What Is The Chemical Makeup Of A Fluorescent Molecule?

Agents that emit lite after excitation by light

A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that tin re-emit low-cal upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with several π bonds.^[ane]

Fluorophores are sometimes used alone, equally a tracer in fluids, every bit a dye for staining of certain structures, as a substrate of enzymes, or equally a probe or indicator (when its fluorescence is afflicted by environmental aspects such as polarity or ions). More than generally they are covalently bonded to a macromolecule, serving every bit 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, via its amine-reactive isothiocyanate derivative fluorescein isothiocyanate (FITC), has been i of the nearly pop fluorophores. From antibody labeling, the applications have spread to nucleic acids cheers to carboxyfluorescein (FAM), TET, ...). Other historically mutual fluorophores are derivatives of rhodamine (TRITC), coumarin, and cyanine.^[2] Newer generations of fluorophores, many of which are proprietary, often perform amend, beingness more than photostable, brighter, and/or less pH-sensitive than traditional dyes with comparable excitation and emission.^{[three] [4]}

Fluorescence [edit]

The fluorophore absorbs light free energy of a specific wavelength and re-emits lite at a longer wavelength. The captivated wavelengths, energy transfer efficiency, and fourth dimension before emission depend on both the fluorophore construction and its chemical surround, every bit the molecule in its excited land interacts with surrounding molecules. Wavelengths of maximum absorption (≈ excitation) and emission (for instance, 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 across 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 low-cal into the near infrared region.

Master characteristics of fluorophores are:

• Maximum excitation and emission wavelength (expressed in nanometers (nm)): corresponds to the peak in the excitation and emission spectra (commonly 1 peak each).
• Molar absorption coefficient (in Molar⁻¹cm⁻¹): links the quantity of absorbed light, at a given wavelength, to the concentration of fluorophore in solution.
• Breakthrough 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 footing state. It refers to the time taken for a population of excited fluorophores to decay to one/due east (≈0.368) of the original corporeality.
• Stokes shift: difference between the maximum excitation and maximum emission wavelengths.
• Night fraction: proportion of the molecules agile in fluorescence emission. For quantum dots, prolonged single-molecule microscopy showed that 20-90% of all particles never emit fluorescence.^[5] On the other manus, conjugated polymer nanoparticles (Pdots) bear witness almost no dark fraction in their fluorescence.^[6] Fluorescent proteins tin can have a dark fraction from poly peptide misfolding or lacking chromophore formation.^[7]

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

Fluorophores can likewise be used to quench the fluorescence of other fluorescent dyes (meet article Quenching (fluorescence)) or to relay their fluorescence at fifty-fifty longer wavelength (run into commodity Förster resonance free energy transfer (FRET)).

See more on fluorescence principle.

Size (molecular weight) [edit]

Virtually fluorophores are organic minor molecules of 20 - 100 atoms (200 - 1000 Dalton - the molecular weight may exist higher depending on grafted modifications, and conjugated molecules), just there are also much larger natural fluorophores that are proteins: dark-green fluorescent protein (GFP) is 27 kDa and several phycobiliproteins (PE, APC...) are ≈240kDa. In 2020, the smallest known fluorophore was claimed to be 3-hydroxyisonicotinaldehyde, a compound of 14 atoms and but 123 Da.^[8]

Fluorescence particles like breakthrough dots: ii-10 nm diameter, 100-100,000 atoms, are also considered fluorophores.^[9]

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

Families [edit]

Fluorescence of different substances nether UV light. Green is a fluorescein, cerise is Rhodamine B, yellow is Rhodamine 6G, blue is quinine, purple is a mixture of quinine and rhodamine 6g. Solutions are nearly 0.001% concentration in water.

Fluorophore molecules could be either utilized alone, or serve every bit a fluorescent motif of a functional system. Based on molecular complexity and synthetic methods, fluorophore molecules could exist generally classified into four categories: proteins and peptides, small organic compounds, synthetic oligomers and polymers, and multi-component systems.^{[ten] [xi]}

Fluorescent proteins GFP (green), YFP (yellow) and RFP (carmine) tin exist attached to other specific proteins to grade a fusion protein, synthesized in cells afterward transfection 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
• Squaraine derivatives and ring-substituted squaraines, including Seta and Square dyes
• Squaraine rotaxane derivatives: See Tau dyes
• 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: pour blue, etc.
• Oxazine derivatives: Nile red, Nile bluish, 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
• Dipyrromethene derivatives: BODIPY, aza-BODIPY

These fluorophores fluoresce due to delocalized electrons which tin jump a band and stabilize the free energy captivated. Benzene, ane of the simplest aromatic hydrocarbons, for case, is excited at 254 nm and emits at 300 nm.^[12] This discriminates fluorophores from quantum dots, which are fluorescent semiconductor nanoparticles.

They tin can exist attached to protein to specific functional groups, such as - amino groups (active ester, carboxylate, isothiocyanate, hydrazine), carboxyl groups (carbodiimide), thiol (maleimide, acetyl bromide), organic azide (via click chemical science or non-specifically (glutaraldehyde)).

Additionally, diverse functional groups can exist present to alter its properties, such as solubility, or confer special properties, such equally boronic acrid 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'south polarity (solvatochromic), hence called environment-sensitive. Often dyes are used within cells, which are impermeable to charged molecules, as a result of this the carboxyl groups are converted into an ester, which is removed past esterases inside the cells, eastward.grand., fura-2AM and fluorescein-diacetate.

The post-obit dye families are trademark groups, and do not necessarily share structural similarities.

• CF dye (Biotium)
• DRAQ and CyTRAK probes (BioStatus)
• BODIPY (Invitrogen)
• EverFluor (Setareh Biotech)
• Alexa Fluor (Invitrogen)
• Bella Fluor (Setareh Biotech)
• 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, RPEPerCP, Phycobilisomes) (Columbia Biosciences)
• APC, APCXL, RPE, BPE (Phyco-Biotech, Greensea, Prozyme, Flogen)
• Vio Dyes (Miltenyi Biotec)

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

Examples of ofttimes encountered fluorophores [edit]

Reactive and conjugated dyes [edit]

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 Bluish	403	455	406	Maleimide
Pacific Orange	403	551
3-Hydroxyisonicotinaldehyde	385	525	123	QY 0.15; pH sensitive
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-Colour, Breakthrough Cerise
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
G-Dye100	498	524		suitable for protein labeling and electrophoresis
Thou-Dye200	554	575		suitable for protein labeling and electrophoresis
Thousand-Dye300	648	663		suitable for protein labeling and electrophoresis
G-Dye400	736	760		suitable for protein labeling and electrophoresis
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.v	581	594;(640)	1102	QY 0.xv
Cy5	(625);650	670	792	QY 0.28
Cy5.5	675	694	1272	QY 0.23
Cy7	743	767	818	QY 0.28
TRITC	547	572	444	TRITC
Ten-Rhodamine	570	576	548	XRITC
Lissamine Rhodamine B	570	590
Texas Red	589	615	625	Sulfonyl chloride
Allophycocyanin (APC)	650	660	104 grand
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 [edit]

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	Deoxyribonucleic acid
Chromomycin A3	445	575		CG-selective
Mithramycin	445	575
YOYO-1	491	509	1271
Ethidium Bromide	210;285	605	394	in aqueous solution
GelRed	290;520	595	1239	Non-toxic substitute for Ethidium Bromide
Acridine Orange	503	530/640		DNA/RNA
SYTOX Green	504	523	~600	DNA
TOTO-i, TO-PRO-one	509	533		Vital stain, TOTO: Cyanine Dimer
TO-PRO: Cyanine Monomer
Thiazole Orange	510	530
CyTRAK Orange	520	615	-	(Biostatus) (red excitation nighttime)
Propidium Iodide (PI)	536	617	668.4
LDS 751	543;590	712;607	472	Deoxyribonucleic acid (543ex/712em), RNA (590ex/607em)
seven-AAD	546	647		7-aminoactinomycin D, CG-selective
SYTOX Orangish	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 [edit]

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

Fluorescent proteins [edit]

Dye	Ex (nm)	Em (nm)	MW	QY	BR	PS	Notes
GFP (Y66H mutation)	360	442
GFP (Y66F mutation)	360	508
EBFP	380	440		0.xviii	0.27		monomer
EBFP2	383	448			20		monomer
Azurite	383	447			xv		monomer
GFPuv	385	508
T-Sapphire	399	511		0.threescore	26	25	weak dimer
Cerulean	433	475		0.62	27	36	weak dimer
mCFP	433	475		0.xl	13	64	monomer
mTurquoise2	434	474		0.93	28		monomer
ECFP	434	477		0.15	3
CyPet	435	477		0.51	18	59	weak dimer
GFP (Y66W mutation)	436	485
mKeima-Reddish	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.ix	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	twoscore		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.seven		dimer, (Evrogen)
ZsYellow1	529	539		0.65	xiii		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[13]
mKO	548	559		0.sixty	31	122	monomer
TurboRFP	553	574	26 chiliad	0.67	62		dimer, (Evrogen)
tdTomato	554	581		0.69	95	98	tandem dimer
TagRFP	555	584			l		monomer (Evrogen)
DsRed monomer	556	586	~28k	0.one	iii.v	xvi	monomer, (Clontech)
DsRed2 ("RFP")	563	582	~110k	0.55	24		(Clontech)
mStrawberry	574	596		0.29	26	15	monomer
TurboFP602	574	602	26 thou	0.35	26		dimer, (Evrogen)
AsRed2	576	592	~110k	0.21	13		tetramer, (Clontech)
mRFP1	584	607	~30k	0.25			monomer, (Tsien lab)
J-Cherry	584	610		0.20	eight.eight	13	dimer
R-phycoerythrin (RPE)	565 >498	573	250 kDa	0.84			heterotrimer[thirteen]
B-phycoerythrin (BPE)	545	572	240 kDa	0.98			heterotrimer[thirteen]
mCherry	587	610		0.22	16	96	monomer
HcRed1	588	618	~52k	0.03	0.six		dimer, (Clontech)
Katusha	588	635			23		dimer
P3	614	662	~10,000 kDa				phycobilisome complex[13]
Peridinin Chlorophyll (PerCP)	483	676	35 kDa				trimer[xiii]
mKate (TagFP635)	588	635			xv		monomer (Evrogen)
TurboFP635	588	635	26 thou	0.34	22		dimer, (Evrogen)
mPlum	590	649	51.4 thousand	0.x	4.one	53
mRaspberry	598	625		0.15	13		monomer, faster photobleach than mPlum
mScarlet	569	594		0.70	71	277	monomer[14]

Abbreviations:

• Ex (nm): Excitation wavelength in nanometers
• Em (nm): Emission wavelength in nanometers
• MW: Molecular weight
• QY: Quantum yield
• BR: Effulgence: Tooth absorption coefficient * quantum yield / 1000
• PS: Photostability: time [sec] to reduce brightness past l%

Applications [edit]

Fluorophores have particular importance in the field of biochemistry and protein studies, e.g., in immunofluorescence simply besides in jail cell analysis,^[15] e.thousand. immunohistochemistry^{[3] [16]} and pocket-size molecule sensors.^{[17] [xviii]}

Uses outside the life sciences [edit]

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

• Multi-ton scale usages in cloth dyeing and optical brighteners in laundry detergents
• Avant-garde cosmetic formulations; safety equipment and article of clothing
• Organic low-cal-emitting diodes (OLED)
• Fine arts and design (posters and paintings)
• Synergists for insecticides and experimental drugs
• Equally a dye in highlighters to give off a glow-like issue
• Solar panels to collect more low-cal / wavelengths

See also [edit]

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

References [edit]

1. ^ Juan Carlos Stockert, Alfonso Blázquez-Castro (2017). "Affiliate 3 Dyes and Fluorochromes". Fluorescence Microscopy in Life Sciences. Bentham Science Publishers. pp. 61–95. ISBN978-1-68108-519-vii . Retrieved 24 December 2017.
2. ^ Rietdorf J (2005). Microscopic Techniques. Advances in Biochemical Engineering / Biotechnology. Berlin: Springer. pp. 246–9. ISBNiii-540-23698-8 . Retrieved 2008-12-13 .
3. ^ ^{a b} Tsien RY; Waggoner A (1995). "Fluorophores for confocal microscopy". In Pawley JB (ed.). Handbook of biological confocal microscopy. New York: Plenum Press. pp. 267–74. ISBN0-306-44826-2 . Retrieved 2008-12-13 .
4. ^ Lakowicz, JR (2006). Principles of fluorescence spectroscopy (3rd ed.). Springer. p. 954. ISBN978-0-387-31278-1.
5. ^ Pons T, Medintz IL, Farrell D, Wang 10, Grimes AF, English DS, Berti 50, Mattoussi H (2011). "Unmarried-molecule colocalization studies shed light on the idea of fully emitting versus dark single quantum dots". Small. 7 (xiv): 2101–2108. doi:10.1002/smll.201100802. PMID 21710484.
6. ^ Koner AL, Krndija D, Hou Q, Sherratt DJ, Howarth M (2013). "Hydroxy-terminated conjugated polymer nanoparticles have near-unity bright fraction and reveal cholesterol-dependence of IGF1R nanodomains". ACS Nano. vii (2): 1137–1144. doi:10.1021/nn3042122. PMC3584654. PMID 23330847.
7. ^ Garcia-Parajo MF, Segers-Nolten GM, Veerman JA, Greve J, van Hulst NF (2000). "Real-time light-driven dynamics of the fluorescence emission in single green fluorescent protein molecules". PNAS. 97 (13): 7237–7242. Bibcode:2000PNAS...97.7237G. doi:10.1073/pnas.97.13.7237. PMC16529. PMID 10860989.
8. ^ Cozens, Tom (2020-12-16). "Fluorescent molecule breaks size tape for light-green-emitting dyes". chemistryworld.com . Retrieved 2021-12-03 .
9. ^ Li Z, Zhao X, Huang C, Gong 10 (2019). "Recent advances in green fabrication of luminescent solar concentrators using nontoxic quantum dots equally fluorophores". J. Mater. Chem. C. 7 (40): 12373–12387. doi:x.1039/C9TC03520F.
10. ^ Liu, J.; Liu, C.; He, Westward. (2013), "Fluorophores and Their Applications as Molecular Probes in Living Cells", Curr. Org. Chem., 17 (6): 564–579, doi:10.2174/1385272811317060003
11. ^ Juan Carlos Stockert, Alfonso Blázquez-Castro (2017). "Chapter four Fluorescent Labels". Fluorescence Microscopy in Life Sciences. Bentham Scientific discipline Publishers. pp. 96–134. ISBN978-one-68108-519-vii . Retrieved 24 Dec 2017.
12. ^ Omlc.ogi.edu
13. ^ ^{a b c d eastward} Columbia Biosciences
14. ^ Bindels, Daphne Due south.; Haarbosch, Lindsay; van Weeren, Laura; Postma, Marten; Wiese, Katrin Eastward.; Mastop, Marieke; Aumonier, Sylvain; Gotthard, Guillaume; Royant, Antoine; Hink, Marker A.; Gadella, Theodorus West. J. (January 2017). "mScarlet: a bright monomeric red fluorescent protein for cellular imaging". Nature Methods. 14 (1): 53–56. doi:ten.1038/nmeth.4074. ISSN 1548-7105. PMID 27869816. S2CID 3539874.
15. ^ Sirbu, Dumitru; Luli, Saimir; Leslie, Jack; Oakley, Fiona; Benniston, Andrew C. (2019). "Enhanced in vivo Optical Imaging of the Inflammatory Response to Acute Liver Injury in C57BL/6 Mice Using a Highly Brilliant Well-nigh-Infrared BODIPY Dye". ChemMedChem. 14 (10): 995–999. doi:10.1002/cmdc.201900181. ISSN 1860-7187. PMID 30920173. S2CID 85544665.
16. ^ Taki, Masayasu (2013). "Chapter 5. Imaging and sensing of cadmium in cells". In Astrid Sigel; Helmut Sigel; Roland Thousand. O. Sigel (eds.). Cadmium: From Toxicology to Essentiality. Metal Ions in Life Sciences. Vol. 11. Springer. pp. 99–115. doi:ten.1007/978-94-007-5179-8_5. PMID 23430772.
17. ^ Sirbu, Dumitru; Butcher, John B.; Waddell, Paul G.; Andras, Peter; Benniston, Andrew C. (2017-09-xviii). "Locally Excited Country-Charge Transfer Country Coupled Dyes as Optically Responsive Neuron Firing Probes" (PDF). Chemistry - A European Journal. 23 (58): 14639–14649. doi:10.1002/chem.201703366. ISSN 0947-6539. PMID 28833695.
18. ^ Jiang, Xiqian; Wang, Lingfei; Carroll, Shaina L.; Chen, Jianwei; Wang, Meng C.; Wang, Jin (2018-08-20). "Challenges and Opportunities for Small-Molecule Fluorescent Probes in Redox Biology Applications". Antioxidants & Redox Signaling. 29 (6): 518–540. doi:10.1089/ars.2017.7491. ISSN 1523-0864. PMC6056262. PMID 29320869.

External links [edit]

• The Database of fluorescent dyes
• Table of fluorochromes
• The Molecular Probes Handbook - a comprehensive resource for fluorescence technology and its applications.

Source: https://en.wikipedia.org/wiki/Fluorophore

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