ASTM E2719-09(2022)

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: E2719 − 09 (Reapproved 2022)
Standard Guide for
Fluorescence—Instrument Calibration and Qualification
This standard is issued under the fixed designation E2719; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 2. Referenced Documents
2 4
1.1 This guide (1) lists the available materials and methods 2.1 ASTM Standards:
for each type of calibration or correction for fluorescence E131 Terminology Relating to Molecular Spectroscopy
instruments (spectral emission correction, wavelength E388 Test Method for Wavelength Accuracy and Spectral
accuracy, and so forth) with a general description, the level of Bandwidth of Fluorescence Spectrometers
quality, precision and accuracy attainable, limitations, and E578 Test Method for Linearity of Fluorescence Measuring
useful references given for each entry. Systems
E579 Test Method for Limit of Detection of Fluorescence of
1.2 The listed materials and methods are intended for the
Quinine Sulfate in Solution
qualification of fluorometers as part of complying with regu-
latory and other quality assurance/quality control (QA/QC)
3. Terminology
requirements.
3.1 Definitions (2):
1.3 Precision and accuracy or uncertainty are given at a 1 σ
3.1.1 absorption coeffıcient (α), n—a measure of absorption
confidence level and are approximated in cases where these
of radiant energy from an incident beam as it traverses an
values have not been well established.
-αb
absorbing medium according to Bouguer’s law, I/I = e ,
o
1.4 The values stated in SI units are to be regarded as
where I and I are the transmitted and incident intensities,
o
standard. No other units of measurement are included in this
respectively, and b is the path length of the beam through the
standard.
sample. E131
3.1.1.1 Discussion—Note that transmittance T = I/I and
1.5 This standard does not purport to address all of the
o
absorbance A = –log T.
safety concerns, if any, associated with its use. It is the
responsibility of the user of this standard to establish appro-
3.1.2 absorptivity (a), n—the absorbance divided by the
priate safety, health, and environmental practices and deter-
product of the concentration of the substance and the sample
mine the applicability of regulatory limitations prior to use.
pathlength, a = A/bc. E131
1.6 This international standard was developed in accor-
3.1.3 Beer-Lambert law, n—relates the dependence of the
dance with internationally recognized principles on standard-
absorbance (A) of a sample on its path length (see absorption
ization established in the Decision on Principles for the
coeffıcient, α) and concentration (c), such that A =abc.
Development of International Standards, Guides and Recom-
3.1.3.1 Discussion—Also called Beer’s law or Beer-
mendations issued by the World Trade Organization Technical
Lambert-Bouquer law. E131
Barriers to Trade (TBT) Committee.
3.1.4 calibrated detector (CD), n—opticalradiationdetector
whose responsivity as a function of wavelength has been
This guide is under the jurisdiction of ASTM Committee E13 on Molecular
determined along with corresponding uncertainties (3).
Spectroscopy and Separation Science and is the direct responsibility of Subcom-
3.1.5 calibrated diffuse reflector (CR), n—Lambertian re-
mittee E13.01 on Ultra-Violet, Visible, and Luminescence Spectroscopy.
Current edition approved Nov. 1, 2022. Published November 2022. Originally
flector whose reflectance as a function of wavelength has been
approved in 2009. Last previous edition approved in 2014 as E2719–09 (2014).
determined along with corresponding uncertainties (4).
DOI: 10.1520/E2719-09R22.
The boldface numbers in parentheses refer to the list of references at the end of
this standard.
3 4
Certain commercial equipment, instruments, or materials are identified in this For referenced ASTM standards, visit the ASTM website, www.astm.org, or
guide to foster understanding. Such identification does not imply recommendation contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
or endorsement by ASTM International nor does it imply that the materials or Standards volume information, refer to the standard’s Document Summary page on
equipment identified are necessarily the best available for the purpose. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2719 − 09 (2022)
3.1.6 calibrated optical radiation source (CS), n—optical quantum efficiency to be dependent on the absorbance,
radiation source whose radiance as a function of wavelength concentration, and excitation and emission path lengths of the
hasbeendeterminedalongwithcorrespondinguncertainties (5, sample (9, 10).
6).
3.1.19 Lambertian reflector, n—surface that reflects optical
3.1.7 calibration, n—set of procedures that establishes the radiation according to Lambert’s law, that is, the optical
relationship between quantities measured on an instrument and
radiation is unpolarized and has a radiance that is isotropic or
the corresponding values realized by standards. independent of viewing angle.
3.1.8 certified reference material (CRM), n—material with
3.1.20 limit of detection, n—estimate of the lowest concen-
properties of interest whose values and corresponding uncer-
tration of an analyte that can be measured with a given
tainties have been certified by a standardizing group or
technique, often taken to be the analyte concentration with a
organization. E131
measured signal-to-noise ratio of three.
3.1.9 certified value, n—value for which the certifying body
3.1.21 noise level, n—peak-to-peak noise of a blank.
has the highest confidence in its accuracy in that all known or
3.1.22 photobleaching, n—loss of emission or absorption
suspected sources of bias have been investigated or accounted
intensity by a sample as a result of exposure to optical
for by the certifying body (7).
radiation.
3.1.10 diffuse scatterer, n—material that scatters optical
3.1.22.1 Discussion—Thislosscanbereversibleorirrevers-
radiation in multiple directions; this includes diffuse reflectors,
ible with the latter typically referred to as photodegradation or
whichareoftenLambertian,andscatteringsolutions,whichare
photodecomposition.
not Lambertian.
3.1.23 qualification, n—process producing evidence that an
3.1.11 fluorescenceanisotropy(r),n—measureofthedegree
instrument consistently yields measurements meeting required
of polarization of fluorescence, defined as r=(I – I )/(I +
ll ' ll
specifications and quality characteristics.
2I ), where I and I are the observed fluorescence intensities
' ll '
3.1.24 quantum counter, n—photoluminescent emitter with
when the fluorometer’s emission polarizer is oriented parallel
a quantum efficiency that is independent of excitation wave-
and perpendicular, respectively, to the direction of the polar-
length over a defined spectral range.
ized excitation.
3.1.24.1 Discussion—When a quantum counter is combined
3.1.12 fluorescence band, n—region of a fluorescence spec-
with a detector to give a response proportional to the number
truminwhichtheintensitypassesthroughamaximum,usually
of incident photons, the pair is called a quantum counter
corresponding to a discrete electronic transition.
detector.
3.1.13 fluorescence lifetime, n—parameter describing the
3.1.25 quasi-absolute fluorescence intensity scale,
time decay of the fluorescence intensity of a sample compo-
n—fluorescence intensity scale that has been normalized to the
nent; if a sample decays by first-order kinetics, this is the time
intensity of a fluorescent reference sample or artifact under a
required for its fluorescence intensity and corresponding ex-
fixed set of instrumental and experimental conditions.
cited state population to decrease to 1/e of its initial value.
3.1.25.1 Discussion—This artifact should be known to yield
3.1.14 fluorescence quantum effıciency, n—ratio of the num-
a fluorescence intensity that is reproducible with time and
beroffluorescencephotonsleavinganemittertothenumberof
between instruments under the fixed set of conditions.
photons absorbed.
3.1.26 Raman scattering, n—inelasticscatteringofradiation
3.1.15 fluorescence quantum yield (Φ), n—probabilitythata
(the wavelengths of the scattered and incident radiation are not
molecule or species will fluoresce once it has absorbed a
equal) by a sample that occurs because of changes in the
photon.
polarizability of the relevant bonds of a sample during a
3.1.15.1 Discussion—This quantity is an innate property of
molecular vibration. (See Terminology E131, Raman spec-
the species and is typically calculated for a sample as the ratio
trum.)
of the number of molecules that fluoresce to the number of
3.1.26.1 Discussion—Theradiationbeingscattereddoesnot
molecules that absorbed.
have to be in resonance with electronic transitions in the
3.1.16 flux (or radiant flux or radiant power), n—rate of
sample, unlike fluorescence (11).
propagation of radiant energy typically expressed in Watts.
3.1.27 Rayleigh scattering, n—elasticscatteringofradiation
3.1.17 grating equation, n—relationship between the angle
byasample,thatis,thescatteredradiationhasthesameenergy
ofdiffractionandwavelengthofradiationincidentonagrating,
(same wavelength) as the incident radiation.
that is, mλ = d(sinα + sinβ), where d is the groove spacing on
3.1.28 responsivity, n—ratio of the photocurrent output and
thegrating; αand βaretheanglesoftheincidentanddiffracted
the radiant power collected by an optical radiation detection
wavefronts, respectively, relative to the grating normal; and m
system.
is the diffraction order, which is an integer (8).
3.1.29 sensitivity, n—measure of an instrument’s ability to
3.1.18 inner filter effects, n—decrease in the measured
detect an analyte under a particular set of conditions.
quantum efficiency of a sample as a result of significant
absorptionoftheexcitationbeam,reabsorptionoftheemission 3.1.30 spectral bandwidth (or spectral bandpass or
of the sample by itself, or both, and this causes the measured resolution), n—measure of the capability of a spectrometer to
E2719 − 09 (2022)
separate radiation or resolve spectral peaks of similar wave- cuvette. To check the spectral transmission characteristics,
lengths. (See Terminology E131, resolution.) measure a cuvette’s transmittance in a UV/Vis
spectrophotometer, after filling it with a solvent of interest.
3.1.31 spectral flux (or spectral radiant flux or spectral
Check to insure that the cuvettes being used transmit energy
radiant power), n—flux per unit spectral bandwidth typically
through the entire analytical wavelength range. Many organic
expressed in W/nm.
solvents dissolve plastic, so plastic cuvettes should not be used
3.1.32 spectral responsivity, n—responsivity per unit spec-
in these cases. Standard cuvettes have inner dimensions of
tral bandwidth.
10 mm by 10 mm by 45 mm. If only a small amount of sample
3.1.33 spectral slit width, n—mechanical width of the exit
is available, then microcuvettes can be used. Black self-
slitofaspectrometerdividedbythelineardispersionintheexit
masking quartz microcuvettes are recommended since they
slit plane. E131
require no masking of the optical beam. Cuvette caps or
3.1.34 traceability, n—linking of the value and uncertainty stoppers should be used with volatile or corrosive solvents.
5.2.1 Handling and Cleaning—For highest quality work,
of a measurement to the highest reference standard or value
through an unbroken chain of comparisons, where highest windows should never be touched with bare hands. Clean,
refers to the reference standard whose value and uncertainty powder-free, disposable gloves are recommended. Cuvettes
shouldberinsedseveraltimeswithsolventafteruseandstored
are not dependent on those of any other reference standards,
and unbroken chain of comparisons refers to the requirement wet in the normal solvent system being used. For prolonged
storage, cuvettes should be stored dry, wrapped in lens tissue
that any intermediate reference standards used to trace the
measurement to the highest reference standard must have their and sealed in a container. To clean a cuvette more thoroughly,
it should be filled with an acid, such as 50 % concentrated
values and uncertainties linked to the measurement as well
(12). nitric acid, and allowed to sit for several hours. It should then
be rinsed with deionized water several times to remove all
3.1.35 transfer standard, n—reference standard used to
traces of acid.
transfer the value of one reference standard to a measurement
or to another reference standard. 5.3 Selection of Solvent—Solvents can change the spectral
shape, cause peak broadening, and alter the wavelength posi-
3.1.36 transition dipole moment, n—oscillating dipole mo-
tionofafluorophore (13).Checktoinsurethatthesolventdoes
ment induced in a molecular species by an electromagnetic
not itself absorb or contain impurities at the analytical wave-
wave that is resonant with an energy transition of the species,
length(s). As standard practice, when optimizing a procedure,
for example, an electronic transition.
oneshouldfirstscanthesolventusingtheanalyticalparameters
3.1.36.1 Discussion—Its direction defines that of the transi-
to see if the solvent absorbs or fluoresces in the analytical
tion polarization and its square determines the intensity of the
wavelength range. This will also identify the position of the
transition.
Raman band of the solvent and any second order bands from
the grating. It is essential to examine the quality of solvents
4. Significance and Use
periodically since small traces of contaminants may be enough
4.1 By following the general guidelines (Section 5) and
to produce high blank values.
instrument calibration methods (Sections6–16) in this guide,
5.3.1 Water is the most common solvent and deionized-
usersshouldbeabletomoreeasilyconformtogoodlaboratory
distilled water should always be employed. All other reagents
and manufacturing practices (GXP) and comply with regula-
used in the assay should be carefully controlled and high
tory and QA/QC requirements, related to fluorescence mea-
quality or spectrophotometric grades are recommended.
surements.
5.3.2 Solvents should not be stored in plastic containers
4.2 Each instrument parameter needing calibration (for
sinceleachingoforganicadditivesandplasticizerscanproduce
example, wavelength, spectral responsivity) is treated in a
high blank values.
separate section.Alist of different calibration methods is given
5.3.3 Reagent blanks should be measured during the ana-
for each instrument parameter with a brief usage procedure.
lytical procedure and the actual value of the blank determined
Precautions, achievable precision and accuracy, and other
before the instrument is zeroed.
useful information are also given for each method to allow
5.4 Other Contaminants:
users to make a more informed decision as to which method is
5.4.1 Soaking glassware in detergent solutions is a general
the best choice for their calibration needs. Additional details
method of cleaning. Some commercial preparations are
for each method can be found in the references given.
5. General Guidelines
TABLE 1 Spectral Transmission Characteristics of
5.1 General areas of concern and precautions to minimize
Cuvette Materials
errors for fluorescence measurements are given by topic. All
Wavelength Range (nm)
topics applicable to a user’s samples, measurements and
Glass 350 to 2500
analysis should be considered.
Near Infrared Quartz 220 to 3800
Far UV Quartz 170 to 2700
5.2 Cuvettes—Various types of cuvettes or optical “cells”
Polystyrene 400 to 1000
are available. They vary in material composition and in size.
Acrylic 280 to 1000
The former will determine the effective spectral range of the
E2719 − 09 (2022)
TABLE 2 Summary of Methods for Determining Wavelength Accuracy
Precision, Established
Sample λ Region Drop-In Off-Shelf Limitations Refs.
Accuracy Values
Pen Lamp UV-NIR (EM) Maybe Y ±0.1 nm alignment Y Test Method
or better E388
Dy-YAG crystal 470 nm–760 nm (EM) Y Y ±0.1 nm Y 14
255 nm–480 nm (EX)
Eu glass 570 nm–700 nm (EM) Y Y ±0.2 nm N 15
360 nm–540 nm (EX)
Anthracene in PMMA 380 nm–450 nm (EM) Y Y ±0.2 nm limited range N
310 nm–380 nm (EX)
Ho O + DR 330 nm–800 nm (EM or EX) Maybe Y ±0.4 nm need blank Y 16-18
2 3
Xe Source 400 nm–500 nm (EX) Y Y ±0.2 nm limited range, calibration N 19
Xe Source + DR UV-NIR Maybe Y ±0.2 nm one mono Y 19
must be calibrated
Water Raman UV-blue Y Y ±0.2 nm one mono N 20
must be calibrated
strongly fluorescent. Before use, the fluorescence characteris- interference by other components including solvent. In some
ticsofadilutesolutionofthedetergentshouldbemeasured,so cases, a lesser absorbing wavelength is selected to eliminate
that the user knows if detergent contamination is a cause for interferences from other compounds that absorb at the same
concern. wavelength or to avoid photobleaching.
5.4.2 Stopcock grease is another common contaminant with
5.7 Selection of Spectral Bandwidth—Ideally, one would
strong native fluorescence.
like to select the widest slit possible to give the greatest signal
5.4.3 The growth of micro-organisms in buffer or reagent
to noise ratio while maintaining spectral selectivity.
solutionswillaffectblankvaluesbyboththeirfluorescenceand
light scattering properties.
6. Wavelength Accuracy
5.4.4 Filter paper and lab wipes can be sources of contami-
6.1 Methods for determining the accuracy of the emission
nation due to fluorescent residues. These should be checked
(EM) or excitation (EX) wavelength for a fluorescence instru-
before use.
ment are given here and summarized in Table 2 with an
5.5 WorkingwithDiluteSolutions—Itiscommonpracticeto
emphasis on monochromator (mono) based wavelength selec-
store concentrated stock solutions and make dilutions to
tion.
produce working standards. It is always better to confirm the
6.2 Low-Pressure Atomic Lamps (see Test Method E388)—
concentration of the stock solution spectrophotometrically
These low-pressure atomic lamps, often referred to as pen
before the calibration curve is prepared. Final solutions are
lamps because of their size and shape, should be placed at the
always very dilute and should never be stored for long periods.
sample position and pointed toward the detection system for
Standards should be measured in duplicate or triplicate to
EM wavelength accuracy determination. The EM wavelength
insure accuracy.
selector (λ -selector) is then scanned over the wavelength
5.5.1 Adsorption—Loss of fluorophore by adsorption onto
EM
range of interest (see Fig. 1). High accuracy is only achieved
thewallsofthecontainercanoccuratlowconcentrationlevels.
when the light from the lamp is aligned properly into the
Glasssurfacesshouldbethoroughlycleanedinacidbeforeuse.
wavelengthselector,forexample,theopticalradiationmustfill
5.5.2 Photo-Decomposition and Oxidation—Since fluores-
cence intensity is directly proportional to the intensity of the entrance slit of the monochromator. Atomic lines that are
too close to each other to be resolved by the instrument should
incident light, fluorescence instruments employ intense light
sources to produce high sensitivity. In some cases the level of not be used. Although these lamps can be placed at the EX
source position for EX wavelength accuracy determination,
incident light may be sufficient to decompose the sample under
investigation. This should be checked and samples should be weaker signals are typically observed, for example, by a
reference detector, and alignment is more difficult than for the
measured as quickly as possible. The presence of trace oxidiz-
EM wavelength accuracy determination.
ing agents, for example, dissolved oxygen or traces of
peroxides, can reduce fluorescence intensity.
6.3 Dysprosium-Yttrium Aluminum Garnet (Dy-YAG) Crys-
5.6 Selection of Optimal Wavelength—To choose an appro- tal (14)—This sample is available in standard cuvette format,
priate analyte excitation band, scan the analyte with a UV/Vis so it can simply be inserted into a cuvette holder, referred to as
spectrophotometer to determine the absorbance maxima and to “drop in” in the tables. An EX or EM spectrum is then
see if there is any interfering compound or scattering at the collectedforanEXorEMwavelengthaccuracydetermination,
analytical wavelength. The optimal wavelength is usually that respectively (see Fig. 2). Peaks that are too close to each other
which shows the strongest absorbance and is free from to be resolved by the instrument should not be used.
E2719 − 09 (2022)
FIG. 1 Hg Pen Lamp Spectrum
FIG. 2 EM Spectrum of a Dy-YAG Crystal Excited at 352.7 nm
E2719 − 09 (2022)
FIG. 3 EM Spectrum of a Eu-Ion-Doped Glass Excited at 392 nm
6.4 Europium (Eu)-Doped Glass (15) or Polymethylmeth- scanned with and without the Ho O sample in place, and the
2 3
acrylate (PMMA)—This sample is available in standard cu- ratio of the two intensities is calculated to obtain an effective
vette format, so it can simply be inserted into a cuvette holder. transmittance spectrum with dips in the intensity ratio corre-
An EX or EM spectrum is then collected for an EX or EM sponding to absorption peaks of the sample (see Fig. 5).
wavelength accuracy determination, respectively (see Fig. 3).
6.7 Xenon (Xe) Source Lamp (19)—This method is for
Accurate peak positions for this glass have not been well
fluorometers that use a high-pressure Xe arc lamp as an EX
established, and the positions of peaks can change somewhat
source.Afew peaks between 400 nm and 500 nm can be used,
depending on the particular glass matrix used and sample
but most of these are a result of multiple lines, so their
temperature. For these reasons, a one time per sample deter-
positions are not well established (see Fig. 6). For this reason,
mination of these peak positions using another wavelength
a determination of these peak positions (one time per lamp)
calibration method is recommended.
using another wavelength calibration method is recommended.
6.5 Anthracene-Doped PMMA —This sample is available For EX wavelength calibration, the EX wavelength selector
in standard cuvette format, so it can simply be inserted into a (λ -selector) is scanned while collecting the reference detec-
EX
cuvette holder.An EX or EM spectrum is then collected for an tor signal. If this is used for EM wavelength calibration, a
EX or EM wavelength accuracy determination, respectively diffuse reflector or scatterer shall be placed at the sample
(see Fig. 4). position and the λ -selector shall be removed or set to zero
EX
order.
6.6 Holmium Oxide (Ho O ) Solution or Doped Glass with
2 3
Diffuse Reflector, Scatterer, or Fluorescent Dye (16-18)—This
6.8 Instrument Source with Diffuse Reflector or Scatterer
sample is available in standard cuvette format, so it can simply
(19)—A dilute scattering solution in a standard cuvette or a
be inserted into a cuvette holder. An EX or EM spectrum is
solid diffuse reflector set at 45° relative to the EX beam can be
then collected for an EX or EM wavelength accuracy
used to scatter the EX beam into the detection system. One
determination, respectively. The wavelength selector not being
wavelength selector is fixed at a wavelength of interest and the
scanned shall be removed or set to zero order, that is, in this
other scans over the fixed wavelength (see Fig. 7). The
position a grating behaves like a mirror reflecting all wave-
differencebetweenthefixedwavelengthandtheobservedpeak
lengths. The diffuse reflector, scatterer, or fluorescent dye is
position is the wavelength bias between the two wavelength
selectors at that wavelength. Either the EX or the EM wave-
5 length selector shall have a known accuracy at the desired
Other rare earth doped glasses have narrow EX and EM transitions, but
Eu-doped glass is the only one listed because it is one of the most commonly used wavelengths to use this method to calibrate the unknown side.
and most readily available.
6 6.9 Water Raman (20)—Deionized water is used. One
Other polyaromatic hydrocarbon-doped PMMAs have narrow EX and EM
transitions, including those with ovalene, p-terphenyl, and naphthalene. wavelength selector is fixed at a wavelength of interest and the
E2719 − 09 (2022)
FIG. 4 EM Spectrum of Anthracene-Doped PMMA Excited at 360 nm
FIG. 5 Effective Transmittance Spectrum of a Ho O -Doped Glass with Diffuse Reflector
2 3
other is scanned (see Fig. 8).The water Raman peak appears at weak to use this method when going into the visible region.
–1
a wavelength that is about 3400 cm lower in energy than the Either the EX beam or the EM wavelength selector shall have
EX wavelength (21). The Raman scattering intensity is pro- a known accuracy at the desired wavelengths to use this
–4
portional to λ , so the Raman intensity quickly becomes too method to calibrate the unknown side.
E2719 − 09 (2022)
FIG. 6 Xe Source Lamp (High-Pressure, 450 W) Spectrum in a Spectral Region Containing Peak Structure
FIG. 7 EX Source Profile with EX Wavelength Fixed at 404.3 nm (EX Bandwidth of 1.0 nm) and EM Monochromator Scanned
(EM Bandwidth of 0.1 nm)
7. Spectral Slit Width Accuracy bandwidth,takentobethefullwidthathalfthepeakmaximum
(FWHM), of a single line of a pen lamp, using the same setup
7.1 Spectral slit width accuracy of the EM or EX wave-
and with the same precautions described in 6.2 (see Test
length selector can be determined by measuring the spectral
E2719 − 09 (2022)
FIG. 8 Water Raman Spectrum with EX Wavelength Set at 350 nm and EX and EM Bandwidths at 5 nm
Method E388). For fluorescence spectrometers with both EX described in 11.3. In this case, solutions with a low concentra-
and EM monochromators, an alternative method may be used tion (A < 0.05 at 1 cm path length) should be used and
in which one monochromator is scanned over the position of fluorophore adsorption to cuvette walls may affect measure-
the other using the setup described in 6.8 (19). The uncertain- ments at very low concentrations (see Test Method E578). In
ties involved in either method have not been well established, addition, fluorophores are needed that are not prone to reab-
but a 60.5 nm uncertainty or better is estimated here based on sorption effects and that do reveal concentration-independent
what has been reported. emissionspectra.Usersshallinsurethatthefluorescencesignal
intensities of samples are reproducible and do not decrease
8. Linearity of the Detection System
over the time period they are being excited and measured
because the organic dyes typically used can be prone to
8.1 Several methods can be used to determine the linear
photobleaching and other degradation over time.
intensity range of the detection system. They can be separated
into three types based on the tools used to vary the intensity of
9. Spectral Correction of Detection System Responsivity
optical radiation reaching the detector: (1) double aperture, (2)
optical filters, polarizers or both, and (3) fluorophore concen- 9.1 Calibration of the relative responsivity of the EM
trations. The double-aperture method is the most well estab- detection system with EM wavelength, also referred to as
lishedandprobablythemostaccuratewhendonecorrectly,but spectral correction of emission, is necessary for successful
it is also the most difficult to perform (22, 23). A variety of quantification when intensity ratios at different EM wave-
methods using optical filters, polarizers, or a combination of lengths are being compared or when the true shape or peak
the two have been reported (19, 24). These methods require maximum position of an EM spectrum needs to be known.
high-quality, often costly, components, and some user exper- Such calibration methods are given here and summarized in
tise. The third method is the most popular and easiest to Table 3. This type of calibration is necessary because the
implement. It uses a set of solutions obtained by serial dilution relative spectral responsivity of a detection system can change
of a fluorescent stock solution, similar to that used for significantly over its useful wavelength range (see Fig. 9). It is
obtaining calibration curves for analyte concentration, as highly recommended that the linear range of the detection
TABLE 3 Summary of Methods for Determining Spectral Correction of Detection System Responsivity
Precision, Certified
Sample λ Region Drop-In Off-Shelf Limitations Refs.
Accuracy Values
CS UV-NIR N Y <±5 % difficult setup Y 19, 24-27
CD + CR UV-NIR N Maybe ±10 % difficult setup Y 19, 25, 26, 28
CRMs UV-NIR Y Y ±5 % Y 29-31
E2719 − 09 (2022)
FIG. 9 Example of the Relative Spectral Responsivity of an EM Detection System (Grating Monochromator-PMT Based) (19) for Which a
Correction Needs to be Applied to a Measured EM Spectrum to Obtain Its True Spectral Shape (Relative Intensities)
system be determined (see Section 8) before spectral calibra- 9.3 Calibrated Detector (CD) with CR (19, 25, 26, 28)—
tion is performed and appropriate steps are taken (for example, This is a two-step method. The first step uses a CD to measure
the use of attenuators) to insure that all measured intensities the flux of the EX beam as a function of EX wavelength, as
during this calibration are within the linear range. Also note described in 10.2. Alternatively, a quantum counter solution
that when using an EM polarizer, the spectral correction for can be used instead of a CD, as described in 10.3. The second
emission is dependent on the polarizer setting. step uses a CR with reflectance R to reflect a known fraction
CR
of the flux of the EX beam into the detection system. This is
9.2 Calibrated Optical Radiation Source (CS)–Tungsten
done by placing the CD at the sample position at a 45° angle
Lamp (19, 24-27)—The optical radiation from a CS is directed
relative to the excitation beam, assuming a right-angle detec-
into the EM detection system by placing the CS at the sample
tion geometry relative to the excitation beam, and synchro-
position. If the CS is too large to be placed at the sample
nously scanning both the λ - and λ -selectors over the EM
EX EM
position, a calibrated diffuse reflector (CR) may be placed at
region of interest while collecting both the signal output (S’)
the sample position to reflect the optical radiation from the CS
andthereferenceoutput(Rf’).Thismethodenablestherelative
intotheEMdetectionsystem.The λ -selectorisscannedover
EM
correction factor (C ) to be calculated using the equation
CD
theEMregionofinterest,usingthesameinstrumentsettingsas
C =(C R Rf’)/S’. See Section 3 for definitions of terms.
CD R CR
that used with the sample, and the signal channel output (S")is
collected. The known radiance of the CS incident on the 9.4 Certified Reference Materials (CRMs) (29-31)—The
detection system (L) can be used to calculate the relative CRMs presently available are either organic dye solutions or
correction factor (C ), such that C = L/S". The corrected solid, inorganic glasses released by national metrology insti-
CS CS
EM intensity is equal to the product of the signal output of the tutes(NMIs)withcertifiedrelativefluorescencespectra,thatis,
sample (S) and C . relativeintensityanduncertaintyvaluesaregivenasafunction
CS
of EM wavelength at a fixed EX wavelength. They have been
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
Other types of calibrated lamps can be used, but tungsten is ideal in the visible responsivityisknown.Theformeristypicallythemoreaccurateofthetwo,because
range due to its broad, featureless spectral profile and high intensity. the integrating sphere insures spatially uniform illumination of the photodiode.
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designedtoresemblecloselytypicalsamples.ACRMisplaced counter solution is placed at the sample position in a quartz
at the sample position and its spectrum is collected and cuvette. If front face detection is possible, then a standard
compared to the certified spectrum according to the instruc- cuvette can be used with the EX beam at normal incidence. If
tions given on the accompanying certificate, yielding spectral 90° detection only is possible, then a right-triangular cuvette
correctionfactorsfortheinstrument.ThecorrectedEMspectra can be used with the excitation beam at 45° incidence to the
of some commonly used dyes have also been reported recently hypotenuse side and one of the other sides facing the detector.
in the literature (32, 33). Scan the EX wavelength over the region of interest with the
EM wavelength fixed at a position corresponding to the
10. Spectral Correction of Excitation Beam Intensity
long-wavelength tail of the EM band and collect the signal
intensity(S ).Theinstrument’sreferencedetectorcanalsobe
10.1 Calibration of the EX intensity with EX wavelength is QC
used to measure the intensity of the EX beam by measuring its
necessary for successful quantification when intensity ratios at
output (Rf ) simultaneously with S . Then, the correction
differentEXwavelengthsarebeingcomparedorthetrueshape QC QC
factor for the responsivity of the reference detector C = S
or peak maximum position of an EX spectrum needs to be R QC
/Rf is calculated. Note that each quantum counter has a
known. Such calibration methods are given here and summa-
CD
limited range. For instance, Rhodamine B can achieve the
rized in Table 4. This type of calibration is necessary because
specified uncertainty from 250 nm to 600 nm. Beyond this
the relative spectral flux of an EX beam at the sample can
range, the intensity falls off and uncertainties increase. Also
change significantly over its wavelength range (see Fig. 10).
note that S will be proportional to the quantum flux at the
The neglect of EX intensity correction factors can often cause
QC
sample, not the flux in power units. In addition, a quantum
greater errors than that of EM correction factors (19, 34).
counter is prone to polarization and geometry effects that are
Fortunately, many fluorescence instruments have a built-in
concentrationdependent.Thespectralrangeandcorresponding
reference detection system to monitor the intensity of the EX
uncertainty of a quantum counter should be known and not
beam. This is commonly done using a photodiode or a
assumed.
photomultiplier tube (PMT) or a quantum counter detector to
measureafractionoftheEXbeamthatissplitofffromtherest
10.4 Si Photodiode (Uncalibrated) (19)—Thisisusedinthe
of the beam. The collected reference signal can be used to
same way as a calibrated Si photodiode (see 9.2), except its
correct the fluorescence signal for fluctuations caused by
spectral responsivity is not known. A Si photodiode is some-
changes in the EX beam intensity. Reference detectors are
times erroneously assumed to have a responsivity that is
often not calibrated with EX wavelength, introducing errors,
qualitatively flat over its effective range. In fact, using its
which can be particularly large over longer EX wavelength
output to correct an EX spectrum can lead to quantitatively
ranges (for example, greater than 50 nm) or in a wavelength
significant errors, particularly over a large EX range and in the
region in which the EX intensity changes rapidly with EX
UV region. That said, using an uncalibrated Si photodiode for
wavelength, such as the ultraviolet (UV).Also note that, when
correction will in most cases yield a more accurate spectrum
using an EX polarizer, the spectral correction for EX intensity
than using no correction.
is dependent on the polarizer setting.
11. Calibration Curves for Concentration
10.2 Calibrated Detector-Si Photodiode (CD-Si) (19,
24)—A CD is put at the sample position with the excitation
11.1 Guidelines—Calibration curves of fluorescence
beamincidentonit.TheoutputoftheCD(S )ismeasuredas
CD
intensity, that is, instrument responsivity, as a function of
afunctionofEMwavelengthbyscanningthe λ -selectorover
EX
fluorophore concentration can be determined for a particular
the EX region of interest using the same instrument settings as
instrument and fluorophore. Reference materials composed of
that used with the sample. The known responsivity of the CD
the fluorophore of interest shall be used. The highest accuracy
(R ) is used to calculate the flux of the EX beam (φ ), such
CD x is obtained when the fluorophore in both the standard and the
that φ =S /R .Theinstrument’sreferencedetectorcanalso
x CD CD
sample experience the same microenvironment. For example,
be used to measure the intensity of the EX beam by measuring
they are dissolved in the same solvent or attached to the same
itsoutput(Rf )simultaneouslywith S .Then,thecorrection
CD CD
biomolecules. This type of calibration enables concentrations
factor for the responsivity of the reference detector C =
R
and amounts of fluorophores to be compared over time and
φ /Rf .
x CD
between instruments without determining the absolute respon-
sivity of the instrument (see Section 9).
10.3 Quantum Counters (27, 35, 36)—A quantum counter
solution is a concentrated dye solution that absorbs all of the 11.1.1 Concentration Range of the Standards—The concen-
photons incident on it and has an EM spectrum whose shape tration of the highest and lowest standards should bracket the
andintensitydonotchangewithEXwavelength.Thequantum concentrations of the unknowns that are being measured. For
TABLE 4 Summary of Methods for Determining Spectral Correction of EX Beam Intensity
Precision, Certified
Sample λ Region Drop-In Off-Shelf Limitations Refs.
Accuracy Values
CD - Si UV-NIR N Y ±2 % difficult setup Y 19, 24
Quantum Counter UV-NIR Y Y ±5 % limited range N 27, 35
Photodiode - Si UV-NIR N Y #±50 % N 19
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FIG. 10 Example of the Relative Flux of an EX Beam (Xe Lamp-Grating Monochromator Based) (19) for Which a Correction Needs to be
Applied to a Measured EX Spectrum to Obtain Its True Spectral Shape (Relative Intensities)
best precision and accuracy, the concentrations of the analyte 11.2 Fluorophores with Specified Purity and Uncertainty
should be low enough that the absorbance at the excitation (37)—Ifthepurityofafluorophore(forexample,ahigh-purity,
wavelength is less than 0.05 to prevent inner filter effects. A organic dye powder) is known, then it can be put in the same
test for concentration quenching is to dilute the sample in half. microenvironment (for example, solvent) as an unknown
If the resultant signal is not half the previous value, then sampletoproduceastandardsample.Severalstandardsamples
concentration quenching is occurring and one needs to work at should be produced to cover the concentration range of
a lower initial sample concentration. interest. These standard samples are measured under the same
11.1.2 Measurements: conditions as that of any unknowns and the fluorescence
11.1.2.1 Make up a stock standard solution. Verify the intensities are recorded. Fluorescence intensity versus standard
concentration of the solution, if possible, using a UV/Vis sample concentration is plotted and the points are fitted to a
spectrophotometer. polynomial, typically a straight line. The concentration of an
11.1.2.2 Use concentrations covering the range of interest unknown is determined by using the fitted polynomial along
for the unknown samples and which produce acceptable with the measured intensity of the unknown to find the
fluorescence values. When making standards, individual ali- corresponding concentration.
quots can be used to make the standards, instead of a serial
11.3 Fluorophore Solutions with Specified Concentration
dilution, as a check of accuracy.
and Uncertainty (38) (see Test Method 578)—Standard solu-
11.1.2.3 A calibration curve using at least three standards,
tions with known concentrations can be used in the same way
that is, three data points, should be used.
as a standard fluorophore (see 10.2). In this case, the fluoro-
11.1.2.4 The highest and lowest standard should bracket the
phores are in solution, so they are ready to use or they can be
concentration level of the analytical assay.
dilutedtoproducestandardsolutionsoflowerconcentration.In
11.1.2.5 Measure the fluorescence of standard at the ana-
both cases, the solvent used in the standard and unknown
lytical wavelength.
solutions should be the same.
11.1.2.6 Make a plot of the fluorescence signal as the
ordinate and the concentration as the abscissa. 11.4 Molecules of Equivalent Soluble Fluorophore (MESF)
11.1.2.7 Handling of Standards—Always insure that the (39-42)—Thematchingofmicroenvironmentsbetweensample
samples are handled in the same way as the standards, and standard solutions, as emphasized in 10.2 and 10.3, cannot
particularly for extraction procedures and filtration because of always be achieved. This is of particular concern when the
errors due to partition coefficients. sample contains immobilized fluorophores, for example, those
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attached to a cell. In many such cases, it is very difficult or advisable to use a solvent that does not absorb significantly in
impossible to determine the concentration of fluorophores in a the excitation region of the analyte.
candidate standard solution. MESF units are used, particularly 11.5.8 Aggregation—If aggregation occurs, select a solvent
in flow cytometry, to quantify such complex systems. These to minimize aggregation of fluorophores.
11.5.9 Scattering—Filter the sample to minimize particu-
unitsexpressthefluorescenceintensityofafluorescentanalyte,
for example several immobilized fluorophores bound to a lates.
11.5.10 Adsorption—Measure the sample as quickly as
microbead or cell, as the corresponding number of free
fluorophores of the same type in a standard solution with the possible after preparation and thoroughly clean all glassware
and cuvettes (see 5.5.1).
same intensity. The MESF scale for a particular fluorophore is
11.5.11 Instrumental Noise—Work at concentrations and
determined using the same procedure as that given in 10.3.
use instrumental parameters (for example, integration time and
This scale is transferred from a conventional fluorometer to a
spectral bandwidth) that provide an acceptable signal to noise
flow cytometer using fluorophore-labeled microbead suspen-
ratio.
sions with predetermined MESF values.
11.5 Errors—A number of sources of error can be intro-
12. Day-to-Day and Instrument-to-Instrument Intensity
duced into the system from sample preparation, instrumental
12.1 Thedeterminationofthestabilityofaninstrumentover
limitationsandchemicalinterferences,causingdeviationsfrom
time and comparability between instruments of fluorescence
the Beer-Lambert law. An awareness of these potential prob-
intensityismadepossiblebyperformancevalidationstandards.
lems is important.
The fluorescence intensity of such standards can be monitored
11.5.1 Weighing Error—Gravimetric and volumetric errors
over time and between instruments, enabling an absolute
associated with weighing and diluting of the sample.
intensity scale to be established without performing absolute
11.5.2 Non-Linearity—The proportional relationship be-
fluorescence measurements. These standards shall emit a
tween light absorption and fluorescence emission is only valid
fluorescence intensity that does not change with time or
forcaseswheretheabsorptionissmall.Astheconcentrationof
irradiation. Even though such standards do not need to be
fluorophore increases, deviations occur and the plot of emis-
certified,theirlongtermstabilityandrelateduncertaintiesneed
sion versus concentration becomes non-linear. This is due to
to be known. Another possibility is that they be single-use
inner filter effects. In cases where it is necessary to work at
standards that can be made with a highly reproducible fluores-
high concentrations, it is possible to increase the linear
cenceintensity.Inthiscase,theuncertaintiesintroduc
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