TFS1065 BD00033053
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`TFS1065
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`A Brilliant New Addition to the Fiuorescent Probe
`Toolbox
`
`Stephen C. De Rosa’?"*
`
`%
`
`Key terms
`fluorescent probe; quantum yield; extinction coefficient;
`violet
`
`laser;
`
`FLUORESCENT probesarecritical for flow cytometry and flu-
`orescent microscopy. Although new probes are developed and
`introduced commercially each vear, there are only a selected
`few that are exceptional in what we commonly refer to as
`brightness. One such probe, phycoerythrin (PE), was first
`reported 30 years ago in 1982 for use in cell analysis as an anti-
`body conjugate (1). The use of a second probe, allophycocya-
`nin (APC), was reported a year later (2). It was not until 1998
`that another probe of comparable brightness,
`the quantum
`dot, was reported. Unique from all others, the quantum dotis
`inorganic (3). Quantumdot probes have been especially useful
`because they are optimaily excited by ultraviolet and violet
`light, a spectral region with limited coverage from other bight
`fluorescent probes. Now, a new probe (Brilliant Violet, BV) of
`a different class but of comparable brightness and excited by
`violet light has heen developed as reported by Chattopadhyay
`et al. in this issue (page 456).
`Although just 10 years ago flow cytometry experiments
`measuring more than six parameters or four “colors” were
`limited to relatively few advanced laboratories, this capability
`is now much more widely distributed, including even some
`
`resource limited settings. One reason for this increased accessi-
`bility is introduction of off-the-shelf and customm-modified
`instrumentation with greater capacity for multicolor detection
`
`Dapartnent-of Laboratery Medicine-University-of Washington:
`seacte, Washingion $8195:
`NWaccine and intestous Disease Dansion Fred Hishinson Cancer
`Research Center Seattle: Wiashingtor: 98104
`
`SHIN Vacsing THals:N ebork (EVEN) Seatve: Washington: Sb1ng
`
`Raceived 10 Februgry 2012; Accepted 14 Pe aruary 2042
`
`Grant saonsor HAE Vaccme: tials Network Laooratory: Progra:
`Grant number UME Algsed 1a.
`Grant sponsor: universiy of Washington Canter tor AIDS Research:
`Grantnuimber PSo Algaera?
`
`Cytometry Part A @ 81A: d45--446, 2012
`
`and another reason is the growing commercial availability of
`reagents consisting of conjugates to less common fhrorescent
`probes. The expansion of multicolor technology makes use of
`fhaorescent probes that are excited at a variety of wavelengths,
`and to allowfor this, instrumentation has been modified by add-
`ing additional lasers of different colors. In addition to the nearly
`universal blue and red lasers, a viclet laser exciting at about 405
`ont is now commonlyincluded in manyinstruments (4).
`Luitially, there were very few fluorescent probes excited by
`the violet laser and they were disrnal
`in terms of brightness.
`Soon, better probes and commercial conjugates became avail-
`able although none rivaled the brightness of PE and APC.
`Therefore, the introduction of quantum dots caused a great
`amount of excitement not only because of their brightness, but
`also because of the selection ofdifferent colors all excited by the
`violet laser. Although quantum dots continue to he extremely
`useful, the initial enthusiasm has been tempered by some prac-
`tical issues such as shelflife
`sensitivity to heavy metal contami-
`nants, tendency to form aggregates, and cross-laser compensa-
`tion requirements that became more evident as they were
`included in multicolor panels (3). Thus, alternate violet-excited
`bright fluorescent probes are kely to be of benefit to many
`researchers (6).
`The intrinsic brightness of a fluorescent compound is a
`function of the quantumyield and the molar extinction coefii-
`cient. The quantum vield is the probability of emitting a pho-
`ton once a photon of light is absorbed and thus is a measure
`of the efficiency of energy conversion. High quantum yields
`are optimal and the quantum yield for BV is comparable to
`manyother bright fluorochromes (Table 1). The molar extinc-
`
`“Corrasnondenee to: Stephen ff De Rosa: Pred: Hutchinson Cancer
`Research: Genter: 168 Falivisw Ave: NE CE200) Beathe AAOH OS:
`LSA:
`
`f-maik sderosa@iicraiorg
`
`Puoiished online 6 Aonk 2012 in Witey:-Oniing Liorary
`iwiteyontinelheary.cam)
`
`DOE HO 106/ogty.3 20036
`
`©: 212 international Society for Advancennnt of Gytameatry
`
`BD00033054
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`RCPS RE leee
`
`Table 1. Quantum yield and extinction coefficient for commonly-usad fluorescent probes
`
`FLUOROCHROME
`QUANTUMYIELD
`MOLAR EXTINCTION COFFFICIENT*
`EXCITATION/ABSORBANCE
`
`Brilliant Violet
`R-PE
`APC
`Quantum Dot 655
`Quantum Dot 5&5
`AlexaFluor 660
`Fluorescein
`AlexaFlior 488
`Pacific Blue
`
`0.69
`O.82
`9.608
`ae. 3"
`‘
`
`
`
`0.78
`
`2,506,000
`1,960,000
`700,000
`5,700,000
`2,200,000
`132,000
`86,000
`71,000
`46,000)
`
`Violet (405 nm)
`Blue (496 nm}
`Red (656 nm}
`Violet (405 nm)
`Violet (405 nm)
`Red (663 nm}
`Blue (488 nm)
`Blue (495 nm)
`Violet (405 nm)
`
`“Moasured at the indicated excitation/absorbance wavelength (cm
`bouantum yields generally increase for larger quantum dots (14).
`
`mo),
`
`tion coefficient is a measure of the probability of absorbing a
`photon oflight at the wavelength of excitation and this is the
`measure that is exceptional for PE, APC, the quantum dots,
`and now BV. Other factors also affect the brightness of anti-
`body conjugates such as the number of fluorophores conju-
`gated to each antibody, but these intrinsic factors have a domi-
`nant effect (7}.
`The newfluorescent probe reported in this issue of Cyto-
`metry is a unique type offluorescent compound with a fasci-
`
`nating pedigree stemming from a discovery that earned the
`
`Nobel Prize in chemistry in 2000. The discovery and de
`Lop-
`ment of conductive polymers has yielded many applications
`already in use in commercial products. The BV fluorescent
`probe resulted from chemical modifications to a polymer that
`yielded the appropriate excitation spectrum, water solubility,
`conjugation ability, and minimal nonspecific binding. Addi-
`tional violet-exctted fluorescent probes with differing emission
`spectra have been developed bycreating tandem dyes of BV
`conjugated with more traditional fluorophores such as Cy3
`(the BV570 tandem dye reported in this issue).
`In this issue, Chattopadhyay and colleagues report on the
`use of these new dyes as antibody conjugates and as a strepta-
`vidin conjugate used with pMHCI multimers. The compari-
`son of the BV421 conjugates with the Pacific Blue conjugates
`demonstrates their remarkable brightness, with the CD8 con-
`
`jugate about 10-fold brighter as quantified by the stain index.
`Thus,
`the exceptional molar extinction coefficient translates
`into bright cell staining reagents. The authors also demon-
`strate suitable photostability and utility in fluorescence mi-
`croscopy. Although onky one tandem dye was tested, it is likely
`that a series of bright tandemdyes based on the Brilliant Vio-
`let backbone are forthcoming and will be a welcome addition
`to the ever-growing arsenal of fluorescent probes available to
`cytometrists.
`Multicolor flow cytometry continues to be a cornerstone
`of inumunological and celbular proGling. Althoughit is inevita-
`
`ble that newtechnologies will enter the field, such as nonfluor-
`escent based flow cytometry (CyTOF) using mass spectrome-
`try, where reagent labels are distinguished by mass and there is
`much less expertise required to assemble and optimize cell
`staimng panels exarnining large numbers of markers (8),
`it is
`unlikely that they will replace fluorescent-based flow cytome-
`try, at least in the near future. Thus, the art and science of
`creating staining panels remain important (9,10) and Brilliant
`Violet and its tandem dyes are useful additions to the toolbox
`of fluorescent probes. Indeed, there is a bright future for Brilli-
`ant Violet.
`
`ACKNOWLEDGMENTS
`
`The author thanks Stephen Voght for help with editing.
`
`LITERATURE CITED
`
`i,
`O81 VT, Glazer AN, §
`L. Fluorescent phyocbiliprotein conjugates for analyses of
`cells and reolecules. J
`Biol 1982;93:981--986,
`
`nson L, Withams 'M. Strom TB. Laumanciluores-
`
`2. Shapiro HM, Glazer AN, Ch:
`cence measurement ina flo
`neter using low-power helinm-neon laser excita -
`
`tion. Cytometry 1983;4:27
`
`
`isatos AP. Semiconductor nanccrys-
`3. Bruchez M, Jr, Moronne M, Gin P, Weiss &, Ali
`
`4:2013-Z016,
`
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`cence 1998;
`tals as fiuorescent biclegical fa
`4. Shapiro HM, Perlniatter NG. Violet laser diedes as light sources for cytometry. Cyto-
`metry 200154.
`3-136.
`
`wa
`
`
`Chattopad
`°K. Quantumdot technology in flow cytoaetry, Methods Cell Biel
`2014;102
`7,
`ited fluorochromes by
`6. Telford WG, Hawley
`
`
`S4A48-55,
`flow cytometryusing a violet laser diode.
`
`
`
`ILougda
`d RP.The Landbwok: A Guide to Fluorescent Probes and Labeling Technolo-
`
`enc, OR: Invitrogen Corporalion; 2005.
`gies. £
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`7.
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`of differential
`Single-cell mass cytemeiry
`amed &, Treio A, Ormatsky O], et al.
`iramune and drug responses across a human hematopoietic continuum. Science
`32:6R7-696,
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`9. Maboke Y
`
`atlopadhivay B, Roederer
`M. Publication of upiimrized mrakticolor im-
`
`munofluor
`~ytometry Part A 2010;77A:814--818.
`
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`10. Matike YD, Re
`¢ M, Optimizing a multicolor inmunophenotyping assay. Clin
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`Lab Med 200
`69-485, v.
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`
` enger O, Deter-
`il. Grabolle M, Spieles M, Le.
`kV, Gaponik. N, Eychraulter A, Resch
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`minalion of the fluorescence
`quantum yield of quantum dois:
`lable procedures
`and achievable uncertainties. Anal Chem 2009;81:6285-6294.
`
`446
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`Fluorescent Probe Toalbox
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`BD00033055
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