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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- 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.