019: the microscope is now Legacy status. The MetaMorph #4646 license can be used on the PC by prior arrangement with George...

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