NISTIR 7457 

Recommendations and Guidelines for 

Standardization of Fluorescence Spectroscopy 

Paul C. DeRose 

Biochemical Science Division 

National Institute of Standards and Technology 

Gaithersburg, MD 20899-8312 

1 
NISTIR 7457 

Recommendations and Guidelines for 

Standardization of Fluorescence Spectroscopy 

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 
Table of Contents 

1. INTRODUCTION ................................................................................... 4

2.

........................................................................................ 6

QUALITATIVE AND QUANTITATIVE FLUORESCENCE 

MEASUREMENTS

2.1 Qualitative Fluorescence Measurements............................................................ 6 

2.2 Quantitative Fluorescence Measurements .......................................................... 7 

3. FACTORS AFFECTING QUANTIFICATION ..................................... 7

3.1 Instrument-Based Factors ................................................................................... 7 

3.2 Sample-Based Factors......................................................................................... 8 

4. APPARATUS COMPONENTS.............................................................. 9

4.1 Excitation Light Source .................................................................................... 10 

4.2 Excitation Wavelength Selector........................................................................ 10 

4.3 Sampling Device............................................................................................... 10 

4.4 Emission Wavelength Selector......................................................................... 10 

4.5 ............................................................................................................. 11 Detector

5. CALIBRATION OF FLUORESCENCE INSTRUMENTS ................. 11

5.1 Analyte Concentration – Calibration Curves.................................................... 11 

5.2 Emission Wavelength and Spectral Slit Width................................................. 12 

5.3 Excitation Wavelength and Spectral Slit Width ............................................... 12 

5.4 Linearity of the Detection System .................................................................... 13 

5.5 Signal Level (Relative Emission) ..................................................................... 13 

5.6 Reference Signal Level (Relative Excitation)................................................... 15 

5.7 Intensity & Sensitivity ...................................................................................... 17 

6.

.......................................................................................... 18

QUALIFICATION AND VERIFICATION OF FLUORESCENCE 

INSTRUMENTS

6.1 Wavelength Accuracy....................................................................................... 18 

6.2 Signal Level (Relative Emission) ..................................................................... 19 

6.3 Reference Signal Level (Relative Excitation)................................................... 19 

6.4 Intensity, Sensitivity & Performance Verification............................................ 19 

6.5 Fluorescence Lifetimes..................................................................................... 20 

7. METHOD VALIDATION..................................................................... 20

8. DEFINITION OF TERMS .................................................................... 20

REFERENCES ............................................................................................. 23

3 
1. INTRODUCTION 

Fluorescence is a two-step process requiring an initial absorption of light, followed by 

emission. Fluorescence spectroscopy is an electronic spectroscopic technique related to 

ultraviolet-visible-near infrared (UV-VIS-NIR) absorbance spectroscopy, but it is also a 

background-free technique with light emitted from the sample in all directions, similar to 

Raman spectroscopy. The initial absorption of a photon by a molecule in the sample 

promotes an electron to an excited state. The excited electron returns to the ground 

electronic state by emitting a photon. If the emission arises from an “allowed” transition, 

typically having a short lifetime between 1 ns and 10 ns, then it is called fluorescence. If 

the emission arises from a “forbidden” transition, typically having a long lifetime 

between 1 ms and 1 s, then it is called phosphorescence. Phosphorescence is usually less 

intense than fluorescence under similar conditions. Fluorescence spectroscopy is 

specifically discussed in what follows, but many of these issues also apply to 

phosphorescence. The basic concepts behind fluorescence spectroscopy have been well 

established [1, 2], but its applications [3, 4] and standardization are still expanding and 

progressing, making it a developing, not yet mature, technique. 

0

0.2

0.4

0.6

0.8

1

390 440 490 540 590

Wavelength (nm)

Relative Intensity

Excitation

Emission

Fig. 1: Fluorescence excitation and emission spectra for fluorescein in 

borate buffer with the wavelength axis being excitation and emission 

wavelength, respectively. 

The most common type of fluorescent sample is a dilute, transparent solution that absorbs 

light following the Beer-Lambert law and emits a corresponding fluorescence intensity 

directly proportional to the concentration, the absorption coefficient and the fluorescence 

quantum yield of the fluorescent species or fluorophore. A conventional fluorescence 

spectrometer has both excitation and emission wavelength selectors and a spectrum is 

collected by fixing the wavelength of one of the selectors and scanning the other 

wavelength selector over a range. When the excitation wavelength is fixed and the 

emission wavelength is scanned, the resulting spectrum is termed an emission spectrum. 

When the emission wavelength is fixed and the excitation wavelength is scanned, the 

4 
resulting spectrum is termed an excitation spectrum. (see Fig. 1) The fluorescence 

spectrum is plotted as relative intensity or counted photons of fluorescence versus 

wavelength. The appearance of a fluorescence spectrum is much like a UV-VIS-NIR 

absorbance spectrum. In fact, the shape or contour of an excitation spectrum is often 

identical to that of the corresponding absorbance spectrum for an organic dye in solution 

over the same wavelength range. 

Polyatomic fluorophores in condensed media, e.g., solutions, thin films and solids, at 

room temperature exist in the ground or excited electronic state in a broad distribution of 

vibrational energy levels, which causes homogeneous broadening of excitation or 

emission spectra, respectively. A microenvironment or shell also surrounds each 

fluorophore in condensed media and differences in the structure of this shell between 

individual fluorophores causes inhomogeneous broadening. These two types of 

broadening cause fluorescence spectra to be broader than some other types of spectra 

(e.g., IR absorbance or Raman), with the typical width of a fluorescence band being 

between 10 nm and 100 nm. Once electronically excited, a polyatomic fluorophore 

experiences vibrational relaxation before emitting a photon, causing a red shift, or Stokes 

shift, of the fluorescence spectrum relative to the wavelength at which it was excited. 

Few naturally-occurring biological compounds fluoresce strongly. This apparent 

disadvantage has been turned into an advantage by the synthesis of fluorescent probes 

designed to bind to target analytes only. A large number of analyte-specific fluorophores 

are now commercially available, which bind to a comparable number of biological target 

molecules. [5, 6] Many of these fluorescent probes, such as fluorescein, rhodamine and 

their derivatives, have relatively large absorption coefficients and quantum yields close to 

one, i.e., they fluoresce nearly as many photons as they absorb. Fluorescence techniques 

are also termed “background-free,” since very little excitation light reaches the detector. 

These advantages make fluorescence detection highly sensitive, down to single molecule 

detection in some cases. Specificity and sensitivity are two of the most significant 

strengths of fluorescence techniques. Fluorescence spectroscopy is also typically not 

destructive to the sample and measurements can be made quickly, on the order of seconds 

to minutes. 

EX 

EM

EX EM

EX 

EM 

a) b) c)

Fig. 2: Schematic of the excitation beam (EX) and detected emission (EM) orientations for 

a) 0°/90° right angle transmitting, b) front face and c) 0°/180° transmitting geometries. 

5 
A right angle or 0°/90° geometry is typically used to measure dilute solutions and other 

transparent samples, where the excitation beam is normal to the sample and fluorescence 

is detected at a 90° angle relative to the beam (see Fig. 2a). A front-face geometry is used 

to measure optically dense samples, where the excitation beam is incident on the sample 

at less than 90° and the fluorescence is collected at an angle ≤ 90° (see Fig. 2b). The 

epifluorescence geometry is a special case of the front-face geometry that is often used in 

fluorescence microscopy and optical fiber-based fluorometers, where the excitation beam 

and collected fluorescence are both normal to and on the same side of the sample, i.e., a 

0°/0° geometry. A 0°/180° transmitting geometry is also often used in microscopy (see 

Fig. 2c). 

The number of chemical assays and screening techniques using fluorescence detection 

continues to increase rapidly and has resulted in a corresponding increase in the need for 

standardization of fluorescence measurements. Only a few standard methods [7-9] and 

reference materials [10-14] have been well established and are readily available, at 

present, for the characterization of fluorescence measuring systems. National metrology 

institutes and international standards organizations are working to provide new 

fluorescence standard materials and methods in the near future. This document briefly 

discusses the major issues that should be considered by users of fluorescence instruments 

who aim to achieve high quality measurements. Standard methods and materials are also 

described where appropriate and references are given where more detailed explanations 

can be found. A few guideline/recommendation documents have appeared recently [15-

17], but this one aims to be most useful to non-expert users of fluorescence spectrometers 

in the bioanalytical, pharmaceutical and clinical communities. 

2. QUALITATIVE AND QUANTITATIVE FLUORESCENCE 

MEASUREMENTS 

There are two general classes of measurements that are commonly performed by 

fluorescence spectrometry: qualitative and quantitative. 

2.1 Qualitative Fluorescence Measurements 

Qualitative fluorescence measurements are used to detect the presence of particular 

analytes, yielding a positive or negative answer. The excitation and emission wavelengths 

are often fixed at the peak maximum of the fluorophore to be detected. The observation 

of a fluorescence intensity at the peak position that is greater than a set threshold, 

typically based on the noise level, e.g., the limit of detection, indicates a positive result. 

For more complex systems, e.g., where multiple fluorophores are present, the shape of 

the fluorescence emission spectrum or the value of the fluorescence lifetime can be used 

to determine which of the fluorophores of interest are present. 

6 
2.2 Quantitative Fluorescence Measurements 

Quantitative fluorescence measurements are used to determine amounts or concentrations 

of analytes in unknown samples. These quantities may be determined in absolute units, 

such as moles or moles per liter, or in relative units, such as the ratio of the 

concentrations of two fluorescent analytes contained in a single unknown solution. These 

determinations use the following proportionality relating fluorescence signal (S) at a 

given pair of excitation and emission wavelengths (λex , λem) to fluorescent analyte 

concentration (c):

S ∝ I0 ΩRR

dα Φ c (1) 

where I0 is the intensity of the excitation beam, Ω is the fraction of the fluorescence 

collected by the detection system, RR

d is the responsivity of the detection system, and α, 

Φ, and c are the absorption coefficient, fluorescence quantum yield and concentration of 

the fluorescent analyte, respectively. This linear proportionality with concentration 

applies to optically dilute samples, e.g., solutions with an absorbance of less than 0.05 at 

a pathlength of 1 cm. 

3. FACTORS AFFECTING QUANTIFICATION 

3.1 Instrument-Based Factors [18-23]

Measurements on a fluorescence instrument require that instrument parameters, such as 

wavelengths, bandwidths and detector gain, be set. All of these parameters can be set 

with varying degrees of repeatability and accuracy, depending on the instrument used. 

This can introduce a measurement uncertainty or bias that is particularly significant when 

measured values are compared between instruments. For instance, the measured peak 

positions of the emission bands of two analytes may differ between instruments due to a 

wavelength bias. A corresponding bias between instruments could be introduced in the 

results of an assay that depends on the ratio of the fluorescence intensities at the two 

specified emission wavelengths. 

The intensity of the excitation beam can change significantly with excitation wavelength 

or with time, due to the wavelength-dependence of the intensity of the light source and 

the transmittance of the excitation wavelength selector or the time-dependence of the 

light source intensity, respectively. It is therefore advisable to monitor the excitation 

beam intensity and correct the measured fluorescence intensity for these fluctuations. 

This can be particularly important when excitation spectra are being collected, because 

the excitation intensity often has sharp peaks and valleys with wavelength when lamp 

sources, such as a Xe lamp, are being used. 

The responsivity of a detection system is not linear with intensity at all intensities, 

making it important to know the linear intensity range of the detection system being used. 

7 
The linear range for most detection systems is from its limit of detection up to a threshold 

intensity, above which the responsivity becomes increasingly non-linear with increasing 

intensity. The linear range of the fluorescence detection system should be established 

before attempting to calibrate the responsivity of the detection system. 

The responsivity of the detection system is also wavelength dependent, due to the 

wavelength dependence of both the transmittance of the emission wavelength selector 

and the responsivity of the detector. These factors can affect the shape of a measured 

emission spectrum. 

The diffraction efficiency of gratings and the responsivity of detectors is often 

polarization dependent. This can result in changes in the excitation intensity and the 

responsivity of the detection system as a result of changing excitation and emission 

polarization settings. Even when polarizers are not used, the excitation beam may be 

polarized and the responsivity of the detection system may be polarization dependent and 

both of these factors will be different for different instruments. Not only can this cause 

intensity differences, but spectral correction factors will likely change with emission 

polarization as well. 

The passing of multiple wavelengths by a diffraction grating can introduce unexpected 

sharp peaks into a fluorescence spectrum. A particular wavelength is chosen using a 

diffraction grating by setting the angle of the grating with respect to the incident light, 

such that the incident light is diffracted at the desired wavelength according to the grating 

equation. The value of mλ, not λ, is fixed according to this equation, where m is an 

integer, termed the “diffraction order.” Therefore, the grating equation can be satisfied by 

more than one wavelength for a single grating position. For instance, if a grating in an 

emission monochromator is set to pass 500 nm light at first order, it will also pass 250 nm 

light at second order. As a result, the scattered light from a 250 nm excitation beam will 

be detected as a peak at an emission wavelength of 500 nm.

3.2 Sample-Based Factors [24-26]

The fluorescence intensity of optically dense samples (e.g., A>0.05 at a pathlength of 1 

cm) does not increase linearly with concentration due to significant absorption of the 

excitation beam and/or the emission (reabsorption) by the sample. These “inner filter 

effects” can also greatly reduce the amount of fluorescence that reaches the detector, 

especially for a right angle transmitting geometry. The fluorescence intensity can also 

become strongly dependent on sample position and optical geometry. At even higher 

concentrations (e.g., A>>0.05 at a pathlength of 1 cm) aggregation of fluorophores often 

occurs, causing the shape of the fluorescence spectrum to be different than that of a dilute 

sample, along with non-linear concentration behavior. 

The fluorescence intensity of a sample may decrease with exposure time to light, due to 

photobleaching and photodegradation. This is particularly true of most organic dyes, 

which are the most widely used fluorescent probes. The light exposure time of such 

8 
samples may need to be limited to obtain reproducible fluorescence intensities and in 

some cases even reproducible spectral shapes. 

The fluorescence intensity of fluorophores is temperature dependent. Typically, the rates 

of fluorescence quenching processes, such as collisional quenching in solutions, increase 

with temperature, causing a corresponding decrease in fluorescence intensity. 

Temperature coefficients for fluorescence intensity for particular fluorophores can be 

used to correct for this temperature dependence. 

The absorbance and consequently the intensity of fluorescence from a sample are 

dependent on the orientation of the sample’s absorption transition dipole with respect to 

that of the polarization of the excitation light. The polarization of fluorescence is parallel 

to the direction of polarization of the fluorescent species’ emission transition dipole. 

Fluorescence anisotropy (r) is used to describe the extent of polarization of emission and 

is defined by 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. A sample whose 

fluorophores are oriented non-randomly and have a rotational period that is long 

compared to their fluorescence lifetime will emit anisotropic fluorescence. The spectral 

shape and intensity of such fluorescence is dependent on the viewing angle and the 

polarization factors of the instrument. 

It is common for the fluorescence intensity and peak position, and sometimes even the 

spectral shape, of a fluorophore to be environment-dependent. This includes changes due 

to the solvent used, the pH of a solution or the species to which a fluorophore is bound. 

This can be of particular concern when reference samples containing the same 

fluorophores as an unknown sample, but in a different environment, are used to quantify 

the amount of analyte in the unknown. 

Raman signal can introduce peaks into the fluorescence spectrum. The Raman peaks of 

the sample’s solvent or matrix being those most commonly encountered. For instance, the 

Raman peak of water, found red-shifted about 3400 cm-1 from the chosen excitation 

wavelength, would typically be observed in the fluorescence spectrum of any aqueous 

solution excited by UV or blue light. 

4. APPARATUS COMPONENTS 

All modern fluorescence measurements involve irradiating a sample with a light source, 

selecting the excitation wavelength, collecting the resulting fluorescence, rejecting the 

Rayleigh-scattered light, selecting the emission wavelength and detecting the 

fluorescence signal. The following functions will be discussed individually along with the 

equipment used to achieve these functions in commercial instruments: 

1. Excitation Light Source 

2. Excitation Wavelength Selector 

9 
3. Sampling Device 

4. Emission Wavelength Selector 

5. Detector 

4.1 Excitation Light Source 

A variety of lamps, lasers and light-emitting diodes (LEDs) are used as an excitation 

source. Continuous and pulsed versions of these sources are used for steady-state and 

time-resolved instruments, respectively. The Xe lamp is the most commonly used of 

these, due to its relatively high intensity and broad wavelength range (UV to NIR). Lasers 

are the highest intensity sources and are used in applications where short collection times 

and small amounts of sample are required, such as those using flow cytometry or 

microarrays. 

4.2 Excitation Wavelength Selector 

The intensity of scattered light at the excitation wavelength can be comparable to or 

greater than that of the fluorescence at the sample. Therefore, it is important that the 

excitation wavelength profile does not overlap with the emission wavelength region 

being detected. This is achieved for lamps by using an excitation wavelength selector 

(e.g., a filter or a monochromator) between the lamp and sample with a known peak 

transmission wavelength and bandwidth. The inherent bandwidth of the radiation from a 

laser or an LED is often narrow enough that an excitation wavelength selector is not 

necessary. This selector also enables fluorescence excitation spectra to be resolved. 

4.3 Sampling Device 

The sampling device includes all optics and other equipment needed to deliver the 

excitation beam to the sample, collect the emission from the sample and hold the sample 

in place. Sample formats include cuvettes, microwell plates, microarrays, microscope 

slides and flow systems and can be accompanied by a variety of optical delivery and 

collection systems, including conventional transmitting, front face and epifluorescence 

systems and fiber optic-based probes. 

4.4 Emission Wavelength Selector 

Similar to that for excitation, the emission wavelength selector helps to insure that the 

emission wavelength region being detected does not overlap with the excitation 

wavelength profile. In addition, it enables individual fluorescence bands to be detected 

when multiple bands are present and fluorescence emission spectra to be resolved. 

Emission wavelength selectors are also important for the rejection of stray light. Filters, 

10 
monochromators and grating polychromators are most often used for emission 

wavelength selection. 

4.5 Detector 

For the detection of emission, a photomultiplier tube (PMT) or a CCD array is typically 

placed after the emission wavelength selector. The detection of the excitation beam for 

monitoring its intensity is commonly done by a quantum counter detector or a photodiode 

before the sample, where a small fraction of the excitation beam is split off from the rest. 

5. CALIBRATION OF FLUORESCENCE INSTRUMENTS [15, 16, 18-

23]

Two types of fluorescence instrument calibrations are used. The first is analyte-specific, 

where the relationship between the response of the instrument (fluorescence intensity) 

and the concentration or amount of a specific analyte is determined for the sample of 

interest. This is the most commonly used type of calibration for fluorescence. The second 

is analyte-independent and intended for spectral instruments. In this case, the wavelength 

accuracy for emission and/or excitation and the spectral responsivity of the detection 

system are calibrated across the entire or a continuous part of the wavelength range of the 

instrument. 

5.1 Analyte Concentration – Calibration Curves 

Calibration curves of instrument response (fluorescence intensity) versus analyte 

concentration are determined using reference materials containing the analyte of interest. 

For instance, the fluorescence intensities of a set of solutions at different, known analyte 

concentrations, which covers a desirable concentration range, can be measured and 

plotted versus concentration. The plot is then fitted to a polynomial, typically a straight 

line. The resulting calibration curve is both analyte and instrument specific and can be 

used to determine analyte concentrations of unknown samples. [8, 14] Note that this 

method may not be accurate when the microenvironment surrounding the fluorophore is 

different in the reference and unknown samples. In addition, 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. 

It is not always possible to use this method effectively. Firstly, organic dyes, which are 

used as fluorescent probes, are often not commercially available at a known, high purity 

to enable reference solutions to be produced. Secondly, in complex systems where 

fluorophores are bound to large molecules, cells or microbeads, the concentration of 

bound fluorophores in a solution or suspension may be very difficult to determine. For 

the latter case, the molecules of equivalent soluble fluorophore (MESF) scale [27-29] has 

11 
been proposed as an alternative way to use calibration curves to quantify fluorescence 

intensity for a particular analyte. 

5.2 Emission Wavelength and Spectral Slit Width 

A variety of reference samples have been proposed for use in the determination of 

emission wavelength accuracy, including several lamps, and inorganic and organic 

fluorophores. The most widely used and best characterized of these are low pressure 

atomic lamps, commonly referred to as “pen” lamps. In this case, the type of pen lamp 

(e.g., Hg, Ne, Kr, etc.) is chosen, so that its radiated atomic lines are within the desired 

wavelength range. The lamp is placed at the sample position, such that its light is 

centered in the optical path of the detection system of the instrument. Note that the 

accuracy of this method may decrease if the pen lamp is not properly aligned. The 

emission wavelength selector with detector then measures the signal over the wavelength 

range of interest. The measured wavelength positions of the resulting sharp peaks are 

then compared with the known positions to determine wavelength accuracy. [7, 16] 

Spectral slit width accuracy of the emission 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. [7] 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. [18] 

5.3 Excitation Wavelength and Spectral Slit Width 

Many of the reference samples that can be used for determining emission wavelength 

accuracy can also be used for excitation wavelength accuracy. For instance, a pen lamp 

can be placed at the excitation light source position and detected after the excitation 

wavelength selector using the instrument’s reference detector. [19] In this case, a 

relatively weak signal that limits the number of useful atomic lines is likely to be 

observed and alignment of the pen lamp is more critical than for the emission wavelength 

accuracy determination. 

Once the accuracy of the emission wavelength selector has been determined, a diffuse 

scatterer, e.g., a scattering solution or a diffuse reflector, at the sample can be used to 

scatter a fraction of the excitation beam into the detection system to determine excitation 

wavelength accuracy. One wavelength selector is set at a fixed wavelength while the 

other is tuned over the same wavelength to obtain a spectrum. The wavelength bias 

between the two wavelength selectors is equal to the difference between the set 

wavelength position and the observed peak position of the collected spectrum. This 

method [7, 18] can be used at any wavelength, unlike many other methods that depend on 

a limited number of set excitation wavelengths determined by the reference material 

chosen. Methods similar to those used for spectral slit width accuracy of the emission 

12 
wavelength selector may also be used for spectral slit width accuracy of the excitation 

wavelength selector. [7, 18] 

5.4 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. [30, 31] A variety of methods using optical filters, polarizers or a 

combination of the two have been reported. [18, 19] 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 

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. [8] 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. 

5.5 Signal Level (Relative Emission) [21, 23 , 18-20]

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. 3). The 

degree of photometric precision required for the successful outcome of quantitative 

methods should be known. It is highly recommended that the wavelength accuracy (see 

section 5.2) and linear range of the detection system be determined (see section 5.4) 

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 8). 

13 
There are two preferred methods for calibrating photometric responsivity. One (method 

A) uses a calibrated light source (CS) and the other (method B) uses certified reference 

materials (CRMs). Both give results that are traceable to national metrology institutes 

(NMIs). A calibrated tungsten white light source is most commonly used for method A, 

covering the wavelength range from about 350 nm into the NIR. NIST SRMs [10-12] and 

BAM CRMs [13] are currently available for use in method B. Corrected emission spectra 

of some commonly used dyes have also been reported recently in the literature. [32, 33] 

Method A is more difficult to implement than method B and requires periodic 

recertification of the CS, which is more expensive over time. A third method, method C, 

can be used, implementing a calibrated detector and a calibrated diffuse reflector. This 

method typically has larger uncertainties associated with it than methods A and B, [18] 

but is recommended in wavelength regions in the UV and NIR not covered by the other 

two methods. 

0

0.2

0.4

0.6

0.8

1

300 400 500 600 700 800

Wavelength (nm)

Relative Responsivity

Fig. 3: Example of the relative spectral responsivity of an emission 

detection system (grating monochromator-PMT based), [18] for which 

a correction needs to be applied to a measured emission spectrum to 

obtain its true spectral shape (relative intensities). 

Method A - The light from a calibrated source (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 

emission wavelength 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) 

nd CCS. 

n on 

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

a

Method B - The fluorescence standard is placed at the sample position. Its spectrum is 

collected and compared to the certified spectrum according to the instructions give

th

14 
Method C - This is a two-step method. The first step uses a calibrated detector (CD) at 

the sample position to measure the flux of the excitation beam as a function of excitation 

wavelength. The second step uses a calibrated diffuse reflector (CR) 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 excitation and emission wavelength 

selectors over the emission region of interest while collecting both the signal output and 

the reference output. This method enables the relative correction factor to be calculated. 

Note that this method has larger uncertainties than those for methods A or B and is 

pically more difficult to implement. 

.6 Reference Signal Level (Relative Excitation) [22, 23 , 18-20]

izer, the spectral correction for excitation 

tensity is dependent on the polarizer setting. 

ty

5

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. 4). The neglect of excitation intensity correction factors can cause even 

greater errors than that of emission correction factors. [18, 34] 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 5.3) and linear 

range of the detection system (see section 5.4) used to measure the excitation beam be 

determined 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 5.5). 

Also note that when using an excitation polar

in

15 
0

0.2

0.4

0.6

0.8

1

200 300 400 500 600 700 800

Wavelength (nm)

Relative Flux

tation spectrum to obtain its true spectral 

shape (relative intensities). 

alibrated detector (CD), has fewer caveats associated with it 

an the other two methods. 

 the 

orrection factor for the responsivity of the reference detector can also be calculated. 

Fig. 4: Example of the relative flux of an excitation beam (Xe lampgrating monochromator based), [18] for which a correction needs to be 

applied to a measured exci

When a reference detector is not built into an instrument, a spectral correction for the 

reference channel is to be determined, or an independent spectral correction of excitation 

intensity is desired, 1) a calibrated detector, 2) a calibrated diffuse reflector or 3) a 

quantum counter [20, 35, 36] may be placed at the sample position, with the latter two 

methods using the instrument’s fluorescence detection system as a detector. For methods 

1 and 2, the detector and diffuse reflector must be calibrated for responsivity and 

reflectance, respectively, as a function of wavelength. For method 2, excitation and 

emission wavelength selectors must be scanned synchronously and the spectral correction 

for the emission channel (see section 5.5) must be applied to the measured intensities. 

[18] Method 3 should only be used in the quantum counter’s effective wavelength range, 

where a wavelength-independent response can be achieved. This method is probably the 

least expensive of the three to implement, as no calibrations and re-calibrations are 

required. Method 1, using a c

th

Method 1 - A CD is put at the sample position with the excitation beam incident on it. 

The output of the CD is measured as a function of emission wavelength by scanning the 

excitation wavelength selector over the excitation region of interest using the same 

instrument settings as that used with the sample. The known responsivity of the CD is 

used to calculate the flux of the excitation beam. If the instrument’s reference detector is 

used to measure the intensity of the excitation beam simultaneously with the CD, then

c

16 
5.7 Intensity & Sensitivity 

As explained in the Quantitative Fluorescence Measurements section, the absolute value 

of the fluorescence signal measured by the detection system is not only dependent on the 

sample itself, but also on the excitation intensity at the sample and the optical geometry 

of the instrument. Therefore, it is non-trivial to determine the instrument-independent 

fluorescence intensity of any sample or the absolute responsivity of any detection system 

in terms of the intensity coming out of the sample or being measured by the detector, 

respectively, relative to the excitation intensity at the sample. 

The most accurate way to calibrate an instrument for absolute intensity is to use 

conventional physical transfer standard-based methods, such as those employing a 

calibrated light source or a calibrated detector in combination with a calibrated reflector. 

[18, 19] These methods are difficult to perform and require a certain degree of user skill 

and knowledge. The certification and recertification, typically annual, of such standards 

is also expensive. In addition, these standards tend to be bulky and are not compatible 

with many instruments. More simple, easy-to-use, alternative standards and methods are, 

therefore, used by most researchers. 

One approach is to relate fluorescence signal to analyte concentration, using calibration 

curves or MESF units (see section 5.1). Another approach is to measure the intensity of a 

standard sample that can be expected to always give the same fluorescence intensity 

under the same conditions. 

Organic dyes, such as those used as fluorescent probes, are not generally good choices for 

intensity standards, due to issues with photobleaching, stability and reproducible 

concentration. If organic dyes are to be used in this way, then those with known high 

purity and known shelf-life, such as those produced by NMIs are recommended for 

single-time use, i.e., a fresh solution should be used every time. 

A better alternative is to use fluorescent samples that are stable over time even when 

exposed to light. For example, fluorescent, inorganic glasses with well characterized 

photostability and spectral properties and long shelf-lives are commercially available. 

[10, 11 , 24] Such materials can be used for determining a quasi-absolute intensity scale 

by measuring fluorescent signal at fixed wavelength values within their recommended 

range, using a specified set of experimental parameters, such as bandwidths, excitation 

intensity and temperature. 

The sensitivity of a fluorescence instrument can also be determined by measuring the 

signal-to-noise ratio of the fluorescence signal of intensity standards. The Raman line of 

water is often used to measure sensitivity in a similar way, [37-39] but the Raman signal 

is typically only strong enough to be useful in the UV region. Organic dye solutions can 

also be used to measure instrument sensitivity or limit of detection with all of the same 

caveats as when used as intensity standards. [9] 

17 
The methods outlined above enable a quasi-absolute intensity scale to be defined that is 

expected to be instrument independent for instruments with similar optical geometries 

and designs, potentially enabling the sensitivity of different fluorescence instruments to 

be compared, but such comparisons should be approached with caution due to the 

relatively large uncertainties involved, which are difficult to quantify. [18] 

6. QUALIFICATION AND VERIFICATION OF FLUORESCENCE 

INSTRUMENTS 

Instrument qualification is an essential part of method validation, when the results of the 

method are dependent on the performance of the instrument. Instrument qualification 

should be done at well-defined, periodic intervals or after significant changes or 

maintenance on the instrument has been carried out. In between qualifications, more 

frequent (possibly daily) performance verifications should be done to demonstrate that 

the instrument is performing at a consistent quality level. 

Many of the methods in the Calibration section may be appropriate for qualification and 

verification of fluorescence instruments. The instrumental variables and desired accuracy 

of the quantities to be determined will define the particular types of test methods needed. 

The criteria for acceptable instrument performance given in the following subsections are 

applicable for general use unless specified otherwise. Two general types of instrumental 

measurements will be differentiated here, spectral, i.e., those that measure intensity 

versus wavelength, and fixed, i.e., those that measure intensity at a fixed wavelength and 

bandwidth. 

6.1 Wavelength Accuracy 

The confidence level of measured peak positions is defined by wavelength accuracy for 

spectral measurements. Determining and calibrating the wavelength accuracy (see 

sections 5.2 and 5.3) at a single point (one for emission and one for excitation) is often 

sufficient to qualify an instrument for this parameter, as it is common for the relative 

wavelength accuracy to not change dramatically, e.g., on the order of 1 nm, across an 

instruments effective wavelength range. Determination of the accuracy of many 

wavelengths across the desired wavelength range demonstrates if further calibration, 

beyond a single point, is needed. Multi-point calibration involves measuring wavelength 

biases at multiple wavelengths and correcting for the wavelength dependence of the bias. 

A wavelength uncertainty of ± 0.2 nm has been demonstrated for a scanning fluorescence 

spectrometer. [18] A single point calibration can often be applied to the wavelength axis 

in an instrument’s software before data is collected. Whereas, a multi-point calibration 

may require that the correction be applied to spectra after they are collected. 

For fixed measurements, it is most important that the wavelength position and bandwidth 

be reproducible. For filter-based wavelength selection, this just requires that the same 

filter be used when comparing data over time. If a different filter has to be used, for 

18 
instance when data is compared across instruments and laboratories, then the 

transmission curves of the filters must be compared. 

6.2 Signal Level (Relative Emission) 

Correction for the relative responsivity of the detection system with emission wavelength 

(see section 5.5) is important for comparing spectral shapes and integrated intensities of 

emission spectra, and intensities at different emission wavelengths. Note that the linearity 

of the detection system must be determined before a calibration of spectral emission is 

performed (see section 5.4). Corrected emission spectra are required for obtaining 

accurate results for assays that depend on such intensity values and for quantum yield 

determinations. Spectral correction is particularly needed in emission regions where the 

responsivity of the detection system is highly sloped, typically in the UV and red/NIR. 

For filter-based wavelength selection, the detected spectral range is determined by the 

transmission profile of the filter, which changes from one filter to another. 

6.3 Reference Signal Level (Relative Excitation) 

Correction for the relative spectral flux of the excitation beam at the sample (see section 

5.6) is critical for accurate determination of fluorescence intensities at different excitation 

wavelengths and the shape and peak positions of excitation spectra. The spectral profile 

of a broadband excitation source, such as a high pressure atomic lamp, may, in fact, 

exhibit even more dramatic spectral changes with wavelength than typical detection 

systems. This problem is compounded by possible changes of the source intensity with 

time, even at a fixed wavelength. The relative spectral flux of a source or the relative 

spectral responsivity of a reference detector (see section 5.6), should be determined 

periodically, along with correction factors possessing the accuracy and precision required 

for the application. 

6.4 Intensity, Sensitivity & Performance Verification 

The use of photostable, day-to-day intensity standards (see section 5.7) is the easiest way 

to verify the performance of an instrument. If the measured intensity does not change 

from that observed when the instrument was qualified, then it can be assumed that the 

instrument performance has not changed and is, therefore, still qualified. Using such 

standards to determine an artifact-based or quasi-absolute intensity scale potentially 

enables measured intensities and instrument sensitivity to be compared over time or 

between instruments. Note that it is important to demonstrate that intensity measurements 

are being measured within the linear range of the instrument’s detection system before 

intensity comparisons are attempted (see section 5.4). 

For instruments with filter-based wavelength selection, fluorescence standards for 

spectral correction can be used to determine expected intensity differences caused by 

19 
filters with different transmission profiles. By compensating for these intensity 

differences due to spectral mismatch, a quasi-absolute intensity scale can potentially be 

determined for these instruments, as well. Again, note that instrument-to-instrument 

comparisons should be approached with particular caution due to the relatively large 

uncertainties involved, which are difficult to quantify. [18] 

6.5 Fluorescence Lifetimes 

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

measurements used to determine fluorescence lifetimes. [40] Conventional instruments 

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

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. [41] Almost all of the 

candidates mentioned in the literature for use as lifetime standards [42] 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. 

7. METHOD VALIDATION 

Validation of an analytical method demonstrates that the result yielded from the method 

is valid within a specified, acceptable uncertainty budget. For fluorescence measurements 

this involves the consideration of one or more of the topics already discussed. Instrument 

qualification (see section 6), which may also involve instrument calibration (see section 

5), is usually part of the process. Sample related errors may also need addressing (see 

section 3.2). These can arise due to the effects of concentration, anisotropy, photostability 

and shape of the sample in combination with the optical geometry of the instrument on 

measured quantities, such as fluorescence intensity. All suspected errors are quantified 

and combined to give a total estimated error that must be less than the method-specific, 

acceptable limit. [43] 

8. DEFINITION OF TERMS [44, 45]

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 

20 
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 

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

wavelength has been determined along with corresponding uncertainties. [46] 

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

has been determined along with corresponding uncertainties. [47, 48] 

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

function of wavelength has been determined along with corresponding uncertainties. [49] 

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. 

21 
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 

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

which is an integer.[50] 

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. [51, 

52] 

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 

22 
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 

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

the sample, unlike fluorescence. [53] 

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. 

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