Fluorophore

A fluorophore, in analogy to a chromophore, is a component of a molecule which causes a molecule to be fluorescent. It is a functional group in a molecule which will absorb energy of a specific wavelength and re-emit energy at a different (but equally specific) wavelength. The amount and wavelength of the emitted energy depend on both the fluorophore and the chemical environment of the fluorophore. This technology has particular importance in the field of biochemistry and protein studies, e.g. in immunofluorescence and immunohistochemistry.^[1]

Fluorescein isothiocyanate (FITC), a reactive derivative of fluorescein, has been one of the most common fluorophores chemically attached to other, non-fluorescent molecules to create new fluorescent molecules for a variety of applications. Other historically common fluorophores are derivatives of rhodamine (TRITC), coumarin, and cyanine.^[2] Newer generations of fluorophores such as the CF dyes, the FluoProbes dyes, the DyLight Fluors, the Oyester dyes, the Atto dyes, the HiLyte Fluors, and the Alexa Fluors are claimed to be perform better (more photostable, brighter, and/or less pH-sensitive,...) than other standard dyes of comparable excitation and emission.^[1]^[3]^[4]

Size

The size of the fluorophore might sterically hinder the tagged molecule:

quantum dot: 2-10 nm (diameter), 100-100,000 atoms
protein: Green fluorescent protein (GFP) 26 kDa
small molecule: fluorescein: about 20 atoms

Families

Several fluorescent protein exist and are attached to a specific protein in a plasmid to form a fusion protein. Fluorophores 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).

These fluorophores are either quantum dots or small molecules. The former are fluorescent semiconductor nanoparticles. The latter molecules which fluoresce thanks to delocalized electrons which can jump a band and stabilize the energy absorbed, hence all fluorophores are aromatic or conjugated. Benzene, one of the simplest aromatic hydrocarbons, for example, is excited at 254 nm and emits at 300 nm^[5].

Additionally, various functional groups can be present to alter its proprieties, 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.

Common dye families (and their manufacturers) are:

Xanthene derivatives: fluorescein, rhodamine, Oregon green, eosin, Texas red etc.
Cyanine derivatives: cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine and merocyanine
Naphthalene derivatives (dansyl and prodan derivatives)
Coumarin derivatives
oxadiazole derivatives: pyridyloxazole, nitrobenzoxadiazole and benzoxadiazole
Pyrene derivatives: cascade blue etc.
BODIPY (Invitrogen)
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
CF dye (Biotium)
Alexa Fluor (Invitrogen) - a trade grouping and do not share structures
Atto and Tracy (Sigma Aldrich) - a trade grouping and do not share structures
FluoProbes (Interchim) - a trade grouping and do not share structures
Tetrapyrrole derivatives: porphin, phtalocyanine, bilirubin
others: cascade yellow, azure B, acridine orange, DAPI, Hoechst 33258, lucifer yellow, piroxicam, quinine and anthraqinone, squarylium, oligophenylenes, etc.

A few examples of commonly used dyes:

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.

Reactive and conjugated probes

Probe	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		Peridinin chlorphyll 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
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		PharRed

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

Alexa Fluor dyes [antibody conjugates] (Molecular Probes)

Probe	Ex (nm)	Em (nm)	MW	Quencher
Alexa Fluor 350	343	442	410
Alexa Fluor 405	401	421	1028
Alexa Fluor 430	434	540	702
Alexa Fluor 488	499	519	643	QY 0.92
Alexa Fluor 500	503	525	700
Alexa Fluor 514	517	542	714
Alexa Fluor 532	530	555	724	QY 0.61
Alexa Fluor 546	561	572	1079	QY 0.79
Alexa Fluor 555	553	568	1250	QY 0.1
Alexa Fluor 568	579	603	792	QY 0.69
Alexa Fluor 594	591	618	820	QY 0.66
Alexa Fluor 610	610	629	1285
Alexa Fluor 633	632	648	1200
Alexa Fluor 647	652	668	1300	QY 0.33
Alexa Fluor 660	663	691	1100
Alexa Fluor 680	680	702	1150
Alexa Fluor 700	696	719	1400
Alexa Fluor 750	752	776	1300
Alexa Fluor 790	782	804	1750

Cy Dyes (GE Heathcare)

Probe	Ex (nm)	Em (nm)	MW	Quencher
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

Nucleic acid probes

Probe	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
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	647	681,697	413	(Biostatus) (usable excitation down to 488)

Cell function probes

Probe	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
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^[6]

Probe	Ex (nm)	Em (nm)	MW	QY	BR	PS	Notes
Y66H	360	442
Y66F	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
Y66W	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
S65A	471	504
Midoriishi Cyan	472	495		0.9	25		dimer (MBL)
Wild Type GFP	396,475	508	26k	0.77
S65C	479	507
TurboGFP	482	502	26 k	0.53	37		dimer, (Evrogen)
TagGFP	482	505			34		monomer (Evrogen)
S65L	484	510
Emerald	487	509		0.68	39	0.69	weak dimer, (Invitrogen)
S65T	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	1.65		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
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
mCherry	587	610		0.22	16	96	monomer
HcRed1	588	618	~52k	0.03	0.6		dimer, (Clontech)
Katusha	588	635			23		dimer
mKate (TagFP635)	588	635			15		monomer (Evrogen)
TurboFP635	588	635	26 k	0.34	22		dimer, (Evrogen)
mPlum	590	649		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%

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

References

^ ^a ^b Tsien RY, Waggoner Aeditor=Pawley JB (1995). "Fluorophores for confocal microscopy". Handbook of biological confocal microscopy. New York: Plenum Press. pp. 267–74. ISBN 0-306-44826-2. Retrieved 2008-12-13.
^ Rietdorf J (2005). Microscopic Techniques. Advances in Biochemical Engineering / Biotechnology. Berlin: Springer. pp. 246–9. ISBN 3-540-23698-8. Retrieved 2008-12-13.
^ Lakowicz, J.R., Principles of fluorescence spectroscopy. 3rd ed. 2006, New York: Springer. xxvi, 954 p.
^ http://www.invitrogen.com/site/us/en/home/References/Molecular-Probes-The-Handbook/Introduction-to-Fluorescence-Techniques.html
^ http://omlc.ogi.edu/spectra/PhotochemCAD/html/benzene.html
^ http://pingu.salk.edu/flow/fluo.html

External links

[Pawley-1] Tsien RY, Waggoner Aeditor=Pawley JB (1995). "Fluorophores for confocal microscopy". Handbook of biological confocal microscopy. New York: Plenum Press. pp. 267–74. ISBN 0-306-44826-2. Retrieved 2008-12-13.

[2] Rietdorf J (2005). Microscopic Techniques. Advances in Biochemical Engineering / Biotechnology. Berlin: Springer. pp. 246–9. ISBN 3-540-23698-8. Retrieved 2008-12-13.

[3] Lakowicz, J.R., Principles of fluorescence spectroscopy. 3rd ed. 2006, New York: Springer. xxvi, 954 p.

[4] ttp://www.invitrogen.com/site/us/en/home/References/Molecular-Probes-The-Handbook/Introduction-to-Fluorescence-Techniques.html

[5] ttp://omlc.ogi.edu/spectra/PhotochemCAD/html/benzene.html

[6] ttp://pingu.salk.edu/flow/fluo.html

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