NISTIR 7458 

Standard Guide to Fluorescence 

- Instrument Calibration 

and Validation 

Paul C. DeRose 

Biochemical Science Division 

National Institute of Standards and Technology 

Gaithersburg, MD 20899-8312 

1 
NISTIR 7458 

Standard Guide to Fluorescence 

- Instrument Calibration 

and Validation 

Paul C. DeRose 

Biochemical Science Division 

National Institute of Standards and Technology 

Gaithersburg, MD 20899-8312 

October 2007 

U.S. DEPARTMENT OF COMMERCE 

Carlos M. Gutierrez,Secretary 

TECHNOLOGY ADMINISTRATION 

Robert C. Cresanti,Under Secretary of Commerce for Technology 

NATIONAL INSTITUTE OF STANDARDS AND TECHNOLOGY 

James M. Turner, Acting Director

2 
3 

Table of Contents 

1. Scope........................................................................................................ 4

2. Wavelength Accuracy.............................................................................. 4

2.1 Low Pressure Atomic Lamps.............................................................................. 5 

2.2 Dy-YAG crystal.................................................................................................. 5 

2.3 Eu-doped glass or PMMA .................................................................................. 5 

2.4 Anthracene-doped PMMA ................................................................................. 7 

2.5 Ho2O3 solution or doped glass with diffuse reflector, scatterer or fluorescent 

dye ............................................................................................................................ 7 

2.6 Xe Source Lamp ................................................................................................. 7 

2.7 Instrument Source with diffuse reflector or scatterer ......................................... 9 

2.8 Water Raman ...................................................................................................... 9 

3. Spectral Slit Width Accuracy................................................................. 10

4. Linearity of the Detection System ......................................................... 10

5. Spectral Correction of Detection System Responsivity......................... 11

5.1 Calibrated Light Source (CS) – Tungsten Lamp .............................................. 12 

5.2 Calibrated Detector (CD) with Calibrated Diffuse Reflector (CR) .................. 12 

5.3 Certified Reference Materials (CRMs)............................................................. 13 

6. Spectral Correction of Excitation Beam Intensity................................. 13

6.1 Calibrated Detector - Si Photodiode (CD – Si)................................................. 14 

6.2 Quantum Counters............................................................................................ 15 

6.3 Si Photodiode (uncalibrated)............................................................................. 15 

7. Calibration Curves for Concentration.................................................... 15

7.1 Fluorophores with specified purity & uncertainty............................................ 15 

7.2 Fluorophore Solutions with specified concentration & uncertainty ................. 16 

7.3 Molecules of Equivalent Soluble Fluorophore (MESF)................................... 16 

8. Day-to-Day and Instrument-to-Instrument Intensity............................. 16

8.1 Cuvette Format.................................................................................................. 17 

8.2 Microwell Plate Format .................................................................................... 17 

8.3 Microarray Format............................................................................................ 17 

9. Limit of Detection and Sensitivity......................................................... 17

10. Lifetimes ................................................................................................ 17

11. Fluorescence Quantum Yield................................................................. 18

11.1 Absolute methods.............................................................................................. 18 

11.1.1 Optically Dilute Samples (A < 0.05) ........................................................ 18 

11.1.2 Optically Dense Samples.......................................................................... 19 

11.1.3 Integrating Sphere at Sample .................................................................... 19 

11.2 Relative methods............................................................................................... 20 

12. DEFINITION OF TERMS .................................................................... 20

13. Other Guideline/Recommendation Documents..................................... 23

References..................................................................................................... 23
1. Scope 

To list the available materials and methods for each type of calibration or correction for 

fluorescence instruments (spectral emission correction, wavelength accuracy, etc.) with a 

general description, the level of quality, precision and accuracy attainable, caveats, and 

useful references given for each entry. The listed materials and methods are intended for 

the qualification of fluorometers as part of complying with regulatory and other QA/QC 

requirements. Precision and accuracy or uncertainty are given at a 1 σ confidence level 

and are approximated in cases where these values have not been well established.1

2. Wavelength Accuracy 

Methods for determining the accuracy of the emission (EM) or excitation (EX) 

wavelength for a fluorescence instrument are given here with an emphasis on 

monochromator (mono) based wavelength selection. An example spectrum, which has 

been spectrally corrected, is given for each method. 

Table 1 : Summary of Methods for Determining Wavelength Accuracy 

N

Y

N

Y

N

N

Y

Y

Established

Values

limited 

range

380nm-450nm (EM) Y Y ± 0.2 nm

310nm-380nm (EX)

Anthracene

in PMMA

limited 12

range,

calibration

Xe Source 400nm-500nm (EX) Y Y ± 0.2 nm

EX mono 12

must be 

calibrated

Xe Source + UV-NIR Maybe Y ± 0.2 nm

DR

EX mono 13

must be 

calibrated

Water UV-blue Y Y ± 0.2 nm

Raman

need blank

alignment

Caveats

9-11

5, 6

3

1, 2

Ref.

330nm-800nm Maybe Y ± 0.4 nm

(EM or EX)

Ho2O3 + DR 

570nm-700nm (EM) Y Y ± 0.2 nm

360nm-540nm (EX)

Eu glass

470nm-760nm (EM) Y Y ± 0.1 nm

255nm-480nm (EX)

Dy-YAG 

crystal

± 0.1 nm or 

better

Pen Lamp UV-NIR (EM) Maybe Y

Off- Uncertainty

Shelf

Sample λ region Drop-In

N

Y

N

Y

N

N

Y

Y

Established

Values

limited 

range

380nm-450nm (EM) Y Y ± 0.2 nm

310nm-380nm (EX)

Anthracene

in PMMA

limited 12

range,

calibration

Xe Source 400nm-500nm (EX) Y Y ± 0.2 nm

EX mono 12

must be 

calibrated

Xe Source + UV-NIR Maybe Y ± 0.2 nm

DR

EX mono 13

must be 

calibrated

Water UV-blue Y Y ± 0.2 nm

Raman

need blank

alignment

Caveats

9-11

5, 6

3

1, 2

Ref.

330nm-800nm Maybe Y ± 0.4 nm

(EM or EX)

Ho2O3 + DR 

570nm-700nm (EM) Y Y ± 0.2 nm

360nm-540nm (EX)

Eu glass

470nm-760nm (EM) Y Y ± 0.1 nm

255nm-480nm (EX)

Dy-YAG 

crystal

± 0.1 nm or 

better

Pen Lamp UV-NIR (EM) Maybe Y

Off- Uncertainty

Shelf

Sample λ region Drop-In

 

1

 Certain commercial equipment, instruments, or materials are identified in this paper to foster 

understanding. Such identification does not imply recommendation or endorsement by the National 

Institute of Standards and Technology, nor does it imply that the materials or equipment identified are 

necessarily the best available for the purpose. 

4 
2.1 Low Pressure Atomic Lamps [1, 2]

0

0.2

0.4

0.6

0.8

1

290 310 330 350 370 390 410 430

Wavelength (nm)

Relative Intensity

These low pressure atomic lamps, often referred to as pen lamps due to their size and 

shape, should be placed at the sample position and pointed toward the detection system 

for emission wavelength accuracy determination. The emission wavelength selector 

(λEM-selector) is then scanned over the wavelength range of interest. (see Fig. 1) High 

accuracy is only achieved when the lamp is aligned properly into the wavelength selector, 

e.g., the light fills the entrance slit of the monochromator. Atomic lines that are too close 

to other atomic lines to be resolved by the instrument should not be used, i.e., may not be 

appropriate for low resolution instruments. Although these lamps can be placed at the 

excitation source position for excitation wavelength accuracy determination, weaker 

signals are typically observed, e.g., by a reference detector, and alignment is more 

difficult than for the emission wavelength accuracy determination. 

Fig. 1: Hg pen lamp spectrum 

2.2 Dy-YAG crystal [3]

This sample is available in standard cuvette format, so it can simply be dropped into a 

cuvette holder. An excitation or emission spectrum is then collected for an excitation or 

emission wavelength accuracy determination, respectively. (see Fig. 2) Peaks that are too 

close to other peaks to be resolved by the instrument should not be used, i.e., may not be 

appropriate for low resolution instruments. 

2.3 Eu-doped glass [4] or PMMA [5, 6]

This sample is available in standard cuvette format, so it can simply be dropped into a 

cuvette holder. An excitation or emission spectrum is then collected for an excitation or 

emission wavelength accuracy determination, respectively. (see Fig. 3) Accurate peak 

5 
0

0.2

0.4

0.6

0.8

1

450 500 550 600 650 700 750 800

Wavelength (nm)

Relative Intensity

positions for this glass have not been well established and the positions of peaks can 

change somewhat depending on the particular glass matrix used. For these reasons, a onetime per sample determination of these peak positions using another wavelength 

calibration method is recommended. 

Fig. 2: Emission spectrum of a Dy-YAG crystal excited at 352.7 nm. 

0

0.2

0.4

0.6

0.8

1

570 620 670 720

Wavelength (nm)

Relative Intensity

Fig. 3: Emission spectrum of a Eu-ion-doped glass excited at 392 nm. 

6 
2.4 Anthracene-doped PMMA [7, 8] 

This sample is available in standard cuvette format, so it can simply be dropped into a 

cuvette holder. An excitation or emission spectrum is then collected for an excitation or 

emission wavelength accuracy determination, respectively. (see Fig. 4) 

0

0.2

0.4

0.6

0.8

1

370 390 410 430 450 470

Wavelength (nm)

Relative Intensity

Fig. 4: Emission spectrum of anthracene-doped PMMA excited at 360 nm. 

2.5 Ho2O3 solution or doped glass with diffuse reflector, scatterer or fluorescent 

dye [9-11]

This sample is available in standard cuvette format, so it can simply be dropped into a 

cuvette holder. An excitation or emission spectrum is then collected for an excitation or 

emission wavelength accuracy determination, respectively. The wavelength selector not 

being scanned must be removed or set to zero order. The diffuse reflector, scatterer or 

fluorescent dye is scanned with and without the Ho2O3 sample in place and the ratio of 

the two intensities is calculated to obtain an effective transmittance spectrum with dips in 

the intensity ratio corresponding to absorption peaks of the sample. (see Fig. 5) 

2.6 Xe Source Lamp [12] 

This method is for fluorometers that use a high pressure Xe arc lamp as an excitation 

source. A few peaks between 400 and 500 nm can be used, but most of these are due to 

multiple lines, so their positions are not well established. (see Fig. 6) For this reason, a 

determination of these peak positions (one-time per lamp) using another wavelength 

calibration method is recommended. For EX wavelength calibration the excitation 

wavelength selector (λEX-selector) is scanned while collecting the reference detector 

7 
0.2

0.4

0.6

310 360 410 460 510 560 610 660

Wavelength (nm)

Intensity Ratio

Fig. 5: Effective transmittance spectrum of a Ho2O3 doped glass with diffuse reflector. 

0.6

0.7

0.8

0.9

1

430 440 450 460 470 480 490 500

Wavelength (nm)

Relative Intensity

Fig. 6: Xe source lamp (high pressure, 450 W) spectrum in a spectral region containing peak structure. 

signal. If this is used for EM wavelength calibration, a diffuse reflector or scatterer must 

be placed at the sample position and the λEX-selector must be removed or set to zero 

order. 

8 
2.7 Instrument Source with diffuse reflector or scatterer [12]

A dilute scattering solution in a standard cuvette or a solid diffuse reflector at 45 degrees 

relative to the excitation beam can be used to scatter the excitation beam into the 

detection system. One wavelength selector is fixed at a wavelength of interest and the 

other scans over the fixed wavelength. (see Fig. 7) The difference between the fixed 

wavelength and the observed peak position is the wavelength bias between the two 

wavelength selectors at that wavelength. Either the excitation or the emission wavelength 

selector must have a known accuracy at the desired wavelengths in order to use this 

method to calibrate the unknown side. 

0

0.2

0.4

0.6

0.8

1

403 404 405 406

Wavelength (nm)

Relative Intensity

Fig. 7: Excitation source profile with excitation wavelength fixed at 404.3 nm (excitation bandwidth of 1.0 

nm) and emission monochromator scanned (emission bandwidth of 0.1 nm) 

2.8 Water Raman [13] 

Deionized water is used. One wavelength selector is fixed at a wavelength of interest and 

the other is scanned. (see Fig. 8) The water Raman peak appears at a wavelength that is 

about 3400 cm-1 lower in energy than the excitation wavelength. [14] The Raman 

scattering intensity is proportional to λ-4, so the Raman intensity quickly becomes too 

weak to use this method when going into the visible region. Either the excitation beam or 

the emission wavelength selector must have a known accuracy at the desired wavelengths 

in order to use this method to calibrate the unknown side. 

9 
0

0.2

0.4

0.6

0.8

1

382 387 392 397 402 407 412

Wavelength (nm)

Relative Intensity

Fig. 8: Water Raman spectrum with excitation wavelength set at 350 nm and excitation and emission 

bandwidths at 5 nm. 

3. Spectral Slit Width Accuracy 

Spectral slit width accuracy of the emission or excitation wavelength selector can be 

determined by measuring the spectral bandwidth, taken to be the full width at half the 

peak maximum (FWHM), of a single line of a pen lamp, using the same setup and with 

the same caveats described in section 2.1. [1] For fluorescence spectrometers with both 

excitation and emission monochromators, an alternative method may be used where one 

monochromator is scanned over the position of the other, using the setup described in 

section 2.7 [12] The uncertainties involved in both methods have not been well 

established, but ± 0.5 nm or better is estimated here based on what has been reported. 

4. Linearity of the Detection System 

Several methods can be used to determine the linear intensity range of the detection 

system. They can be separated into three types, based on the tools used to vary the 

intensity of light reaching the detector: 1) double aperture, 2) optical filters and/or 

polarizers, 3) fluorophore concentrations. The double aperture method is the most well 

established and probably the most accurate when done correctly, but is also the most 

difficult to perform. [15, 16] A variety of methods using optical filters, polarizers or a 

combination of the two have been reported. [12, 17] These methods require high quality, 

often costly, components and some expertise on the part of the user. The third method is 

the most popular and easiest to implement. It uses a set of solutions obtained by serial 

10 
dilution of a fluorescent stock solution, similar to that used for obtaining calibration 

curves for analyte concentration, as described in section 5.1. In this case, solutions with 

low concentration (A<0.05 at 1cm pathlength) should be used and fluorophore adsorption 

to cuvette walls may affect measurements at vey low concentrations. [18] Users must 

insure that the fluorescence intensities of samples are reproducible and do not decrease 

over the time period that they are being excited and measured, because the organic dyes 

typically used can be prone to photobleaching. 

5. Spectral Correction of Detection System Responsivity 

11 

0

0.2

0.4

0.6

0.8

1

300 400 500 600 700 800

Wavelength (nm)

Relative Responsivity

Calibration of the relative responsivity of the emission detection system with emission 

wavelength, also referred to as spectral correction of emission, is necessary for successful 

quantification when intensity ratios at different emission wavelengths are being compared 

or when the true shape or peak maximum position of an emission spectrum needs to be 

known. Such a calibration is necessary because the relative spectral responsivity of a 

detection system can change significantly over its wavelength range (see Fig. 9). It is 

Fig. 9: Example of the relative spectral responsivity of an emission detection system (grating 

monochromator-PMT based), [12] for which a correction needs to be applied to a measured 

emission spectrum to obtain its true spectral shape (relative intensities). 

highly recommended that the wavelength accuracy (see section 2) and linear range of the 

detection system be determined (see section 4) before this calibration is performed and 

that appropriate steps are taken (e.g., the use of attenuators) to insure that all measured 

intensities during this calibration are within the linear range. Also note that when using 

an emission polarizer, the spectral correction for emission is dependent on the polarizer 

setting. In addition, users must be careful to insure that signal intensities and correction 
factors are expressed consistently, either in power units or in photon units. Some 

detectors, such as those with photon-counting capabilities and quantum counter detectors, 

give a signal that is proportional to the number of incident photons, while other detectors 

give a signal that is proportional to the incident power. Energy and photon signals can be 

interconverted easily (see Planck’s constant in section 12). 

Table 2 : Summary of Methods for Determining Spectral Correction of Detection System Responsivity 

Y

Y

Y

Certified

Values

difficult 

setup

difficult 

setup

Caveats

23-25

12, 

20, 21

12, 17 

20-22

Ref.

CRMs UV-NIR Y Y ± 5 %

CD + CR UV-NIR N Maybe ± 10 %

CS UV-NIR N Y < ± 5 %

Off- Uncertainty

Shelf

Sample λ region Drop-In

Y

Y

Y

Certified

Values

difficult 

setup

difficult 

setup

Caveats

23-25

12, 

20, 21

12, 17 

20-22

Ref.

CRMs UV-NIR Y Y ± 5 %

CD + CR UV-NIR N Maybe ± 10 %

CS UV-NIR N Y < ± 5 %

Off- Uncertainty

Shelf

Sample λ region Drop-In

5.1 Calibrated Light Source (CS) – Tungsten [19] Lamp [12, 20, 21, 17, 22]

The light from a CS is directed into the emission detection system by placing the CS at 

the sample position. If the CS is too large to be placed at the sample position, a calibrated 

diffuse reflector (CR) may be placed at the sample position to reflect the light from the 

CS into the emission detection system. The λEM-selector is scanned over the emission 

region of interest, using the same instrument settings as that used with the sample, and 

the signal channel output (S′′) is collected. The known radiance of the CS incident on the 

detection system (L) can be used to calculate the relative correction factor (CCS), such 

that CCS = L / S′′. The corrected emission intensity is equal to the product of the signal 

output of the sample (S) and CCS. 

5.2 Calibrated Detector (CD)2

 with Calibrated Diffuse Reflector (CR) [12, 20, 21]

This is a two-step method. The first step uses a CD to measure the flux of the excitation 

beam as a function of excitation wavelength, as described in section 3.1. The second step 

uses a CR with reflectance RCR to reflect a known fraction of the flux of the excitation 

beam into the detection system. This is done by placing the CD at the sample position at a 

45° angle, assuming a 0°/90° instrument geometry, and synchronously scanning both the 

λEX- and λEM-selectors over the emission region of interest while collecting both the 

signal output (S′) and the reference output (Rf′). This method enables the relative 

correction factor (CCD) to be calculated using the equation CCD = (CR RCR Rf′) / S′, see 

section 3.1 for definition of terms. 

 

2

 It is assumed in what follows that a calibrated detector is either a photodiode mounted inside an 

integrating sphere or a photodiode alone, whose spectral responsivity is known. The former is typically the 

more accurate of the two, because the integrating sphere insures spatially uniform illumination of the 

photodiode. 

12 
5.3 Certified Reference Materials (CRMs) [23-25]

The CRMs presently available are either organic dye solutions or solid, inorganic glasses 

released by national metrology institutes with certified relative fluorescence spectra, i.e., 

relative intensity and uncertainty values are given as a function of emission wavelength at 

a fixed excitation wavelength. They have been designed to closely resemble typical 

samples. A CRM is placed at the sample position and its spectrum is collected and 

compared to the certified spectrum according to the instructions given on the 

accompanying certificate, yielding spectral correction factors for the instrument. The 

corrected emission spectra of some commonly used dyes have also been reported recently 

in the literature. [26, 27] 

6. Spectral Correction of Excitation Beam Intensity 

Calibration of the excitation intensity with excitation wavelength is necessary for 

successful quantification when intensity ratios at different excitation wavelengths are 

being compared or when the true shape or peak maximum position of an excitation 

spectrum needs to be known. Such a calibration is necessary because the relative spectral 

flux of an excitation beam at the sample can change significantly over its wavelength 

range (see Fig. 10). The neglect of excitation intensity correction factors can cause even 

greater errors than that of emission correction factors. [12, 28] Fortunately, many 

fluorescence instruments have a built-in reference detection system to monitor the 

intensity of the excitation beam. This is usually done using a photodiode or a PMT, or a 

quantum counter detector to measure a fraction of the excitation beam that is split off 

from the rest of the beam. The collected reference signal can be used to correct the 

fluorescence signal for fluctuations due to changes in the excitation beam intensity. 

Reference detectors are often not calibrated with excitation wavelength, introducing 

errors, which can be particularly large over longer excitation wavelength ranges (e.g., 

greater than 50 nm) or in a wavelength region where the excitation intensity changes 

rapidly with excitation wavelength, such as the UV. It is highly recommended that the 

wavelength accuracy of the excitation wavelength selector (see section 2) and linear 

range of the detection system used to measure the excitation beam be determined (see 

section 4) before a spectral correction of the excitation beam is performed and that 

appropriate steps are taken (e.g., the use of attenuators) to insure that all measured 

intensities during this calibration are within the linear range. Signals and correction 

factors must be expressed consistently in either power or photon units (see section 5). 

Also note that when using an excitation polarizer, the spectral correction for excitation 

intensity is dependent on the polarizer setting. 

13 
Table 3 : Summary of Methods for Determining Spectral Correction of Excitation Beam Intensity 

N

N

Y

Certified

Values

limited 

range

difficult 

setup

Caveats

12

22, 29

12, 17

Ref.

Photodiode - UV-NIR N Y ≤ ± 50 %

Si

Quantum UV-NIR Y Y ± 5 %

Counter

CD - Si UV-NIR N Y ± 2 %

Off- Uncertainty

Shelf

Sample λ region Drop-In

N

N

Y

Certified

Values

limited 

range

difficult 

setup

Caveats

12

22, 29

12, 17

Ref.

Photodiode - UV-NIR N Y ≤ ± 50 %

Si

Quantum UV-NIR Y Y ± 5 %

Counter

CD - Si UV-NIR N Y ± 2 %

Off- Uncertainty

Shelf

Sample λ region Drop-In

6.1 Calibrated Detector - Si Photodiode (CD – Si) [12, 17]

A CD is put at the sample position with the excitation beam incident on it. The output of 

the CD (SCD) is measured as a function of emission wavelength by scanning the λEXselector over the excitation region of interest using the same instrument settings as that 

used with the sample. The known responsivity of the CD (RCD) is used to calculate the 

flux of the excitation beam (φx), such that φx = SCD / RCD. The instrument’s reference 

detector can also be used to measure the intensity of the excitation beam by measuring its 

output (RfCD) simultaneously with SCD. Then the correction factor for the responsivity of 

the reference detector CR = φx / RfCD. 

0

0.2

0.4

0.6

0.8

1

200 300 400 500 600 700 800

Wavelength (nm)

Relative Flux

Fig. 10: Example of the relative flux of an excitation beam (Xe lamp-grating monochromator 

based), [12] for which a correction needs to be applied to a measured excitation spectrum to 

obtain its true spectral shape (relative intensities). 

14 
6.2 Quantum Counters [22, 29]

A quantum counter solution is a concentrated dye solution that absorbs all of the photons 

incident on it and has an emission spectrum whose shape and intensity do not change 

with excitation wavelength. The quantum counter solution is placed at the sample 

position in a quartz cuvette. If front face detection is possible, then a standard cuvette can 

be used with the excitation beam at normal incidence. If 90-degree detection is only 

possible, then a right-triangular cuvette can be used with the excitation beam at 45-degree 

incidence to the hypotenuse side and one of the other sides facing the detector. Scan the 

excitation wavelength over the region of interest with the emission wavelength fixed at a 

position corresponding to the long-wavelength tail of the emission band and collect the 

signal intensity (SQC). The instrument’s reference detector can also be used to measure 

the intensity of the excitation beam by measuring its output (RfQC) simultaneously with 

SQC. Then the correction factor for the responsivity of the reference detector 

CR = SQC / RfCD is calculated. Note that each quantum counter has a limited range. For 

instance, Rhodamine B can achieve the specified uncertainty from 250 nm to 600 nm. 

Beyond this range the intensity falls off and uncertainties increase. Also note that SQC

will be proportional to the quantum flux at the sample, not the flux in power units. 

6.3 Si Photodiode (uncalibrated) [12]

This is used in the same way as a calibrated Si photodiode (see section 3.1), except its 

spectral responsivity is not known. A Si photodiode is sometimes erroneously assumed to 

have a responsivity that is qualitatively flat over its effective range. In fact, using its 

output to correct an excitation spectrum can lead to quantitatively significant errors, 

particularly over a large excitation range and in the UV region. Although, using an 

uncalibrated Si photodiode for such a correction will almost always yield a more accurate 

spectrum than when using no correction. 

7. Calibration Curves for Concentration 

Calibration curves of fluorescence intensity, i.e., instrument responsivity, as a function of 

fluorophore concentration can be determined for a particular instrument and fluorophore. 

Reference materials composed of the fluorophore of interest must be used. The highest 

accuracy is obtained when the fluorophore in both the standard and the sample 

experience the same microenvironment. For example, they are dissolved in the same 

solvent or attached to the same biomolecules. This type of calibration enables 

concentrations and amounts of fluorophores to be compared over time and between 

instruments without determining the absolute responsivity of the instrument (see section 

V). 

 

7.1 Fluorophores with specified purity & uncertainty [30]

If the purity of a fluorophore (e.g., a high-purity, organic dye powder) is known, then it 

can be put in the same microenvironment (e.g., solvent) as an unknown sample to 

15 
produce a standard sample. Several standard samples should be produced to cover the 

concentration range of interest. These standard samples are measured under the same 

conditions as that of any unknowns and the fluorescence intensities are recorded. 

Fluorescence intensity versus standard sample concentration is plotted and the points are 

fitted to a polynomial, typically a straight line. The concentration of an unknown is 

determined by using the fitted polynomial along with the measured intensity of the 

unknown to find the corresponding concentration.

7.2 Fluorophore Solutions with specified concentration & uncertainty [31, 32]

Standard solutions with known concentrations can be used in the same way as a standard 

fluorophore (see section 4.1). In this case the fluorophores are in solution, so they are 

ready to use or they can be diluted to produce standard solutions of lower concentration. 

In both cases, the solvent used in the standard and unknown solutions should be the same. 

7.3 Molecules of Equivalent Soluble Fluorophore (MESF) [33-35]

The matching of microenvironments between sample and standard solutions, as 

emphasized in the last two sections, cannot always be achieved. This is of particular 

concern when the sample contains immobilized fluorophores, e.g., those attached to a 

cell. In many such cases, it is very difficult or impossible to determine the concentration 

of fluorophores in a candidate standard solution. MESF units are used, particularly in 

flow cytometry, to quantify such complex systems. These units express the fluorescence 

intensity of a fluorescent analyte, e.g., several immobilized fluorophores bound to a 

microbead or cell, as the corresponding number of free fluorophores of the same type in a 

standard solution with the same intensity. The MESF scale for a particular fluorophore is 

determined using the same procedure as that given in section 4.2. This scale is transferred 

from a conventional fluorometer to a flow cytometer, using fluorophore-labeled 

microbead suspensions with pre-determined MESF values. 

8. Day-to-Day and Instrument-to-Instrument Intensity 

The stability of an instrument over time and comparability between instruments of 

fluorescence intensity is made possible by performance validation standards. The 

fluorescence intensity of such standards can be monitored over time and between 

instruments, enabling an absolute intensity scale to be established without performing 

absolute fluorescence measurements. These standards must emit a fluorescence intensity 

that does not change with time or irradiation. Another possibility is that they be singleuse standards that can be made with a highly reproducible fluorescence intensity. It is not 

necessary for these standards to reproduce the exact spectrum of analyte samples, but 

they should be measurable with routine instrument settings, e.g., typical excitation 

intensity, bandwidths and emission wavelengths. 

 

16 
8.1 Cuvette Format [25, 5 , 6 , 8 , 24 , 23]

This is the most commonly used format in conventional, benchtop fluorometers, as well 

as in many portable instruments. Both solid and liquid standards are available for this 

format and most can be used in both 0°/90° and front-face geometries. Standards of this 

type have been released by NMIs and industry, but the most well known of these is high 

purity water, where its Raman line is used as a pseudo-fluorescence signal.[36, 37, 13] 

Unfortunately, the “Water Raman” method is effectively limited to the UV to violet 

region of the spectrum. Inorganic solid standards are the most robust, most photostable, 

longest lasting and easiest-to-use of fluorescent samples available in a cuvette format, 

although organic dyes may more closely resemble the behavior of fluorescent probes. 

8.2 Microwell Plate Format [38, 6 , 39]

Some solid materials, similar to those used to make some of the cuvette standards 

mentioned in section 5.1, have been used to make microwell plate reference materials. 

These have typically been made by taking a microplate-sized piece of the material and 

putting a mask over it that mimics the well boundaries. Organic dye solutions, such as 

those used in section 5.1, can be put into the empty microwells of any plate and used as a 

standard. Of course, a fresh dye solution of known concentration has to be dispensed each 

time such a standard is used. The reference materials described here can be used as dayto-day intensity standards for filter-based instruments if the same filter is always used. 

For instrument-to-instrument comparisons or when filters are changed, the spectral 

differences between filters must be considered. No microwell plate standards have been 

recommended by NMIs or generally accepted by the community at-large to this point. 

8.3 Microarray Format [40-43]

Standard slides containing arrays of fluorescent dye samples with a morphology and 

intensity that is more consistent than that of a typical microarray sample are 

commercially available. No microarray standards have been recommended by NMIs or 

generally accepted by the community at-large to this point. 

9. Limit of Detection and Sensitivity [44, 13]

Methods and reference materials used in section 8 can also be used for determining limit 

of detection and sensitivity by employing samples that approach the limit of detection of 

the instrument, i.e., within two orders of magnitude of the background intensity of a 

blank or of the noise of the detection system, and comparing the sample intensity to the 

background intensity or noise. 

10. Lifetimes [45, 46]

Time-domain and frequency-domain measurements are the two types of fluorescence 

measurements used to determine fluorescence lifetimes. Conventional instruments 

include those based on time-correlated single-photon counting (time-domain) and 

17 
multifrequency phase and modulation (frequency-domain) techniques, which are 

typically used to measure lifetimes from picoseconds to microseconds. More simple, 

time-domain instruments are commonly used to measure lifetimes on the order of 

milliseconds or longer. Criteria for fluorescence lifetime standards include 1) high purity, 

2) a single exponential decay component, and 3) a lifetime independent of excitation and 

emission wavelengths. Possibly, the most thorough comparison of fluorescence lifetime 

candidates was recently performed by nine expert laboratories. [45] Almost all of the 

candidates mentioned in the literature for use as lifetime standards have been liquid, 

organic dye solutions, probably due to the more complex, excited-state kinetics that exist 

in most solid fluorescent samples. Lifetime standards are measured in the same way as 

typical unknown samples. A bias in the measured lifetime or an observed multiexponential decay of the standard indicates the presence of systematic errors in the 

instrument. 

11. Fluorescence Quantum Yield [47]

Defined as the ratio of the number of molecules that fluoresce a photon to the number of 

molecules that absorb a photon from the excitation source, this quantity is an intrinsic 

property of a particular molecular species. Sample effects, such as inner filter effects, 

often introduce errors into measured quantum yield values. Absolute quantum yields can 

be particularly difficult to measure accurately, due to instrumental errors that need to be 

avoided or corrected and experimental setups requiring some expertise on the part of the 

investigator. Because of this, relative quantum yields are much more commonly 

measured than absolute by employing a species with a known quantum yield as a 

reference. This makes the accuracy of the unknown, relative quantum yield dependent on 

the accuracy of the known, reference value. Unfortunately, the fluorescence quantum 

yields of very few species have been well established. Absolute spectroscopic methods 

also use a reference, e.g., a diffuse scatterer, but no reference value needs to be known in 

advance, as the reference has an effective quantum yield of one. Only methods that can 

use a conventional fluorescence spectrometer as a detector are summarized below. All of 

these methods are capable of yielding quantum yield values with an accuracy of ± 10 %, 

when systematic uncertainties are minimized. 

11.1 Absolute methods 

11.1.1 Optically Dilute Samples (A < 0.05) 

This most commonly used absolute method was developed by Weber and Teale.[48] The 

variation of this method described here, using a spectrometer as a detector, was first done 

by Eastman.[49] An optically dilute scattering solution is used as a reference, such as 

colloidal silica or glycogen. A right angle geometry and identical instrument settings 

(e.g., excitation wavelength, emission range, bandwidths, etc.) should be used for both 

sample and reference measurements. In addition, the response of the detector should not 

be polarization dependent and the excitation light should be unpolarized, or correction 

factors should be determined to compensate for these. If all of these conditions are not 

met, then large systematic errors may result. 

18 
Solutions at several optical densities, preferable all with A < 0.05, of both the fluorescent 

sample (f subscript) and the scatterer (s subscript) are made and their absorbances (A) 

and emission spectra with fluorescence signals (S) are measured at the excitation 

wavelength. The values of Sf/Af and Ss/As extrapolated to zero optical density, mf and ms, 

respectively, are calculated. [50] The response of the detection system (Rd) as a function 

of emission wavelength is determined. The polarization (p) and the refractive index (n) of 

the sample and the reference are measured. The quantum yield (φ) is equal to a 

summation over the emission wavelength range of the product Rd (mf / ms) (nf / ns)

2

 

(3+pf)/(3+ps). Alternatively, the emission monochromator can be set to zero-order, so all 

wavelengths reach the detector at once, thereby, removing the summation from the 

equation. This is less tedious and closer to Weber and Teale’s original method where no 

emission monochromator was used, although stray light is more likely to introduce error 

in this case. 

11.1.2 Optically Dense Samples 

This method, first described by Vavilov,[51] is similar to the Weber and Teale method, 

but a solid, diffuse scatterer, e.g., barium sulfate or sintered polytetrafluoroethylene, with 

a known diffuse reflectance is used as a reference. Front face detection with the sample 

parallel to the detector is the best optical geometry. [52] This method requires a detailed 

knowledge of the optical geometry of the instrument used, among other complications. 

Therefore, this method is only recommended when optically dense samples must be used. 

11.1.3 Integrating Sphere at Sample [53]

An integrating sphere can be placed at the sample position of a fluorescence spectrometer 

with the sample placed inside the sphere. This eliminates the need for refractive index, 

polarization, and spatial anisotropy corrections. Excitation and emission ports, facing the 

excitation beam and detection system (right angle geometry), respectively, are open on 

the integrating sphere. The light coming out of the exit port is collected by the detection 

system. If the sample holder in the sphere is able to rotate the sample in and out of the 

direct path of the excitation beam, this enables secondary excitation (reabsorption) and 

emission (reemission) to be subtracted out, and the absorptance (α) of a sample to be 

measured accurately with the same system. The response of the sample-integrating 

sphere-detection system must first be determined as a function of wavelength. The 

quantum yield and absorptance can then be calculated using the following equations: 

α

α φ

empty

in out

f X

E −− )1( E = out

out in

X

− XX

α = (1) 

where Ein and Eout are the integrated emission spectra of the sample after direct (sample in 

the beam path) and secondary (sample out of the beam path) excitation, respectively. X is 

the integrated profile of the excitation, obtained with the integrating sphere-detection 

system. Both the emission spectra and the excitation profile are collected with the 

excitation beam set at a fixed wavelength and the appropriate spectral range being chosen 

19 
or scanned by the detection system. The subscripts empty, in and out refer to an empty 

sphere (no sample), the sample directly in the path of the beam, and the sample in the 

sphere but out of the direct path of the beam, respectively. 

11.2 Relative methods 

A relative method using an optically dilute quantum yield standard (reference) with a 

quantum yield that is known with high accuracy is the most commonly used method for 

quantum yield determination. Absorbance values (A) and emission spectra for the 

unknown sample and standard reference are measured at their corresponding excitation 

wavelengths. A reference detector is used to measure the relative intensity of the 

excitation beam simultaneously with the collection of the emission spectra. The response 

of the detection system (Rd) as a function of emission wavelength is determined and used 

to correct the emission spectra. The following equation is used to calculate the quantum 

yield: 

⎥

⎦

⎤ ⎢

⎣

⎡

⎥

⎦

⎤ ⎢

⎣

⎡

⎥

⎥

⎦

⎤

⎢

⎢

⎣

⎡

⎥

⎥

⎦

⎤

⎢

⎢

⎣

⎡ =

r

f

r

f

ff

rr

ff

rr

rf E

E

n

n

I

I

A

A

)(

)(

)(

)(

λ

λ

λ

λ φφ , (2) 

where n is refractive index at the peak emission wavelength, I(λ) is the relative excitation 

intensity at excitation wavelength λ, E is the integrated area under the corrected emission 

spectrum and subscripts f and r refer to the unknown and reference samples, respectively. 

The fluorescence quantum yields of only a short list of compounds have been 

characterized to the extent necessary to be used as standards [54], with quinine sulfate 

being the most thoroughly established. [30] 

This same procedure can be used to measure the relative quantum yield of optically dense 

samples. In this case, the absorbance ratio in eq. (2) is equal to one, since the excitation 

beam is absorbed completely, thereby simplifying the equation. Optically dense samples 

are used for both the unknown and the reference. Unfortunately there are many 

complications associated with measuring optically dense samples, making this method 

less accurate than its optically dilute counterpart, in most cases. 

12. DEFINITION OF TERMS [55, 56]

absorption coefficient (α) – a measure of absorption of radiation from an incident beam 

as it traverses a sample according to Bouguer’s law, I/Io = e-αb

, where I and I0 are the 

transmitted and incident intensities, respectively, and b is the pathlength of the beam 

through the sample. Note that transmittance T = I/Io and absorbance A = – log T 

Beer-Lambert law (or Beer’s law or Beer-Lambert-Bouquer law) – relates the 

dependence of the absorbance (A) of a sample on its pathlength (see absorption 

coefficient, α) and concentration (c), such that A = αbc 

20 
calibrated detector (CD) - a light detector whose responsivity as a function of 

wavelength has been determined along with corresponding uncertainties. [57] 

calibrated light source (CS) – a light source whose radiance as a function of wavelength 

has been determined along with corresponding uncertainties. [58, 59] 

calibrated diffuse reflector (CR) - a Lambertian reflector whose reflectance as a 

function of wavelength has been determined along with corresponding uncertainties. [60] 

certified reference material (CRM) – a material with properties of interest, whose 

values and corresponding uncertainties have been certified by a standardizing group or 

organization. 

diffuse scatterer – a material that scatters light in multiple directions; this includes 

diffuse reflectors, which are often Lambertian, and scattering solutions, which are not 

Lambertian. 

fluorescence anisotropy (r) – a measure of the degree of polarization of fluorescence, 

defined as r = (Ill – I⊥) / (Ill + 2I⊥), where Ill and I⊥ are the observed fluorescence 

intensities when the fluorometer’s emission polarizer is oriented parallel and 

perpendicular, respectively, to the direction of the polarized excitation.

fluorescence band – a region of a fluorescence spectrum where the intensity passes 

through a maximum, usually corresponding to a discrete electronic transition 

fluorescence lifetime – parameter describing the time decay of the fluorescence intensity 

of a sample component; if a sample decays by first-order kinetics, this is the time 

required for its fluorescence intensity and corresponding excited state population to 

decrease to 1/e of its initial value. 

fluorescence quantum efficiency – the ratio of the number of fluorescence photons 

leaving an emitter versus the number of photons absorbed. 

fluorescence quantum yield (Φ) – the probability that a molecule or species will 

fluoresce once it has absorbed a photon. This quantity is an innate property of the species 

and is typically calculated for a sample as the ratio of the number of molecules that 

fluoresce versus the number of molecules that absorbed. 

flux (or radiant flux) – rate of propagation of radiant energy, typically expressed in 

watts (W); spectral flux is the flux per unit spectral bandwidth, typically expressed in W / 

nm. 

grating equation – describes the relationship between the angle of diffraction and 

wavelength of radiation incident on a grating, i.e., mλ = d (sinα + sinβ), where d is the 

groove spacing on the grating, α and β are the angles of the incident and diffracted 

21 
wavefronts, respectively, relative to the grating normal and m is the diffraction order, 

which is an integer.[61] 

inner filter effects – a decrease in the measured quantum efficiency of a sample due to 

significant absorption of the excitation beam and /or reabsorption of the emission of the 

sample by itself. This causes the measured quantum efficiency to be dependent on the 

absorbance, concentration, and excitation and emission pathlengths of the sample. [62, 

63] 

intensity – a measure of the amount of electromagnetic energy present. This general 

definition, which is used here, is synonymous with or directly proportional to the signal 

output of a photodetector or the flux of a sample or light source. A more specific 

definition, often used in radiometry, is “the radiant flux per unit solid angle from a point 

source,” which is typically expressed in W/sr. 

Lambertian reflector – a surface that reflects light according to Lambert’s law, i.e., the 

light is unpolarized and has a radiance that is isotropic or independent of viewing angle. 

limit of detection – an estimate of the lowest concentration of an analyte that can be 

measured with a given technique, often taken to be the analyte concentration with a 

measured signal to noise ratio of three. 

noise level – the peak-to-peak noise of a blank 

photobleaching – a loss of emission or absorption intensity by a sample due to exposure 

to light. This loss can be reversible or irreversible with the latter typically referred to as 

photodegradation or photodecomposition. 

Planck’s constant (h) - relates the energy (E) of a photon to its frequency (ν), such that 

E = hν. 

quantum counter – a photoluminescent emitter with a quantum efficiency that is 

independent of excitation wavelength over a defined spectral range. When a quantum 

counter is combined with a detector to give a response proportional to the number of 

incident photons, the pair is called a quantum counter detector. 

quasi-absolute fluorescence intensity scale – a fluorescence intensity scale that has 

been normalized to the intensity of a fluorescent reference sample or artifact under a 

fixed set of instrumental and experimental conditions. This artifact should be known to 

yield a fluorescence intensity that is reproducible with time and between instruments 

under the fixed set of conditions. 

Raman scattering – inelastic scattering of radiation (the wavelengths of the scattered 

and incident radiation are not equal) by a sample that occurs because of changes in the 

polarizability of the relevant bonds of a sample during a molecular vibration. The 

22 
radiation being scattered does not have to be in resonance with electronic transitions in 

the sample, unlike fluorescence. [64] 

Rayleigh scattering – elastic scattering of radiation by a sample, i.e., the scattered 

radiation has the same energy (same wavelength) as the incident radiation.

responsivity (spectral) – ratio of the photocurrent output and the radiant power collected 

by a light detection system. Spectral responsivity is the responsivity per unit spectral 

bandwidth. 

sensitivity – a measure of an instrument’s ability to detect an analyte under a particular 

set of conditions 

spectral bandwidth (or spectral bandpass or resolution) – a measure of the capability 

of a spectrometer to separate radiation or resolve spectral peaks of similar wavelengths. 

spectral slit width – the mechanical width of the exit slit of a spectrometer divided by 

the linear dispersion in the exit slit plane. 

transition dipole moment – an oscillating dipole moment induced in a molecular species 

by an electromagnetic wave that is resonant with an energy transition of the species, e.g., 

an electronic transition. Its direction defines that of the transition polarization, and its 

square determines the intensity of the transition. 

13. Other Guideline/Recommendation Documents [40, 65, 66] 

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2. keywords: standard pen lamp 

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23 
7. Other polyaromatic hydrocarbon doped PMMAs have narrow excitation and emission 

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