fluorochromes

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specific fluorochromes

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specific fluorochromes

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1. any of various fluorescent substances used in fluorescence microscopy to stain specimens

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出典(authority):フリー百科事典『ウィキペディア（Wikipedia）』「2014/08/24 09:12:35」(JST)

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For uses and theory of fluorescence, see Fluorescence in the life sciences.

A fluorophore-labeled human cell.

A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or plane or cyclic molecules with several π bonds.

Fluorophores are sometimes used alone, as a tracer in fluids, as a dye for staining of certain structures, as a substrate of enzymes, or as a probe or indicator (when its fluorescence is affected by environment such as polarity, ions,...). But more generally it is covalently bonded to a macromolecule, serving as a marker (or dye, or tag, or reporter) for affine or bioactive reagents (antibodies, peptides, nucleic acids). Fluorophores are notably used to stain tissues, cells, or materials in a variety of analytical methods, i.e., fluorescent imaging and spectroscopy.

Fluorescein, by its amine reactive isothiocyanate derivative FITC, has been one of the most popularized fluorophores. From antibody labeling, the applications have spread to nucleic acids thanks to (FAM(Carboxyfluorescein), TET,...). Other historically common fluorophores are derivatives of rhodamine (TRITC), coumarin, and cyanine. Newer generations of fluorophores, many of which are proprietary, often perform better (more photostable, brighter, and/or less pH-sensitive) than traditional dyes with comparable excitation and emission.

Fluorescence

The fluorophore absorbs light energy of a specific wavelength and re-emits light at a longer wavelength. The absorbed wavelengths, energy transfer efficiency, and time before emission depend on both the fluorophore structure and its chemical environment, as the molecule in its excited state interacts with surrounding molecules. Maximum wavelengths of absorption (≈ excitation) and emission (for example, Absorption/Emission = 485 nm/517 nm) are the typical terms used to refer to a given fluorophore, but the whole spectrum may be important to consider. The excitation wavelength spectrum may be a very narrow or broader band, or it may be all beyond a cutoff level. The emission spectrum is usually sharper than the excitation spectrum, and it is of a longer wavelength and correspondingly lower energy. Excitation energies range from ultraviolet through the visible spectrum, and emission energies may continue from visible light into the near infrared region.

Main characteristics of fluorophores are :

• Maximum excitation and emission wavelength (expressed in nanometers (nm)): corresponds to the peak in the excitation and emission spectra (usually one peak each),
• Extinction Coefficient (or molar absorption, in Mol⁻¹cm⁻¹) : links the quantity of absorbed light, at a given wavelength, to the concentration of fluorophore in solution.
• Quantum yield : efficiency of the energy transferred from incident light to emitted fluorescence (= number of emitted photons per absorbed photons)
• Lifetime (in picoseconds): duration of the excited state of a fluorophore before returning to its ground state. It refers to the time taken for a population of excited fluorophores to decay to 1/e (≈0.368) of the original amount.
• Stokes shift: difference between the max excitation and max emission wavelengths.

These characteristics drive other properties, including the photobleaching or photoresistance (loss of fluorescence upon continuous light excitation). Other parameters should be considered, as the polarity of the fluorophore molecule, the fluorophore size and shape (i.e. for polarization fluorescence pattern), and other factors can change the behavior of fluorophores.

Fluorophores can also be used to quench the fluorescence of other fluorescent dyes (see article Quenching (fluorescence)) or to relay their fluorescence at even longer wavelength (see article FRET)

See more on fluorescence principle.

Size (molecular weight)

Most fluorophores are organic small molecules of 20 - 100 atoms (200 - 1000 Dalton - the molecular weight may be higher depending on grafted modifications, and conjugated molecules), but there are also much larger natural fluorophores that are proteins: Green fluorescent protein (GFP) is 26 kDa and several phycobiliproteins (PE, APC...) are ≈240kDa.

Fluorescence particles are not considered as fluorophores (quantum dot: 2-10 nm diameter, 100-100,000 atoms)

The size of the fluorophore might sterically hinder the tagged molecule, and affect the fluorescence polarity.

Families

Fluorophore molecules could be either utilized alone, or serve as a fluorescent motif of a functional system. Based on molecular complexity and synthetic methods, fluorophore molecules could be generally classified into four categories: proteins and peptides, small organic compounds, synthetic oligomers and polymers, and multi-component systems.

• fluorescent proteins

GFP (green), YFP (yellow) and RFP (red) can be attached to other specific proteins to form a fusion protein, synthesized in cells after tranfection of a suitable plasmid carrier.

Non-protein organic fluorophores belong to following major chemical families:

• Xanthene derivatives: fluorescein, rhodamine, Oregon green, eosin, and Texas red
• Cyanine derivatives: cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, and merocyanine
• Naphthalene derivatives (dansyl and prodan derivatives)
• Coumarin derivatives
• oxadiazole derivatives: pyridyloxazole, nitrobenzoxadiazole and benzoxadiazole
• Anthracene derivatives: anthraquinones, including DRAQ5, DRAQ7 and CyTRAK Orange
• Pyrene derivatives: cascade blue etc.
• Oxazine derivatives: Nile red, Nile blue, cresyl violet, oxazine 170 etc.
• Acridine derivatives: proflavin, acridine orange, acridine yellow etc.
• Arylmethine derivatives: auramine, crystal violet, malachite green
• Tetrapyrrole derivatives: porphin, phthalocyanine, bilirubin

These fluorophores fluoresce thanks to delocalized electrons which can jump a band and stabilize the energy absorbed. Benzene, one of the simplest aromatic hydrocarbons, for example, is excited at 254 nm and emits at 300 nm. This discriminates fluorophores from quantum dots, which are fluorescent semiconductor nanoparticles.

They can be attached to protein to specific functional groups, such as - amino groups (Active ester, Carboxylate, Isothiocyanate, hydrazine) - carboxyl groups (carbodiimide) - thiol (maleimide, acetyl bromide) - azide (via click chemistry or non-specifically (glutaraldehyde)).

Additionally, various functional groups can be present to alter its properties, such as solubility, or confer special proprieties, such as boronic acid which binds to sugars or multiple carboxyl groups to bind to certain cations. When the dye contains an electron-donating and an electron-accepting group at opposite ends of the aromatic system, this dye will probably be sensitive to the environment's polarity (solvatochromic), hence called environment-sensitive. Often dyes are used inside cells, which are impermeable to charged molecules, as a result of this the carboxyl groups are converted into an ester, which is removed by esterases inside the cells, e.g., fura-2AM and fluorescein-diacetate.

The following dye families are trademark groups, and do not necessarily share structural similarities.

• CF dye (Biotium)
• DRAQ and CyTRAK probes (BioStatus)
• BODIPY (Invitrogen)
• Alexa Fluor (Invitrogen)
• DyLight Fluor (Thermo Scientific, Pierce)
• Atto and Tracy (Sigma Aldrich)
• FluoProbes (Interchim)
• Abberior Dyes (Abberior)
• DY and MegaStokes Dyes (Dyomics)
• Sulfo Cy dyes (Cyandye)
• HiLyte Fluor (AnaSpec)
• Seta, SeTau and Square Dyes (SETA BioMedicals)
• Quasar and Cal Fluor dyes (Biosearch Technologies)
• SureLight Dyes (APC, RPE, PerCP, Phycobilisomes)(Columbia Biosciences])
• APC, APCXL, RPE, BPE (Phyco-Biotech)

Bovine Pulmonary Artery Endothelial cell nuclei stained blue with DAPI, mitochondria stained red with MitoTracker Red CMXRos, and F-actin stained green with Alexa Fluor 488 phalloidin and imaged on a fluorescent microscope.

Examples of frequently encountered fluorophores

Reactive and conjugated dyes

Dye	Ex (nm)	Em (nm)	MW	Notes
Hydroxycoumarin	325	386	331	Succinimidyl ester
Aminocoumarin	350	445	330	Succinimidyl ester
Methoxycoumarin	360	410	317	Succinimidyl ester
Cascade Blue	(375);401	423	596	Hydrazide
Pacific Blue	403	455	406	Maleimide
Pacific Orange	403	551
Lucifer yellow	425	528
NBD	466	539	294	NBD-X
R-Phycoerythrin (PE)	480;565	578	240 k
PE-Cy5 conjugates	480;565;650	670		aka Cychrome, R670, Tri-Color, Quantum Red
PE-Cy7 conjugates	480;565;743	767
Red 613	480;565	613		PE-Texas Red
PerCP	490	675	35kDa	Peridinin chlorophyll protein
TruRed	490,675	695		PerCP-Cy5.5 conjugate
FluorX	494	520	587	(GE Healthcare)
Fluorescein	495	519	389	FITC; pH sensitive
BODIPY-FL	503	512
Cy2	489	506	714	QY 0.12
Cy3	(512);550	570;(615)	767	QY 0.15
Cy3B	558	572;(620)	658	QY 0.67
Cy3.5	581	594;(640)	1102	QY 0.15
Cy5	(625);650	670	792	QY 0.28
Cy5.5	675	694	1128	QY 0.23
Cy7	743	767	818	QY 0.28
SeTau-647	649	695	1461	QY 0.61; Lifetime 3.2 ns
TRITC	547	572	444	TRITC
X-Rhodamine	570	576	548	XRITC
Lissamine Rhodamine B	570	590
Texas Red	589	615	625	Sulfonyl chloride
Allophycocyanin (APC)	650	660	104 k
APC-Cy7 conjugates	650;755	767		Far Red

Abbreviations:
Ex (nm): Excitation wavelength in nanometers
Em (nm): Emission wavelength in nanometers
MW: Molecular weight QY: Quantum yield

Nucleic acid dyes

Dye	Ex (nm)	Em (nm)	MW	Notes
Hoechst 33342	343	483	616	AT-selective
DAPI	345	455		AT-selective
Hoechst 33258	345	478	624	AT-selective
SYTOX Blue	431	480	~400	DNA
Chromomycin A3	445	575		CG-selective
Mithramycin	445	575
YOYO-1	491	509	1271
Ethidium Bromide	493	620	394
Acridine Orange	503	530/640		DNA/RNA
SYTOX Green	504	523	~600	DNA
TOTO-1, TO-PRO-1	509	533		Vital stain, TOTO: Cyanine Dimer
TO-PRO: Cyanine Monomer
Thiazole Orange	510	530
CyTRAK Orange	520	615	-	(Biostatus) (red excitation dark)
Propidium Iodide (PI)	536	617	668.4
LDS 751	543;590	712;607	472	DNA (543ex/712em), RNA (590ex/607em)
7-AAD	546	647		7-aminoactinomycin D, CG-selective
SYTOX Orange	547	570	~500	DNA
TOTO-3, TO-PRO-3	642	661
DRAQ5	600/647	697	413	(Biostatus) (usable excitation down to 488)
DRAQ7	599/644	694	~700	(Biostatus) (usable excitation down to 488)

Cell function dyes

Dye	Ex (nm)	Em (nm)	MW	Notes
Indo-1	361/330	490/405	1010	AM ester, low/high calcium (Ca2+)
Fluo-3	506	526	855	AM ester. pH > 6
Fluo-4	491/494	516	1097	AM ester. pH 7.2
DCFH	505	535	529	2'7'Dichorodihydrofluorescein, oxidized form
DHR	505	534	346	Dihydrorhodamine 123, oxidized form, light catalyzes oxidation
SNARF	548/579	587/635		pH 6/9

Fluorescent proteins

Dye	Ex (nm)	Em (nm)	MW	QY	BR	PS	Notes
GFP (Y66H mutation)	360	442
GFP (Y66F mutation)	360	508
EBFP	380	440		0.18	0.27		monomer
EBFP2	383	448			20		monomer
Azurite	383	447			15		monomer
GFPuv	385	508
T-Sapphire	399	511		0.60	26	25	weak dimer
Cerulean	433	475		0.62	27	36	weak dimer
mCFP	433	475		0.40	13	64	monomer
ECFP	434	477		0.15	3
CyPet	435	477		0.51	18	59	weak dimer
GFP (Y66W mutation)	436	485
mKeima-Red	440	620		0.24	3		monomer (MBL)
TagCFP	458	480			29		dimer (Evrogen)
AmCyan1	458	489		0.75	29		tetramer, (Clontech)
mTFP1	462	492			54		dimer
GFP (S65A mutation)	471	504
Midoriishi Cyan	472	495		0.9	25		dimer (MBL)
Wild Type GFP	396,475	508	26k	0.77
GFP (S65C mutation)	479	507
TurboGFP	482	502	26 k	0.53	37		dimer, (Evrogen)
TagGFP	482	505			34		monomer (Evrogen)
GFP (S65L mutation)	484	510
Emerald	487	509		0.68	39	0.69	weak dimer, (Invitrogen)
GFP (S65T mutation)	488	511
EGFP	488	507	26k	0.60	34	174	weak dimer, (Clontech)
Azami Green	492	505		0.74	41		monomer (MBL)
ZsGreen1	493	505	105k	0.91	40		tetramer, (Clontech)
TagYFP	508	524			47		monomer (Evrogen)
EYFP	514	527	26k	0.61	51	60	weak dimer, (Clontech)
Topaz	514	527			57		monomer
Venus	515	528		0.57	53	15	weak dimer
mCitrine	516	529		0.76	59	49	monomer
YPet	517	530		0.77	80	49	weak dimer
TurboYFP	525	538	26 k	0.53	55.7		dimer, (Evrogen)
ZsYellow1	529	539		0.65	13		tetramer, (Clontech)
Kusabira Orange	548	559		0.60	31		monomer (MBL)
mOrange	548	562		0.69	49	9	monomer
Allophycocyanin (APC)	652	657.5	105 kDa	0.68			heterodimer, crosslinked
mKO	548	559		0.60	31	122	monomer
TurboRFP	553	574	26 k	0.67	62		dimer, (Evrogen)
tdTomato	554	581		0.69	95	98	tandem dimer
TagRFP	555	584			50		monomer (Evrogen)
DsRed monomer	556	586	~28k	0.1	3.5	16	monomer, (Clontech)
DsRed2 ("RFP")	563	582	~110k	0.55	24		(Clontech)
mStrawberry	574	596		0.29	26	15	monomer
TurboFP602	574	602	26 k	0.35	26		dimer, (Evrogen)
AsRed2	576	592	~110k	0.21	13		tetramer, (Clontech)
mRFP1	584	607	~30k	0.25			monomer, (Tsien lab)
J-Red	584	610		0.20	8.8	13	dimer
R-phycoerythrin (RPE)	565 >498	573	250 kDa	0.84			heterotrimer
B-phycoerythrin (BPE)	545	572	240 kDa	0.98			heterotrimer
mCherry	587	610		0.22	16	96	monomer
HcRed1	588	618	~52k	0.03	0.6		dimer, (Clontech)
Katusha	588	635			23		dimer
P3	614	662	~10,000 kDa				phycobilisome complex
Peridinin Chlorophyll (PerCP)	483	676	35 kDa				trimer
mKate (TagFP635)	588	635			15		monomer (Evrogen)
TurboFP635	588	635	26 k	0.34	22		dimer, (Evrogen)
mPlum	590	649	51.4 k	0.10	4.1	53
mRaspberry	598	625		0.15	13		monomer, faster photobleach than mPlum

Abbreviations:
Ex (nm): Excitation wavelength in nanometers
Em (nm): Emission wavelength in nanometers
MW: Molecular weight
QY: Quantum yield
BR: Brightness: Extinction coefficient * quantum yield / 1000
PS: Photostability: time [sec] to reduce brightness by 50%

Applications

Fluorophores have particular importance in the field of biochemistry and protein studies, e.g., in immunofluorescence but also in cell analysis, e.g. immunohistochemistry.

See detailed techniques and applications in the article Fluorescence in the life sciences

Uses outside the life sciences

Additionally fluorescent dyes find a wide use in industry, going under the name of "neon colours", such as

• multi-ton scale usages in textile dyeing and optical brighteners in laundry detergents
• advanced cosmetic formulations; safety equipment and clothing
• organic light-emitting diodes (OLED)
• fine arts and design (posters and paintings)
• synergists for insecticides and experimental drugs
• used as a dye in highlighters to give off a glow-like effect
• Solar panels (collect more light / wavelengths)

See also

• Category:Fluorescent dyes
• Fluorescence in the life sciences
• Dark quencher
• Fluorescence recovery after photobleaching (FRAP) - an application for quantifying mobility of molecules in lipid bilayers.

References

External links

• The Database of fluorescent dyes
• Table of fluorochromes
• Fluorescence Tutorials
• Fluorescence SpectraViewer - check the compatibility of your fluorophores when designing multicolor experiments.
• The Molecular Probes Handbook - a comprehensive resource for fluorescence technology and its applications.

UpToDate Contents

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English Journal

• Tailoring and histochemical application of fluorescent homo-dimeric styryl dyes using frozen sections: From peroxidase substrates to new cytochemical probes for mast cells, keratin, cartilage and nucleic acids.

• Krieg R, Eitner A, Halbhuber KJ.SourceInstitute of Anatomy/Anatomy II, Friedrich-Schiller-University Jena, Teichgraben 7, D-07743 Jena, Germany.
• Acta histochemica.Acta Histochem.2011 Nov;113(7):682-702. Epub 2011 Apr 8.
• Homo-dimers of styryl dyes were chemically tailored in order to become specific cytochemical probes for use in the life sciences. Histochemical applications using fixed cryotome sections are discussed. It is concluded, that homo-dimerization of specific styryl substrates of peroxidase (PO) by way of
• PMID 21477847

• Use of SNARF-1 to measure murine T cell proliferation in vitro and its application in a novel regulatory T cell suppression assay.

• Singh Y, Dyson J, Garden OA.SourceRegulatory T Cell Laboratory, Infection and Immunity Research Group, Department of Veterinary Clinical Sciences, The Royal Veterinary College, Camden Campus, Royal College Street, London, NW1 0TU, United Kingdom; T Cell Development Group, Section of Immunobiology, Imperial College London, Hammersmith Campus, Du Cane Road, London, W12 0NN, United Kingdom.
• Immunology letters.Immunol Lett.2011 Oct 30;140(1-2):21-9. Epub 2011 Jun 1.
• The green fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE) has been used to track the proliferation of T cells in vitro. Such assays often incorporate more than one population of cells, but the paucity of alternative, spectrally distinct dyes suitable for measuring proliferatio
• PMID 21664378

Japanese Journal

• Fluorescent-banded karyotype analysis and identification of chromosomes in three improved Indian varieties of grass pea (Lathyrus sativus L.)

• Talukdar Dibyendu
• Chromosome Science 13(1/2), 3-10, 2011
• … Karyotype analysis of three improved grass pea (Lathyrus sativus L.) varieties, namely Nirmal, BioL-203 and BioR-231 was carried out using root tip metaphase cells after staining with orcein and with two DNA base specific fluorochromes, Chromomycin A3 (CMA) and 4-6diamidino 2-phenyle indole (DAPI). …
• NAID 130000904768

• A cytogenetic study of Polytrias amaura (Poaceae)

• Matsuda Yasushi,Kaneko Hiroshi,Murata Tatsuro,Nagano Katsuya,Hoshi Yoshikazu
• Chromosome Botany 6(1), 5-11, 2011
• … The fluorochromes of CMA and DAPI gave reproducible condensation pattern to every prometaphase chromosome, but not metaphse. …
• NAID 130000654624

Related Links

• Fluorochrome Table

This is a table of some characteristics of fluorochromes useful for flow cytometry or fluorescence microscopy. Within groups, roughly in order of excitation wavelength (families excepted). Peak excitation and emission wavelengths often ...

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fluorescent dye、fluorescent probe、fluorogenic substrate