NISTIR 7915

Standard Practice for Determining

the Relative Spectral Correction 

Factors for the Emission Signal of 

Fluorescence Spectrometers

Paul C. DeRose 
NISTIR 7915

Standard Practice for Determining 

the Relative Spectral Correction 

Factors for the Emission Signal of 

Fluorescence Spectrometers

Paul C. DeRose 

Biosystems and Biomaterials Division

Information Technology Laboratory

January 2013

U.S. Department of Commerce 

Rebecca Blank, Acting Secretary

National Institute of Standards and Technology 

Patrick D. Gallagher, Under Secretary of Commerce for Standards and Technology and Director 
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Standard Practice for Determining the Relative Spectral Correction Factors for the 

Emission Signal of Fluorescence Spectrometers

1. Scope

1.1 This practice describes three methods for determining the relative spectral correction factors 

for grating-based fluorescence spectrometers in the ultraviolet-visible spectral range. These 

methods are intended for instruments with a 0°/90° transmitting sample geometry. Each 

method uses different types of transfer standards, including 1) a calibrated light source (CS), 

2) a calibrated detector (CD) and a calibrated diffuse reflector (CR), and 3) certified reference 

materials (CRMs). The wavelength range covered by the different methods is from 250 nm to 

830 nm with some methods having a broader range than others. Extending these methods to 

the near infrared (NIR) beyond 830 nm will be discussed briefly, when appropriate. These 

methods were designed for scanning fluorescence spectrometers with a single channel 

detector, but can also be used with a multiplex detector, such as a diode array or a CCD.

2. Significance and Use (Intro)

2.1 Calibration of the relative responsivity of the detection system for emission (EM) as a function 

of EM wavelength (λEM), also referred to as spectral correction of emission, is necessary for 

successful quantification when intensity ratios at different EM wavelengths are being 

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

known. Such calibration methods are given here and summarized in Table 1. This type of

calibration is necessary because the relative spectral responsivity of a detection system can 

change significantly over its useful wavelength range (see FIG 1). It is highly recommended 

that the wavelength accuracy (see ASTM E388) [1] and the linear range of the detection 
2

system (see ASTM E2719 and ASTM E578) [2, 3] be determined before spectral calibration 

is performed and appropriate steps are taken to insure that all measured intensities during this 

calibration are within the linear range. For example, the use of attenuators before the sample 

may be needed to attenuate the excitation beam, thereby, decreasing the fluorescence intensity.

Also note that when using an EM polarizer, the spectral correction for emission is dependent 

on the polarizer setting. [4]

 Table 1 : Summary of Methods for Determining Spectral Correction of Detection System Responsivity

Method

 

λEM Drop-In

OffShelf Uncertainty CaveatsCertified 

Values Ref.

CS UV-NIR N Y < ± 5 %

difficult 

setup Y 4, 6-9

CD+CR UV-NIR N Maybe ± 10 %

difficult 

setup Y

4, 7-8, 

13

CRMs UV-NIR Y Y ± 5 % Y 18-24

Note: In Table 1, “Drop-In” refers to whether or not the material/hardware can be put in the 

sample holder and used like a conventional sample; “Off-Shelf” refers to whether or not the 

material/hardware can be purchased in an immediately usable format; “Uncertainty” is the 

estimated expanded (k=2) total uncertainty; “Caveats” refer to important information that a 

user should know about the method before attempting to use it; “Certified Values” refers to 

whether or not the material/hardware is supplied with appropriate values as a function of 

emission wavelength and their corresponding total uncertainties; the references (Ref.) give 

examples and more in-depth information for each method.

2.2 When using CCD or diode array detectors with a spectrometer for λEM selection, the spectral 

correction factors are dependent on the grating position of the spectrometer. Therefore, the 
3

0

0.2

0.4

0.6

0.8

1

300 400 500 600 700 800

Wavelength (nm)

Relative Responsivity

spectral correction profile versus λEM must be determined separately for each grating position 

used. [5]

Fig. 1: Example of the relative spectral responsivity of an emission detection system (grating monochromator-PMT based), 

[4] for which a correction needs to be applied to a measured emission spectrum to obtain its true spectral shape (relative 

intensities).

3. Calibrated Optical Radiation Source (CS) Method [4, 6-9]

3.1 Materials

A calibrated tungsten lamp is most commonly used as a CS in the visible region due to its high 

intensity and broad, featureless spectral profile. Its intensity falls off quickly in the ultraviolet 

(UV) region, but can typically be used down to 350 nm or so. It also displays a high intensity in

the near infrared, peaking at about 1000 nm. It gradually decreases beyond 1000 nm, but 

continues to have significant intensity out to about 2500 nm. A calibrated deuterium lamp can be 

used to extend farther into the UV with an effective range from about 200 nm to 380 nm. The 

effective range of a CS is dependent on the intensity of the CS and the sensitivity of the detection 
4

system. This range can be determined by measuring the low-signal regions where the signal 

profile of the light from the CS becomes flat or indistinguishable from the background signal, 

implying that the CS is not able to measure the signal in these λEM regions.

A calibrated reflector (CR) is often used to reflect the light from the CS into the emission 

detection system. A diffuse reflector made out of compressed or sintered polytetrafluoroethylene 

(PTFE) is most commonly used as a CR, due to its lambertian reflectance, which prevents both 

polarization and spatial dependence of the reflectance. In addition, PTFE possesses a reflectance 

profile that is nearly flat, changing by less than 10% from 250 nm to 2500 nm. For a CS and a CR, 

“calibrated” implies that the spectral radiance and the spectral reflectance, respectively, are known 

and traceable to the SI. This is commonly done through certification of these values by a national 

metrology institute (NMI). [10-12]

3.2 Procedure

Direct the optical radiation from a CS into the EM detection system by placing the CS at the 

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

position to reflect the optical radiation from the CS into the EM detection system. Ensure that the

CS is aligned such that its light is centered on all optics it encounters and the entrance slit, before 

and while entering the λEM selector.

Scan the λEM-selector over the EM region of interest, using the same instrument settings as that 

used with the sample, and collect the signal channel output (S′′).
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Use the known radiance of the CS incident on the detection system (L) to calculate the relative 

correction factor (CCS), such that CCS = L/S′′. Note that L may be replaced by the spectral 

irradiance or the spectral radiant flux, since the correction factors are relative, not absolute. The 

corrected EM intensity is equal to the product of the signal output of the sample (S) and CCS. Since 

CCS are relative correction factors, they can be scaled by any constant. For instance, it is often 

useful to scale them with a constant that gives a CCS value of one at a particular λEM.

Note that L is given in power units, not photon units, whereas, the units for S and S′′are either in 

power or photon units depending on whether your detector measures an analog or a digital 

(photon counting) signal, respectively. In either case, the corrected signal will be in power units, 

so a conversion, i.e. dividing the corrected signal by λEM, is necessary if photon units are needed. 

4. Calibrated Detector (CD) with Calibrated Reflector Method [4, 7, 8, 13]

4.1 Materials

A calibrated photodiode, either by itself or mounted inside an integrating sphere, is most 

commonly used as a calibrated detector (CD). The CD with integrating sphere is typically the 

more accurate of the two, because it insures spatially uniform illumination of the photodiode.

A Si photodiode covers the range from 200 nm to 1100 nm. An InGaAs photodiode can be 

used in the NIR from 800 nm to 1700 nm. For a CD, “calibrated” implies that the spectral 

responsivity is known and traceable to the SI. This is typically done through values certified 

by an NMI. [14, 15] A photodiode usually outputs a current or voltage that is proportional to 

the power of the light incident on it.
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Alternatively, a quantum counter solution can be used instead of a CD.[9, 16, 17] This is a 

dye solution at a sufficiently high concentration such that all of the photons incident on it are 

absorbed. In addition, it has an emission (EM) spectrum whose shape and intensity do not 

change with excitation (EX) wavelength. Note that there are several drawbacks to using a 

quantum counter (QC) instead of a CD. Firstly, QCs tend to have a more limited range than 

CDs with uncertainties that are not certified or well known. In addition, a QC is prone to 

polarization and geometry effects that are concentration dependent. It should also be noted 

that the output measured from the QC will be proportional to the quantum flux (number of 

photons per second) at the sample, not the flux in power units.

4.2 Procedure

Unlike the CS method, this is a two-step method. The first step uses a CD (or a QC) placed at the 

sample position, which measures the excitation intensity incident on the sample as a function of 

EX wavelength by scanning the EX wavelength selector over the desired range.

The second step uses a CR with reflectance RCR to reflect a known fraction of the flux of the EX 

beam into the detection system. Follow the procedures in either section 5.2.1 or 5.2.2 depending 

upon whether you are using a CD or a QC, respectively.

4.2.1 Step 1 with Calibrated Detector – Place the CD at the sample position and scan the 

λEX-selector over the EX region of interest while collecting the signal from the 

CD (SCD) as a function of λEX. Be sure to use the same instrument settings as that 

used with the sample. Calculate the flux of the EX beam (φx), using φx = SCD/RCD, 

where RCD is the known responsivity of the CD. Note that if the instrument has its 
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own reference detector with output (Rf) for monitoring the excitation intensity, 

then the correction factor for the responsivity of the reference detector CR = φx/Rf

can be calculated. Multiplying an Rf value by CR at a particular λEX will give a 

corrected Rf value in the same units as φx , typically Watts. 

4.2.2 Step 1 with Quantum Counter- Place the QC solution at the sample position in a quartz 

cuvette. If front face detection is possible, then use a standard cuvette with the EX 

beam at normal incidence. If 90° detection is chosen, then use a right-triangular 

cuvette with the excitation beam at 45° incidence to the hypotenuse side and one of the 

other sides facing the detector. Scan the λEX-selector over the EX region of interest 

with the λEM fixed at a position corresponding to the long-wavelength tail of the 

emission band and collect the signal intensity (SQC) as a function of λEX. Be sure to 

use the same instrument settings for the excitation beam as that used with the 

sample. SQC is the relative quantum flux of the excitation beam at the sample. Note 

that if the instrument has its own reference detector with output (Rf) for 

monitoring the excitation intensity, then the correction factor for the responsivity 

of the reference detector CR = SQC /Rf can be calculated. Multiplying an Rf value by 

CR at a particular λEX will give a corrected Rf value in units of relative quantum 

flux.

4.2.3 Step 2 Using a Calibrated Reflector – Place the CR at the sample position at a 45°

angle relative to the excitation beam, assuming a right-angle detection geometry 

relative to the excitation beam. Use the same instrument settings as that used with 

the sample. Synchronously scan both the λEX- and λEM-selectors over the EM region 

of interest while collecting both the signal output (S′) and the reference output (Rf′). 
8

Calculate the relative correction factor (CCD) using the equation CCD = (CR RCR Rf′)/S′.

If the instrument does not have a reference detector, then use CCD = (φx RCR)/S′ or CCD

= (SQC RCR)/S′, depending upon whether you are using a CD or a QC, respectively, in 

step 1. Since CCD are relative correction factors, they can be scaled by any constant. 

For instance, it is often useful to scale them with a constant that gives a CCD value of 

one at a particular λEM.

5. Certified Reference Material Method

5.1 Materials

Certified reference materials [18] (CRMs) have been released by national metrology institutes 

(NMIs) for the relative spectral correction of fluorescence emission. [19- 25] They are presently 

sold by the NMIs and other secondary manufacturers. These CRMs are supplied with certified 

relative intensity and uncertainty values as a function of λEM at a fixed λEX. Instructions for use 

are also supplied in the accompanying certificate. Unlike the other materials and methods

explained to this point, these can be used easily by non-experts, since they are designed to be 

measured in the same way as typical samples. As an alternative to CRMs, there are other materials 

with corrected relative intensity emission spectra that have been published in the literature.

[26, 27] These should be used with caution, since the uncertainties in the published values are not 

specified in most cases.

5.2 Procedure

Place the CRM at the sample position and scan its emission spectrum using the excitation 

wavelength, emission range and other specifications given in the certificate. Collect the measured 
9

emission signal (Sm) and compare it to the certified spectrum (Sc) according to the instructions 

given on the certificate. If the instrument has a reference channel to monitor the intensity of the 

excitation beam, then simultaneously collect the reference channel output (Rf).

The easiest way to calculate the relative correction factor (CCRM) as a function of λEM in the 

emission range of a single CRM is to use the equation CCRM = (Sc Rf) / Sm. If the instrument does 

not have a reference detector, then the equation becomes CCRM = Sc / Sm. Note again that because 

these are relative correction factors, they can be scaled by any constant. For instance, it is often 

useful to scale them with a constant that gives a CCRM value of one at a particular λEM. More 

complex procedures for determining correction factors may be given in the certificate or may be 

included in software available with the CRM. [28]

6. Documentation and Reporting

Spectral correction factors should be reported as a function of emission wavelength along with 

the instrument settings under which it was measured; e.g., spectral bandwidth of emission, 

emission polarizer setting, emission filter setting, gain on the detector, emission grating position 

(for diode array and CCD based instruments); and the date of measurement. Recommended 

instrument settings and basic advice on documenting results may be given in CRM certificates.

[19-24]
10

 

1. ASTM E 388-04, “Standard Test Method for Spectral Bandwidth and Wavelength Accuracy 

of Fluorescence Spectrometers,” In: Annual book of ASTM standards, vol 03.06 (2004, original 

version 1972 ).

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Qualification,” In: Annual book of ASTM standards, vol 03.06 (2009).

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Measuring True Fluorescence Spectra,” Rev.Sci.Instru., 78, 033107 (2007).

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and Spectral Response Correction of CCD Array Spectrometers,” J.Res.NIST, 114, 215-228 

(2009).

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Monte, C. and Pilz, W., “Traceability in Fluorometry-Part I: Physical Standards,” J.Fluoresc., 

15, 301 (2005). b) Resch-Genger, U., Pfeifer, D., Monte, C., Pilz, W., Hoffmann, A., Spieles, 

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Fluorometry-Part II: Spectral Fluorescence Standards,” J.Fluoresc., 15, 315 (2005).

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in Visible and Ultraviolet Spectrometry, Vol. 2, Standards in Fluorescence Spectrometry, 

J.N.Miller, Ed. (Chapman and Hall, New York, 1981), pp. 54-61.

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2011).

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(U.S. GPO, Washington, D.C., 1987).

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Washington, D.C., 1998).
11

 

13. Lakowicz, J.R., Principles of Fluorescence Spectroscopy, 2nd edition, Kluwer Academic/

Plenum Publishers, New York, 1999, pp.51–52.

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D.C., 2008).

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3, Measurement of Photoluminescence, K.D.Mielenz, Ed., Academic Press, New York (1982) pp 

228-233.

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Plenum Publishers, New York, 1999, p.50.

18. ASTM E 131-10, “Terminology Related to Molecular Spectroscopy,” In: Annual book of

ASTM standards, vol 03.06 (2010, original version 1957).

19. Certificate of Analysis, Standard Reference Material 2944, Relative Intensity Correction

Standard for Fluorescence Spectroscopy: Red Emission. National Institute of Standards and 

Technology (2011). (https://www-s.nist.gov/srmors/view_detail.cfm?srm=2944)

20. Certificate of Analysis, Standard Reference Material 2943, Relative Intensity Correction 

Standard for Fluorescence Spectroscopy: Blue Emission. National Institute of Standards and 

Technology (2009). (https://www-s.nist.gov/srmors/view_detail.cfm?srm=2943)

21. Certificate of Analysis, Standard Reference Material 2942, Relative Intensity Correction 

Standard for Fluorescence Spectroscopy: Ultraviolet Emission. National Institute of Standards 

and Technology (2009). (https://www-s.nist.gov/srmors/view_detail.cfm?srm=2942)

22. Certificate of Analysis, Standard Reference Material 2941, Relative Intensity Correction 

Standard for Fluorescence Spectroscopy: Green Emission. National Institute of Standards and 

Technology (2007). (https://www-s.nist.gov/srmors/view_detail.cfm?srm=2941)

23. Certificate of Analysis, Standard Reference Material 2940, Relative Intensity Correction 

Standard for Fluorescence Spectroscopy: Orange Emission. National Institute of Standards and 

Technology (2007). (https://www-s.nist.gov/srmors/view_detail.cfm?srm=2940)

24. Certificate of Analysis, Certified Reference Materials BAM-F001-BAM-F005, Calibration 

Kit, Spectral Fluorescence Standards. Federal Institute for Materials Research and Testing: 

Berlin (2006).
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25. Certificate of Analysis, Standard Reference Material 936a, quinine sulfate dihydrate. 

National Institute of Standards and Technology (1994).

26. Gardecki, J.A., Maroncelli, M., “Set of Secondary Emission Standards for Calibration of the 

Spectral Responsivity in Emission Spectroscopy,” Appl. Spectr., 52, 1179 (1998).

27. Velapoldi, R.A., Tonnesen, H.H., “Corrected Emission Spectra and Quantum Yields for a 

Series of Fluorescent Compounds in the Visible Spectral Region, J. Fluoresc., 14, 465 (2004).

28. 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.