References
The table of lifetime standards provides you with lifetime data on standard fluorophores that have single-exponential decays. These data can be used to test your lifetime instrumentation for systematic errors. For convenience we have divided them into nanosecond and picosecond standards.
Nanosecond Lifetime Standards | Lifetime (ns) | Conditions for Lifetime Measurement | Excitation (nm) | Emission (nm) | Ref. |
---|---|---|---|---|---|
NADH | 0.4 | 0.1 M PB 7.4, 20 °C | 330 - 370 | 400 - 600 | 1 |
NATA | 3.0 | 0.1 M PB 7.0, 20 °C | 275 | 310 - 400 | 1 |
p-Terphenyl | 1.05 | Ethanol | 280 - 340 | 310 - 412 | 2 |
PPD | 1.20 | Ethanol | 240 - 340 | 310 - 440 | 2 |
PPO | 1.4 | Ethanol | 280 - 350 | 330 - 480 | 2 |
POPOP | 1.35 | Ethanol | 280 - 390 | 370 - 540 | 2 |
Dimethyl-POPOP | 1.45 | Ethanol | 300 - 400 | 390 - 560 | 2 |
2-Aminopurine | 11.34 | Water | 290 | 380 | 2 |
L-Tyrosine | 3.27 | Water | 285 | 300 | 2 |
Anthranilic Acid | 8.67 | Water | 290 | 400 | 2 |
Indole | 4.49 | Water | 290 | 360 | 2 |
Fluorescein, dianion | 4.1 ± 0.1 | NaOH/Water | 400 | 490 - 520 | 3 |
Rhodamine B | 1.74 ± 0.02 | Water, 20 °C | 400 | 583 | 4 |
PB = phosphate buffer
NATA = N-Acetyl-L-tryptophanamide
PPD = 1.5-diphenyl-1,3,4-oxadiazole
PPO = 2.5-diphenyl-oxazole
POPOP = 1, 4-bis(5-phenyloxazole-2-yl)benzene
Picosecond Lifetime Standards | Lifetime (ps) | Conditions for Lifetime Measurement | Excitation (nm) | Emission (nm) | Ref. |
---|---|---|---|---|---|
DMS | 880 | Cyclohexane, 25 °C | 280 - 375 | 375 - 475 | 2 |
DFS | 328 | Cyclohexane, 25 °C | 280 - 375 | 375 - 450 | 2 |
DBS | 176 | Cyclohexane, 25 °C | 280 - 385 | 375 - 475 | 2 |
DCS | 66 | Cyclohexane, 25 °C | 280 - 420 | 300 - 500 | 2 |
Rose Bengal | 519 | Methanol, 25 °C | 575 | 572 | 2 |
DMS = 4-dimethylamino-4-methoxystilbene
DFS = 4-dimethylamino-4-fluorostilbene
DBS = 4-dimethylamino-4-bromostilbene
DCS = 4-dimethylamino-4-cyanostilbene
References
- J.R. Lakowicz
Principles of Fluorescence Spectroscopy, 1st Ed.
Plenum Press, New York, London, 1983. - J.R. Lakowicz
Principles of Fluorescence Spectroscopy, 3rd Ed.
Springer Science+Business Media, LLC, 2006, p. 883-886. - D. Magde, G.E. Rojas, and P. Seybold
Solvent Dependence of the Fluorescence Lifetimes of Xanthene Dyes.
Photochem. Photobiol. 70, 737, 1999. - Boens, N., Qin, W., Basaric, N., Hofkens, J., Ameloot, M., Pouget, J., Lefevre, J-P., Valeur, B., Gratton, E., vandeVen, M., Silva, N.D., Jr., Engelborghs, Y., Willaert, K., Sillen, A., Rumbles, G., Phillips, D., Visser, A.J.W.G., van Hoek, A., Lakowicz, J.R., Malak, H., Gryczynski, I., Szabo, A.G., Krajcarski, D.T., Tamai, N., Miura, A.
Analytical Chemistry, 79(5), p. 2137-2149
All of the following parameters and more may influence the measured lifetime value.
- Temperature
- Buffer
- Presence of Oxygen (a fluorescence quencher)
- Concentration
- Purity of the Fluorophore Dye
- Quality of Equipment Used (Including Cuvettes and Optical Filters)
Check values against literature, and see above references for more detail.
The most frequently used method of determining the quantum yield of a fluorophore is by comparison with a standard of known quantum yield. The table of quantum yield standards lists dyes that are frequently used as standards in such relative quantum yield measurements.
Quantum Yield (QY) Standards | QY (%) | Conditions for QY Measurement | Excitation (nm) | Ref. |
---|---|---|---|---|
Cy3 | 4 | PBS | 540 | 1 |
Cy5 | 27 | PBS | 620 | 1 |
Cresyl Violet | 54 | Methanol, 22 °C | 540 - 640 | 2 |
Fluorescein | 95 ± 3 | 0.1 M NaOH, 22 °C | 496 | 2 |
POPOP | 97 | Cyclohexane | 300 | 2 |
Quinine Sulfate | 57.7 | 0.1 M H2SO4, 22 °C | 350 | 2 |
Rhodamine 101 | 100 | Ethanol | 450 - 465 | 2 |
Rhodamine 6G | 94 | Ethanol | 488 | 2 |
Rhodamine B | 31 | Water | 514 | 3 |
Tryptophan | 13 ± 1 | Water | 280 | 2 |
Tyrosine | 14 ± 1 | Water | 275 | 2 |
PBS = phosphate-buffered saline
POPOP = 1, 4-bis(5-phenyloxazole-2-yl)benzene
References
- R.B. Mujumdar, L.A. Ernst, S.R. Mujumdar, C.J. Lewis, A.S. Waggoner
Cyanine dye labeling reagents: sulfoindocyanine succinimidyl esters.
Bioconj Chem 4, 105-111, 1993. - J.R. Lakowicz
Principles of Fluorescence Spectroscopy, 3rd Ed.
Springer Science+Business Media, LLC, 2006, p. 54. - D. Magde, G.E. Rojas, and P. Seybold
Solvent Dependence of the Fluorescence Lifetimes of Xanthene Dyes.
Photochem. Photobiol. 70, 737, 1999.
The following data tables provide the names and lifetimes of lifetime standards that are recommended for use with LEDs and laser diodes listed below.
LEDs
Center Wavelength (nm) | Standard | Solvent | Lifetime (ns) |
---|---|---|---|
280 | P-Terphenyl | Ethanol | 1.05 |
PPO | Ethanol | 1.46 | |
295 | P-Terphenyl | Ethanol | 1.05 |
PPO | Ethanol | 1.46 | |
300 | P-Terphenyl | Ethanol | 1.05 |
PPO | Ethanol | 1.46 | |
330 | Dimethyl-POPOP | Ethanol | 1.45 |
BBO | Ethanol | 1.24 | |
370 | Dimethyl-POPOP | Ethanol | 1.45 |
BBO | Ethanol | 1.24 | |
460 | Fluorescein | PBS | 4 |
BodipyFL | Water | 5.8 | |
480 | Fluorescein | PBS | 4 |
BodipyFL | Water | 5.8 | |
520 | Rhodamine B | Water | 1.7 |
Rhodamine 590 | Water | 4.1 |
PBS = phosphate-buffered saline
POPOP = 1,4-bis(5-phenyloxazole-2-yl)-benzene
PPO = 2,5-diphenyl-oxazole
BBO = 2,5-bis([1,1'-biphenyl]-4-yl)-oxazole
Laser Diodes
Center Wavelength (nm) | Standard | Solvent | Lifetime (ns) |
---|---|---|---|
370 | Dimethyl-POPOP | Ethanol | 1.45 |
BBO | Ethanol | 1.24 | |
405 | Coumarin 6 | Ethanol | 2.5 |
Dimethyl-POPOP | Ethanol | 1.45 | |
435 | Coumarin 6 | Ethanol | 2.5 |
Fluorescein | PBS | 4 | |
470 | Fluorescein | PBS | 4 |
BodipyFL | Water | 5.8 | |
635 | Cy5 | Water | 1 |
Alexa Fluor 647 | Water | 1 | |
680 | Alexa Fluor 700 | Water | 1 |
Alexa Fluor 750 | Water | 0.66 | |
780 | Indocyanine Green | Water | 0.52 |
The following data table contains the mean lifetimes, absorption and emission maxima of the free and bound forms of important fluorescent probes for ion recognition.
Fluorescent Probes | Mean Lifetime (ns) | Absorption Max (nm) | Emission Max (nm) | |||
---|---|---|---|---|---|---|
free | bound | free | bound | free | bound | |
a) Calcium Probes | ||||||
Fura-2 | 1.09 | 1.68 | 362 | 335 | 500 | 503 |
Indo-1 | 1.4 | 1.66 | 349 | 331 | 482 | 398 |
Ca-Green | 0.92 | 3.66 | 506 | 506 | 534 | 534 |
Ca-Orange | 1.20 | 2.31 | 555 | 555 | 576 | 576 |
Ca-Crimson | 2.55 | 4.11 | 588 | 588 | 610 | 612 |
Quin-2 | 1.35 | 11.6 | 356 | 336 | 500 | 503 |
b) Magnesium Probes | ||||||
Mg-Quin-2 | 0.84 | 8.16 | 353 | 337 | 487 | 493 |
Mg-Green | 1.21 | 3.63 | 506 | 506 | 532 | 532 |
c) Potassium Probe | ||||||
PBFI | 0.52 | 0.59 | 350 | 344 | 546 | 504 |
d) Sodium Probe | ||||||
Sodium Green | 1.13 | 2.39 | 507 | 507 | 532 | 532 |
e) pH Probes | ||||||
SNAFL-1 | 1.19 | 3.74 | 539 | 510 | 616 | 542 |
Carboxy-SNAFL-1 | 1.11 | 3.67 | 540 | 508 | 623 | 543 |
Carboxy-SNAFL-2 | 0.94 | 4.60 | 547 | 514 | 623 | 545 |
Carboxy-SNARF-1 | 1.51 | 0.52 | 576 | 549 | 638 | 585 |
Carboxy-SNARF-2 | 1.55 | 0.33 | 579 | 552 | 633 | 583 |
Carboxy-SNARF-6 | 1.03 | 4.51 | 557 | 524 | 635 | 559 |
Carboxy-SNARF-X | 2.59 | 1.79 | 575 | 570 | 630 | 600 |
Resorufin | 2.92 | 0.45 | 571 | 484 | 587 | 578 |
BCECF | 4.49 | 3.17 | 503 | 484 | 528 | 514 |
Reference:
- J.R. Lakowicz (Editor)
Topics in Fluorescence Spectroscopy (Vol. 4): Probe Design and Chemical Sensing
Plenum Press, New York and London, 1994.
The following data table contains the mean lifetimes, absorption and emission maxima of the free and bound forms of important fluorescent probes for ion recognition.
Fluorescent Proteins | Extinction Coefficient | Q.Y. | Exmax (nm) | Emmax (nm) | pH Dependence EC 50 | Rel. Bleaching Time |
---|---|---|---|---|---|---|
EBEFP | 31,000 | 0.25 | 383 | 445 | 5.8 | 3 |
ECEFP | 26,000 | 0.40 | 434 | 477 | 4.7 | 85 |
EGEFP | 55,000 | 0.60 | 489 | 508 | 5.9 | 100 |
EYEP | 84,000 | 0.61 | 514 | 527 | 6.5 | 35 |
dsRed | 72,500 | 0.68 | 558 | 583 | 4.3 | 145 |
References:
- Patterson et al.
J. Cell Science 114 (5), 837, 2001. - Baird et al.
Proc. Natl. Acad. Sci. USA 97, 11984, 2000. - Matz et al.
Nat. Biotechnol. 17, 969, 1999.
The following data tables provide you with information on the fluorescent lifetimes, excitation and emission wavelengths of Selected fluorescent dyes, probes and labels that are frequently used for biological applications and in biomedical research.
Fluorophore | Lifetime (ns) | Solvent | Exmax (nm) | Emmax (nm) |
---|---|---|---|---|
5-Hydroxytryptamine | 370 - 415 | 520 - 540 | ||
ATTO 565 | 3.4 | Water | 561 | 585 |
ATTO 655 | 3.6 | Water | 655 | 690 |
Acridine Orange | 2.0 | PB pH 7.8 | 500 | 530 |
Acridine Yellow | 470 | 550 | ||
Alexa Fluor 488 | 4.1 | PB pH 7.4 | 494 | 519 |
Alexa Fluor 532 | 530 | 555 | ||
Alexa Fluor 546 | 4.0 | PB pH 7.4 | 554 | 570 |
Alexa Fluor 633 | 3.2 | Water | 621 | 639 |
Alexa Fluor 647 | 1.0 | Water | 651 | 672 |
Alexa Fluor 680 | 1.2 | PB pH 7.5 | 682 | 707 |
BODIPY 500/510 | 508 | 515 | ||
BODIPY 530/550 | 534 | 554 | ||
BODIPY FL | 5.7 | Methanol | 502 | 510 |
BODIPY TR-X | 5.4 | Methanol | 588 | 616 |
Cascade Blue | 375 | 410 | ||
Coumarin 6 | 2.5 | Ethanol | 460 | 505 |
CY2 | 489 | 506 | ||
CY3B | 2.8 | PBS | 558 | 572 |
CY3 | 0.3 | PBS | 548 | 562 |
CY3.5 | 0.5 | PBS | 581 | 596 |
CY5 | 1.0 | PBS | 646 | 664 |
CY5.5 | 1.0 | PBS | 675 | 694 |
Dansyl | 340 | 520 | ||
DAPI | 0.16 | TRIS/EDTA | 341 | 496 |
DAPI + ssDNA | 1.88 | TRIS/EDTA | 358 | 456 |
DAPI + dsDNA | 2.20 | TRIS/EDTA | 356 | 455 |
DPH | 354 | 430 | ||
Erythrosin | 529 | 554 | ||
Ethidium Bromide - no DNA | 1.6 | TRIS/EDTA | 510 | 595 |
Ethidium Bromide + ssDNA | 25.1 | TRIS/EDTA | 520 | 610 |
Ethidium Bromide + dsDNA | 28.3 | TRIS/EDTA | 520 | 608 |
FITC | 4.1 | PB pH 7.8 | 494 | 518 |
Fluorescein | 4.0 | PB pH 7.5 | 495 | 517 |
FURA-2 | 340 - 380 | 500 - 530 | ||
GFP | 3.2 | Buffer pH 8 | 498 | 516 |
Hoechst 33258 - no DNA | 0.2 | TRIS/EDTA | 337 | 508 |
Hoechst 33258 + ssDNA | 1.22 | TRIS/EDTA | 349 | 466 |
Hoechst 33258 + dsDNA | 1.94 | TRIS/EDTA | 349 | 458 |
Hoechst 33342 - no DNA | 0.35 | TRIS/EDTA | 336 | 471 |
Hoechst 33342 + ssDNA | 1.05 | TRIS/EDTA | 350 | 436 |
Hoechst 33342 + dsDNA | 2.21 | TRIS/EDTA | 350 | 456 |
HPTS | 5.4 | PB pH 7.8 | 454 | 511 |
Indocyanine Green | 0.52 | Water | 780 | 820 |
Laurdan | 364 | 497 | ||
Lucifer Yellow | 5.7 | Water | 428 | 535 |
Nile Red | 485 | 525 | ||
Oregon Green 488 | 4.1 | Buffer pH 9 | 493 | 520 |
Oregon Green 500 | 2.18 | Buffer pH 2 | 503 | 522 |
Oregon Green 514 | 511 | 530 | ||
Prodan | 1.41 | Water | 361 | 498 |
Pyrene | > 100 | Water | 341 | 376 |
Rhodamine 101 | 4.32 | Water | 496 | 520 |
Rhodamine 110 | 4.0 | Water | 505 | 534 |
Rhodamine 123 | 505 | 534 | ||
Rhodamine 6G | 4.08 | Water | 525 | 555 |
Rhodamine B | 1.68 | Water | 562 | 583 |
Ru(bpy)3[PF6]2 | 600 | Water | 455 | 605 |
Ru(bpy)2(dcpby)[PF6]2 | 375 | Buffer pH 7 | 458 | 650 |
SeTau-380-NHS | 32.5 | Water | 270 | 480 |
SeTau-404-NHS | 9.3 | Water | 402 | 515 |
SeTau-405-NHS | 9.3 | Water | 405 | 518 |
SeTau-425-NHS | 26.2 | Water | 340 425 |
545 |
SITS | 336 | 438 | ||
SNARF | 480 | 600-650 | ||
Stilbene SITS, SITA | 365 | 460 | ||
Texas Red | 4.2 | Water | 589 | 615 |
TOTO-1 | 2.2 | Water | 514 | 533 |
YOYO-1 no DNA | 2.1 | TRIS/EDTA | 457 | 549 |
YOYO-1 + ssDNA | 1.67 | TRIS/EDTA | 490 | 510 |
YOYO-1 + dsDNA | 2.3 | TRIS/EDTA | 490 | 507 |
YOYO-3 | 612 | 631 |
PBS = phosphate buffered saline pH 7.4
PB = phosphate buffer
TRIS/EDTA (1mM, pH 7.4) = tris(hydroxymethyl)aminomethane/ethylenediamine-tetraacetic acid.
ss = single-stranded
ds = double-stranded
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