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
  1. J.R. Lakowicz
    Principles of Fluorescence Spectroscopy, 1st Ed.
    Plenum Press, New York, London, 1983.
  2. J.R. Lakowicz
    Principles of Fluorescence Spectroscopy, 3rd Ed.
    Springer Science+Business Media, LLC, 2006, p. 883-886.
  3. D. Magde, G.E. Rojas, and P. Seybold
    Solvent Dependence of the Fluorescence Lifetimes of Xanthene Dyes.
    Photochem. Photobiol. 70, 737, 1999.
  4. 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
  1. 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.
  2. J.R. Lakowicz
    Principles of Fluorescence Spectroscopy, 3rd Ed.
    Springer Science+Business Media, LLC, 2006, p. 54.
  3. 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:

  1. 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:

  1. Patterson et al.
    J. Cell Science 114 (5), 837, 2001.
  2. Baird et al.
    Proc. Natl. Acad. Sci. USA 97, 11984, 2000.
  3. 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

NIRS
Near Infrared Spectroscopy for applications to tissues uses excitation wavelengths in the range from 670 nm through 900 nm; at these wavelengths, the absorption properties of tissue are such that a measurable amount of light can pass through large volumes of tissue. Below 650 nm the absorption of hemoglobin increases to the point that no measurable light can travel through the tissue; above 900 nm the absorption of water makes detection of light passing through tissue difficult. Thus, between 670 and 900 nm there is a unique window within which tissues can be probed by near infrared light; the main absorbers of the tissues in the region are the oxygenated and deoxygenated hemoglobin, and to a lesser extent, water, fat and cytochrome oxidase.
FD–NIRS
Frequency Domain Near Infrared Spectroscopy allows to measure and determine the absorption and scattering coefficients of the tissue (rather than making assumptions on their statistical values or using the differential path length factor).
In frequency domain systems, the NIR laser sources are (a.) either an amplitude modulated sinusoidally at frequencies near one hundred megahertz (100 MHz); or (b.) a train of pulses with a repetition rate of the order of 10 - 50 MHz. The instrumentation for FD–NIRS can be implemented following two paths: (a.) using one single modulation frequency for the excitation source and collecting the signal at three or more locations from the injection point (multi-distance approach); or (b.) use multiple modulation frequencies for the excitation source and collect the signal at one location.
In both implementations, three distinct quantities are measured and recorded: the intensity of the detected signal, its modulation ratio with respect to source modulation and the time the signal takes to traverse the tissue (phase shift). From these measurements the absorption and scattering coefficients of the tissue are determined and, hence, the oxy- and deoxy-hemoglobin concentration of the tissue. The main and unique advantages of FD–NIRS is the capability to provide an absolute baseline of the oxygenation level without making any assumptions and to monitor changes in the oxygenation of the tissues with sampling rates up to 50 Hz.
In ISS instrumentation, the light source is modulated at high frequency (110 MHz) and delivered to the subject through the sensor at four different distances from the location of the collector fiber (multi-distance technique); the distances vary from 1.5 cm to 4.0 cm. Three quantities are measured and recorded: the intensity of the detected signal, its modulation ratio with respect to the modulation of the source and the time the signal takes to traverse the tissue (phase shift). From these parameters the absorption and scattering coefficients of the tissue are determined and, hence, the oxy- and deoxy-hemoglobin concentrations. In some instruments the role of the emitters' fibers and collector is reversed: light is injected at one location and it is collected at four different locations. The main and unique advantage of FDNIRS is the capability to provide an absolute baseline of the oxygenation level and to monitor changes in the oxygenation in real time.
fNIRS
Functional Near Infrared Spectroscopy is a technique used to obtain information on brain activity following a stimulus (optical, visual, acoustic, etc.). The activity is monitored through the detection of temporal changes in the local concentration of oxy- and deoxy-hemoglobin due to neuron activation. The localization of the signal is confined to a volume of about 5 mm3; the temporal resolution is of the order of 200 ms.
Imagent uses wavelengths at 690 nm and 830 nm; the fibers are paired so that at each contact location of the emitter fibers photons of both wavelengths are emitted. The headgear allows for the user to probe the subject's head with different montages of sources-detectors patterns.
DOT
Diffuse Optical Tomography uses the fNIRS technique to reconstruct a 3D image of the region affected by changes of the hemodynamics of the tissue under examination. The 2D image reconstruction is sometimes named "Diffuse Optical Topography".
EROS
Event Related Optical Signal is an fNIRS technique that, instead of using changes in absorption due to the hemodynamics to infer the cognitive response to the stimulus, processes the information carried by the scattering component of the optical signal that probes the cerebral cortex. It is presumed that the changes in the signal are due to the changes in the shape of glia and neurons that are associated with neuron firing (which may be due to the movements of water and ions through the membrane) or to changes in the optical parameters of the membrane itself through the activation. As EROS does not use the changes in absorption due to the hemodynamics, it is a more direct measurement of the cellular activity; it is capable of localizing the brain activity within millimeters with a time scale of a few milliseconds.
The Imagent configuration used for EROS detection usually features one excitation wavelength only (830 nm is preferred as it has a better efficiency penetration in the tissue than the 690 nm) and the fibers are not paired. Two parameters are measured: (1.) the amount of light emitted by the source that reaches the detector; (2.) the phase delay (or time delay) of the photons that reach the detector. The event-related measures are recorded by synchronizing the recording to the stimulus presentation. The EROS signal elicited by a given stimulus is analyzed with respect to a pre-stimulus baseline, recorded right before the stimulus presentation.