Report

Formalization of the MESF Unit of FluorescenceIntensity

Abe Schwartz,1 Adolfas K. Gaigalas,2 Lili Wang,2 Gerald E. Marti,3 Robert F. Vogt,4

E. Fernandez-Repollet5

1Center for Quantitative Cytometry, San Juan, Puerto Rico2Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, Maryland

3Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Bethesda, Maryland4Division of Laboratory Sciences, Centers for Disease Control, Atlanta, Georgia

5Department of Pharmacology, University of Puerto Rico School of Medicine, San Juan, Puerto Rico

This report summarizes the work performed during the past two years at the National Institute of Standardsand Technology (NIST) in the refinement and formal definition of the MESF unit of fluorescence intensity. Inaddition to the theory underlying the MESF unit, considerations of error analysis are also presented. Thedetails of this work may be found in the three publications of the NIST Journal of Research (www.nist.gov)listed as the references 2–4. The use of the fluorescence intensity unit provides a tool to comparequantitative fluorescence intensity measurements over time and across platforms. © 2003 Wiley-Liss, Inc.

Key terms: MESF; Molecules of Equivalent Soluble Fluorochrome; quantitation; fluorescence intensity

INTRODUCTIONThe quantitation of fluorescence intensity in biologi-

cal assays is a long-standing goal (1). Over the pasttwenty years, the fluorescence unit of intensity, MESF,has been introduced and utilized in the field of flowcytometry. MESF is an abbreviation for Molecules ofEquivalent Soluble Fluorochrome. The MESF conceptindicates that a sample labeled with a fluorochrome hasthe same fluorescence intensity as an equivalent num-ber of molecules of the fluorochrome free in a solutionunder the same environmental conditions. This fluores-cence unit provided researchers with tool to compareflow cytometry data in a quantitative manner over timeand across instruments. For instance, a search onPubMed (National Library of Medicine) of the literaturesince 1984 found 193 articles related to quantitativefluorescence flow cytometry and 53 articles specificallyusing MESF as the fluorescence unit of measure. Al-though MESF units have been used internationally overthis extended period, a precise definition of the unithad not been presented until recently. The definition ofthe unit was presented in the context of a fluorescencemeasurement model that relates the physical character-istics of the sample and the resulting fluorescence sig-nal (3). The model clearly states what is being com-pared between the standard and analyte samples, andallows a clear demarcation between instrumental biasesand the physical characteristics of the sample. Thefollowing description briefly summarizes the formal def-

inition of the MESF unit that has been published in theJournal of Research of the National Institute of Stan-dards and Technology (NIST) and its application inquantifying bound antibodies in flow cytometric mea-surements (2– 4).

SUMMARY OF THE DERIVATIONAs shown in the second NIST publication (3), the inter-

pretation of all fluorescence intensity measurements isbased on a solution property called the fluorescence yieldthat is the product of the concentration of fluorophoresand the molecular quantum yield. The fluorescence yieldcan be visualized as the total fluorescence emitted by asolution if every fluorophore was initially in the excitedstate. In principle, the fluorescence yield of any two so-lutions can be compared. This fact provides the founda-tion for the application of standards for the quantitation offluorescence intensity. The comparison of fluorescenceyields of any two solutions starts with the measurement of

Research Support: provided by the National Institute of Standards andTechnology and the Centers for Disease Control and Prevention. A.Schwartz is a visiting scientist at the National Institute of Standards andTechnology.

Correspondence to: A. Schwartz, Ph.D., P.O. Box 194344, San Juan,PR 00919-4344.

E-mail: abe@quantcyte.orgReceived 16 June 2003; Accepted 1 September 2003Published online in Wiley InterScience (www.interscience.wiley.com).

DOI: 10.1002/cyto.b.10066

Cytometry Part B (Clinical Cytometry) 57B:1–6 (2004)

© 2003 Wiley-Liss, Inc.

fluorescence intensity from the two solutions, where thefluorescence intensity is understood to be the signal givenby the detection apparatus. The measurement model givesa detailed relation between the measured fluorescenceintensity and the fundamental solution property—the flu-orescence yield. It becomes apparent that comparing flu-orescence yields from the relative fluorescence intensitymeasurements is not easy. Difficulties arise from the inter-dependence of the measured fluorescence intensity onthe instrument parameters, and also on the environmentalfactors which affect the absorption and emission of thefluorochrome. In the regime of low fluorophore concen-tration the dependence of the fluorescence intensity onthe various factors may be summarized in equation 1(measurement model).

iF � [ge��Q(�)s(�)T(�)d�]c (1)

where

iF � measured fluorescence intensity

g � photomultiplier (PMT) gain

e � elementary charge

� � aperture and collection optics

Q(�) � quantum efficiency of the PMT at �

T(�) � filter characteristics at �

ε � molar extinction coefficient at the

excitation wavelength

� � quantum yield

s(�) � normalized emission spectral function at �

c � concentration of the fluorochrome

In comparing the fluorescence intensity from a standardand a unknown solution Eq. 1 suggests that some factorssuch as g, e, � cancel out. These factors describe part ofthe instrument response that is approximately indepen-dent of wavelength and are presumed to be the samewhen measuring the standard and the unknown solution(the standards and the sample must be run on the sameinstrument at the same settings). Now, if the remainingfactors (except for quantum yield) are collected into afactor called X:

X � ε�Q(�)s(�)T(�)d� (2)

Then the expression for the measured fluorescence inten-sity reduces to:

iF � X �c (3)

where c is the concentration of fluorophores in the solu-tion (standard or unknown) and � is the molecular quan-tum yield. Eq.3 suggests that if the factor X can be madethe same for the standard solution and the unknownsolution then the comparison of fluorescence intensitywould be equivalent to a comparison of fluorescenceyields of the two solutions. Examination of the factor Xsuggests that if the excitation (ε(�)) and emission (s(�))spectra of the standard solution match those of the un-known solution, then the factor X is the same for the twosolutions. In summary, if the standard and unknown solu-tions are measured under the same instrument conditionsand the excitation and emission spectra are the same, thenthe comparison of measured fluorescence intensity is thesame as the comparison of fluorescence yield of the twosolutions. The equality of the fluorescence yields of theunknown and standard solutions provides an equivalencerelation between the concentrations of fluorophores inthe two solutions. Thus, one could state that the concen-tration of fluorophore in the unknown solution is equiva-lent to a known concentration of fluorophore in the stan-dard solution. In some special cases it may be possible toensure that the microenvironments of the fluorophoresare the same in the standard and unknown solutions. Insuch cases, the molecular quantum yields are expected tobe the same in the two solutions and the comparison offluorescence yields reduces to a comparison of actualconcentrations of fluorophores. In practice, it is very dif-ficult to ensure that the microenvironments of fluoro-phores in a standard and unknown solutions are identical,therefore, comparisons are limited to that of equivalenceof concentrations.

The above discussion can be extended to the compar-ison of fluorescence intensity of a standard solution and anunknown suspension of microspheres with immobilizedfluorophores. The microspheres can be viewed as verylarge fluorophores and the language used in the previousdiscussion carries over naturally to the case of fluorescingmicrospheres. The only caveat is the use of number con-centration in suspensions and the use of molar concentra-tion in solutions. Avogadro’s Number can be used toconvert the molar concentration in a standard solution toa number concentration, and the equality of fluorescenceyields of the standard solution and unknown suspensionprovides a value of Molecules of Equivalent Soluble Fluo-rochrome per microsphere. The practical value of theMESF units is obvious in flow cytometry since only parti-cles and biological cells, and not solutions, are measuredwith this instrumentation. The expectation is that if MESF

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values can be assigned in a consistent manner to biologicalcells and to labeled antibodies, then the ratio of the MESFvalues of the cell and the antibody would provide anestimate of antibodies immobilized on the cell, a quantityof prime biologic importance.

MESF MEASUREMENTSThere are several important aspects to consider with

regard to the use of MESF units. First it is apparent fromEq. 3, that if the factors X can be evaluated for thestandard and unknown solutions then a comparison offluorescence yields can be made. The factor X can easilybe measured using a good quality spectrofluorimeter anda reference light source. In fact, the assignment of MESFvalues to microspheres requires that the factor X be mea-sured for the microsphere suspension and the standardsolution. However, if one is concerned with the use ofMESF units with cytometers then the measurement of thefactor X in Eq. 3 is not practical. In this case it is best toadopt the strategy that the comparison of fluorescenceintensity is the same as the comparison of fluorescence

yields if both the standard and unknown solutions (or sus-pensions) are measured under the same instrument condi-tions (easy to implement), and both the excitation and emis-sion spectra of the unknown and standard solutions match.In practice, it is almost impossible to match both the exci-tation and emission spectra of the sample and standard if thetwo are not labeled with the same fluorochrome. Therefore,the fluorochrome on the standard microsphere should bethe same as on the unknown particle. At present it is under-stood that the MESF value of a sample is defined in terms ofa specific fluorochrome, and as such must be so indicated. Inother words, 50,000 MESF of fluorescein is not the same as50,000 MESF of R-phycoerythrin.

The third publication in the NIST Journal of Researchdeals with the sources of error in measurements thatutilize MESF units when the same fluorochrome is used forthe standard and the unknown solution (4). The greatestsource of error is changes in the spectral response andquantum efficiency of the fluorochrome due to smallchanges in the micro-environment of the fluorophore. A

FIG. 1. The normalized emission spectra of fluorescein-labeled monoclonal antibodies (thick plots) and leukocytes stained with these antibodies (thinplots) in PBS, pH 7.2, relative to fluorescein in borate buffer, pH 9.1 (solid line): CD3 (dash dot); CD8 (dash); CD45 (dot); CD45RA (short dot). The insetshows the normalized spectra of CD45 stained leukocytes (dot line) and mononuclear cells (dash line) compared to that of fluorescein in solution (solidline). (Reprinted from J Res Natl Inst Stand Technol 2002; 107: 83–91.)

3FORMALIZATION OF THE MESF UNITS

spectral shift can arise across lots of conjugated antibodiesthat are labeled with the same fluorochrome (Fig. 1).These shifts would not be of significance if the emissionspectrum could be measured and the factor X in Eq 3estimated. However, since most flow cytometers use anarrow band pass filter, e.g., 530/30 for fluorescein, thisshift can result in significant differences in the integratedsignal. Moreover, just the binding of the fluorochrome-conjugated antibody to the cell can give rise to a spectralshift that yields a significant variation of the integratedsignal. In addition, the spectral response of microbeadstandards has been found to differ with the length of thelinking molecule. As seen in Figure 2, one could concludethat the best spectral matching with fluorescein labeledantibodies bound to cells would be achieved with micro-spheres with fluorescein immobilized on the microspheresurface via a molecule with seven carbons.

DEVELOPMENT OF FLUORESCENT STANDARDSThe work conducted at NIST during the past two years

on fluorescence intensity measurements has yielded twoimportant milestones in addition to formalizing the defi-

nition of the MESF unit (3). These include NIST issuing afluorescein solution as a Standard Reference Material(SRM1932) and the establishment of a laboratory for themeasurement of fluorescence yield from solutions andsuspensions. The laboratory contains a spectrofluorimeterthat includes two holographic notch filters to suppressscattered light, a Coulter Multisizer 3 for measuring parti-cle concentrations, and a custom-built cytometer to verifythe MESF values assigned to microspheres.

The format of the fluorescein SRM 1932 is a concen-trated solution of fluorescein at a known concentration ina borate buffer. The SRM is designed to be diluted at least� 100 in a buffer for the desired application such that theproperties of the borate buffer will have a negligible affecton the final working solution standard.

The holographic filters in the NIST spectrofluorimeter areessential to eliminate the scatter from microbead suspen-sions that give rise to extremely high backgrounds (Fig. 3).This allows proper evaluation of the total emission spectrum,thus providing better accuracy without concern for spectralshifts due to differences in environment.

FIG. 2. The normalized emission spectra of the microbeads with different linker lengths, as well as leukocytes stained with either CD45 or CD8monoclonal antibody, with respect to fluorescein in solution: Fluorescein (thick solid line); Bead 3 (thick dash dot line); Bead 7 (thick dot line); Bead12 (thick dash line); cell (CD45) (thin solid line); cell (CD8) (thin dash line). The two vertical lines define the emission collection window by the bandpassfilter used in flow cytometers. (Reprinted from J Res Natl Inst Stand Technol. 2002; 107: 83–91.)

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A second Reference Material (RM) has been authorizedby NIST, which will complement the fluorescein SRMsolution. This RM will consist of a set of microbeads withfluorescein attached to the surface via a seven-carbonspacer. These microspheres will be calibrated in MESFunits. This surface labeling yields reasonable spectralmatching with fluorescein on antibodies, thus satisfyingthe requirements for a practical MESF standard.

An additional set of solution and microbead standardshas been proposed for R-phycoerythrin. Before efforts areexpended on development of official reference products,basic questions of stability, consistency and reproducibil-ity of this fluorochrome must be addressed. This will beconducted in the laboratories of Francis Mandy fromHealth Canada.

Success with phycoerythrin will establish that both sim-ple organic fluorochromes (e.g., fluorescein, rhodamineand cyanine) and proteinasous fluorochromes (e.g., phy-coerythrin, and allophycocyanine) can be developed intoquantitative fluorescence MESF standards and hopefullybe offered as reference materials from NIST.

SIGNIFICANCEThe introduction of the MESF unit in the field of flow

cytometry has provided a tool that has helped take the

field from one of enumeration to one of quantitation. Therecent development of a more fundamental basis for theMESF assignment has clarified the interpretation of themeasured fluorescence intensity in terms of MESF values.Quantitative fluorescence data are no longer dependenton the instrument platform or the specific suspensionmedia of the samples. This independence holds so long asthe following criteria are met:

1. The standards and the unknown samples must berun on the same instrument at the same settings.

2. The excitation and emission spectra of the standardsmust match those of the unknown samples (best achievedby having the same for the standard and the unknownsample).

3. The environment of the standards and the unknownsamples must be the same.

The resulting data can be compared among laboratoriesacross the country and around the world over extendedperiods of time.

The application of the MESF concept of fluorescenceintensity measurements can be expanded and applied tofields other than flow cytometry. With the development ofthe appropriate measurement model and MESF standards,

FIG. 3. Emission spectra of microbeads labeled with fluorescein with (solid line) and without (dotted line) two holographic notch filters before theentrance slit of the monochromator. Note that the 488nm background scattering from the excitation laser is essentially eliminated with the use of theseholographic notch filters.

5FORMALIZATION OF THE MESF UNITS

such applications include quantitation of fluorescence mi-croscopy, micro-array intensity data, fluorescence imageanalysis, and can even be used to refine basic solutionspectrofluorometry. With these new developments, theconcentration of fluorochrome can be so high that MESFcan take on the expanded definition of Moles of Equiva-lent Soluble Fluorophore. Thus the development of theMESF unit comes at an important time considering theever-increasing number and types of assays that are basedon fluorescence intensity.

LITERATURE CITED1. Quantitative fluorescence cytometry: an emerging consensus. Lenkei,

R., Mandy, F., Marti, G., Vogt, R., Editors. Cytometry, 1998;33:1–287.2. Gaigalas AK, Li Li, Henderson O, Vogt R, Barr J, Marti G, Weaver J,

Schwartz A. The development of fluorescence intensity standards. JRes Natl Inst Stand Technol 2001;106:381–389.

3. Schwartz A, Wang, L, Early E, Gaigalas AK, Zhang Y, Marti GE, Vogt RF.Quantitating fluorescence intensity from fluorophore: the definition ofMESF assignment. J Res Natl Inst Stand Technol 2002;107:83–91.

4. Wang L, Gaigalas AK, Abbasi F, Marti GE, Vogt RF, Schwartz A.Quantitating Fluorescence intensity from fluorophores: practical use ofMESF Values. J Res Natl Inst Stand Technol 2002;107:339–353.

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