Formalization of the MESF Unit of Fluorescence Intensity

Transcription

1 Cytometry Part B (Clinical Cytometry) 57B:1 6 (2004) Report Formalization of the MESF Unit of Fluorescence Intensity Abe Schwartz, 1 Adolfas K. Gaigalas, 2 Lili Wang, 2 Gerald E. Marti, 3 Robert F. Vogt, 4 E. Fernandez-Repollet 5 1 Center for Quantitative Cytometry, San Juan, Puerto Rico 2 Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, Maryland 3 Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Bethesda, Maryland 4 Division of Laboratory Sciences, Centers for Disease Control, Atlanta, Georgia 5 Department 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 Standards and Technology (NIST) in the refinement and formal definition of the MESF unit of fluorescence intensity. In addition to the theory underlying the MESF unit, considerations of error analysis are also presented. The details of this work may be found in the three publications of the NIST Journal of Research ( listed as the references 2 4. The use of the fluorescence intensity unit provides a tool to compare quantitative fluorescence intensity measurements over time and across platforms Wiley-Liss, Inc. Key terms: MESF; Molecules of Equivalent Soluble Fluorochrome; quantitation; fluorescence intensity INTRODUCTION The quantitation of fluorescence intensity in biological assays is a long-standing goal (1). Over the past twenty years, the fluorescence unit of intensity, MESF, has been introduced and utilized in the field of flow cytometry. MESF is an abbreviation for Molecules of Equivalent Soluble Fluorochrome. The MESF concept indicates that a sample labeled with a fluorochrome has the same fluorescence intensity as an equivalent number of molecules of the fluorochrome free in a solution under the same environmental conditions. This fluorescence unit provided researchers with tool to compare flow cytometry data in a quantitative manner over time and across instruments. For instance, a search on PubMed (National Library of Medicine) of the literature since 1984 found 193 articles related to quantitative fluorescence flow cytometry and 53 articles specifically using MESF as the fluorescence unit of measure. Although MESF units have been used internationally over this extended period, a precise definition of the unit had not been presented until recently. The definition of the unit was presented in the context of a fluorescence measurement model that relates the physical characteristics of the sample and the resulting fluorescence signal (3). The model clearly states what is being compared between the standard and analyte samples, and allows a clear demarcation between instrumental biases and the physical characteristics of the sample. The following description briefly summarizes the formal definition of the MESF unit that has been published in the Journal of Research of the National Institute of Standards and Technology (NIST) and its application in quantifying bound antibodies in flow cytometric measurements (2 4). SUMMARY OF THE DERIVATION As shown in the second NIST publication (3), the interpretation of all fluorescence intensity measurements is based on a solution property called the fluorescence yield that is the product of the concentration of fluorophores and the molecular quantum yield. The fluorescence yield can be visualized as the total fluorescence emitted by a solution if every fluorophore was initially in the excited state. In principle, the fluorescence yield of any two solutions can be compared. This fact provides the foundation for the application of standards for the quantitation of fluorescence intensity. The comparison of fluorescence yields of any two solutions starts with the measurement of Research Support: provided by the National Institute of Standards and Technology and the Centers for Disease Control and Prevention. A. Schwartz is a visiting scientist at the National Institute of Standards and Technology. Correspondence to: A. Schwartz, Ph.D., P.O. Box , San Juan, PR abe@quantcyte.org Received 16 June 2003; Accepted 1 September 2003 Published online in Wiley InterScience ( DOI: /cyto.b Wiley-Liss, Inc.

2 2 SCHWARTZ ET AL. fluorescence intensity from the two solutions, where the fluorescence intensity is understood to be the signal given by the detection apparatus. The measurement model gives a detailed relation between the measured fluorescence intensity and the fundamental solution property the fluorescence yield. It becomes apparent that comparing fluorescence yields from the relative fluorescence intensity measurements is not easy. Difficulties arise from the interdependence of the measured fluorescence intensity on the instrument parameters, and also on the environmental factors which affect the absorption and emission of the fluorochrome. In the regime of low fluorophore concentration the dependence of the fluorescence intensity on the various factors may be summarized in equation 1 (measurement model). where i F [ge ε Q( )s( )T( )d ]c (1) i F 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 standard and a unknown solution Eq. 1 suggests that some factors such as g, e, cancel out. These factors describe part of the instrument response that is approximately independent of wavelength and are presumed to be the same when measuring the standard and the unknown solution (the standards and the sample must be run on the same instrument at the same settings). Now, if the remaining factors (except for quantum yield) are collected into a factor called X: X ε Q( )s( )T( )d (2) Then the expression for the measured fluorescence intensity reduces to: i F X c (3) where c is the concentration of fluorophores in the solution (standard or unknown) and is the molecular quantum yield. Eq.3 suggests that if the factor X can be made the same for the standard solution and the unknown solution then the comparison of fluorescence intensity would be equivalent to a comparison of fluorescence yields of the two solutions. Examination of the factor X suggests that if the excitation (ε( )) and emission (s( )) spectra of the standard solution match those of the unknown solution, then the factor X is the same for the two solutions. In summary, if the standard and unknown solutions are measured under the same instrument conditions and the excitation and emission spectra are the same, then the comparison of measured fluorescence intensity is the same as the comparison of fluorescence yield of the two solutions. The equality of the fluorescence yields of the unknown and standard solutions provides an equivalence relation between the concentrations of fluorophores in the two solutions. Thus, one could state that the concentration of fluorophore in the unknown solution is equivalent to a known concentration of fluorophore in the standard solution. In some special cases it may be possible to ensure that the microenvironments of the fluorophores are the same in the standard and unknown solutions. In such cases, the molecular quantum yields are expected to be the same in the two solutions and the comparison of fluorescence yields reduces to a comparison of actual concentrations of fluorophores. In practice, it is very difficult to ensure that the microenvironments of fluorophores in a standard and unknown solutions are identical, therefore, comparisons are limited to that of equivalence of concentrations. The above discussion can be extended to the comparison of fluorescence intensity of a standard solution and an unknown suspension of microspheres with immobilized fluorophores. The microspheres can be viewed as very large fluorophores and the language used in the previous discussion carries over naturally to the case of fluorescing microspheres. The only caveat is the use of number concentration in suspensions and the use of molar concentration in solutions. Avogadro s Number can be used to convert the molar concentration in a standard solution to a number concentration, and the equality of fluorescence yields of the standard solution and unknown suspension provides a value of Molecules of Equivalent Soluble Fluorochrome per microsphere. The practical value of the MESF units is obvious in flow cytometry since only particles and biological cells, and not solutions, are measured with this instrumentation. The expectation is that if MESF

3 FORMALIZATION OF THE MESF UNITS 3 FIG. 1. The normalized emission spectra of fluorescein-labeled monoclonal antibodies (thick plots) and leukocytes stained with these antibodies (thin plots) 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 inset shows the normalized spectra of CD45 stained leukocytes (dot line) and mononuclear cells (dash line) compared to that of fluorescein in solution (solid line). (Reprinted from J Res Natl Inst Stand Technol 2002; 107: ) values can be assigned in a consistent manner to biological cells and to labeled antibodies, then the ratio of the MESF values of the cell and the antibody would provide an estimate of antibodies immobilized on the cell, a quantity of prime biologic importance. MESF MEASUREMENTS There are several important aspects to consider with regard to the use of MESF units. First it is apparent from Eq. 3, that if the factors X can be evaluated for the standard and unknown solutions then a comparison of fluorescence yields can be made. The factor X can easily be measured using a good quality spectrofluorimeter and a reference light source. In fact, the assignment of MESF values to microspheres requires that the factor X be measured for the microsphere suspension and the standard solution. However, if one is concerned with the use of MESF units with cytometers then the measurement of the factor X in Eq. 3 is not practical. In this case it is best to adopt the strategy that the comparison of fluorescence intensity is the same as the comparison of fluorescence yields if both the standard and unknown solutions (or suspensions) are measured under the same instrument conditions (easy to implement), and both the excitation and emission spectra of the unknown and standard solutions match. In practice, it is almost impossible to match both the excitation and emission spectra of the sample and standard if the two are not labeled with the same fluorochrome. Therefore, the fluorochrome on the standard microsphere should be the same as on the unknown particle. At present it is understood that the MESF value of a sample is defined in terms of a specific fluorochrome, and as such must be so indicated. In other words, 50,000 MESF of fluorescein is not the same as 50,000 MESF of R-phycoerythrin. The third publication in the NIST Journal of Research deals with the sources of error in measurements that utilize MESF units when the same fluorochrome is used for the standard and the unknown solution (4). The greatest source of error is changes in the spectral response and quantum efficiency of the fluorochrome due to small changes in the micro-environment of the fluorophore. A

4 4 SCHWARTZ ET AL. FIG. 2. The normalized emission spectra of the microbeads with different linker lengths, as well as leukocytes stained with either CD45 or CD8 monoclonal antibody, with respect to fluorescein in solution: Fluorescein (thick solid line); Bead 3 (thick dash dot line); Bead 7 (thick dot line); Bead 12 (thick dash line); cell (CD45) (thin solid line); cell (CD8) (thin dash line). The two vertical lines define the emission collection window by the bandpass filter used in flow cytometers. (Reprinted from J Res Natl Inst Stand Technol. 2002; 107: ) spectral shift can arise across lots of conjugated antibodies that are labeled with the same fluorochrome (Fig. 1). These shifts would not be of significance if the emission spectrum could be measured and the factor X in Eq 3 estimated. However, since most flow cytometers use a narrow band pass filter, e.g., 530/30 for fluorescein, this shift can result in significant differences in the integrated signal. Moreover, just the binding of the fluorochromeconjugated antibody to the cell can give rise to a spectral shift that yields a significant variation of the integrated signal. In addition, the spectral response of microbead standards has been found to differ with the length of the linking molecule. As seen in Figure 2, one could conclude that the best spectral matching with fluorescein labeled antibodies bound to cells would be achieved with microspheres with fluorescein immobilized on the microsphere surface via a molecule with seven carbons. DEVELOPMENT OF FLUORESCENT STANDARDS The work conducted at NIST during the past two years on fluorescence intensity measurements has yielded two important milestones in addition to formalizing the definition of the MESF unit (3). These include NIST issuing a fluorescein solution as a Standard Reference Material (SRM1932) and the establishment of a laboratory for the measurement of fluorescence yield from solutions and suspensions. The laboratory contains a spectrofluorimeter that includes two holographic notch filters to suppress scattered light, a Coulter Multisizer 3 for measuring particle concentrations, and a custom-built cytometer to verify the MESF values assigned to microspheres. The format of the fluorescein SRM 1932 is a concentrated solution of fluorescein at a known concentration in a borate buffer. The SRM is designed to be diluted at least 100 in a buffer for the desired application such that the properties of the borate buffer will have a negligible affect on the final working solution standard. The holographic filters in the NIST spectrofluorimeter are essential to eliminate the scatter from microbead suspensions 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 spectral shifts due to differences in environment.

5 FORMALIZATION OF THE MESF UNITS 5 FIG. 3. Emission spectra of microbeads labeled with fluorescein with (solid line) and without (dotted line) two holographic notch filters before the entrance slit of the monochromator. Note that the 488nm background scattering from the excitation laser is essentially eliminated with the use of these holographic notch filters. A second Reference Material (RM) has been authorized by NIST, which will complement the fluorescein SRM solution. This RM will consist of a set of microbeads with fluorescein attached to the surface via a seven-carbon spacer. These microspheres will be calibrated in MESF units. This surface labeling yields reasonable spectral matching with fluorescein on antibodies, thus satisfying the requirements for a practical MESF standard. An additional set of solution and microbead standards has been proposed for R-phycoerythrin. Before efforts are expended on development of official reference products, basic questions of stability, consistency and reproducibility of this fluorochrome must be addressed. This will be conducted in the laboratories of Francis Mandy from Health Canada. Success with phycoerythrin will establish that both simple organic fluorochromes (e.g., fluorescein, rhodamine and cyanine) and proteinasous fluorochromes (e.g., phycoerythrin, and allophycocyanine) can be developed into quantitative fluorescence MESF standards and hopefully be offered as reference materials from NIST. SIGNIFICANCE The 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. The recent development of a more fundamental basis for the MESF assignment has clarified the interpretation of the measured fluorescence intensity in terms of MESF values. Quantitative fluorescence data are no longer dependent on the instrument platform or the specific suspension media of the samples. This independence holds so long as the following criteria are met: 1. The standards and the unknown samples must be run on the same instrument at the same settings. 2. The excitation and emission spectra of the standards must match those of the unknown samples (best achieved by having the same for the standard and the unknown sample). 3. The environment of the standards and the unknown samples must be the same. The resulting data can be compared among laboratories across the country and around the world over extended periods of time. The application of the MESF concept of fluorescence intensity measurements can be expanded and applied to fields other than flow cytometry. With the development of the appropriate measurement model and MESF standards,

6 6 SCHWARTZ ET AL. such applications include quantitation of fluorescence microscopy, micro-array intensity data, fluorescence image analysis, and can even be used to refine basic solution spectrofluorometry. With these new developments, the concentration of fluorochrome can be so high that MESF can take on the expanded definition of Moles of Equivalent Soluble Fluorophore. Thus the development of the MESF unit comes at an important time considering the ever-increasing number and types of assays that are based on fluorescence intensity. LITERATURE CITED 1. Quantitative fluorescence cytometry: an emerging consensus. Lenkei, R., Mandy, F., Marti, G., Vogt, R., Editors. Cytometry, 1998;33: Gaigalas AK, Li Li, Henderson O, Vogt R, Barr J, Marti G, Weaver J, Schwartz A. The development of fluorescence intensity standards. J Res Natl Inst Stand Technol 2001;106: Schwartz A, Wang, L, Early E, Gaigalas AK, Zhang Y, Marti GE, Vogt RF. Quantitating fluorescence intensity from fluorophore: the definition of MESF assignment. J Res Natl Inst Stand Technol 2002;107: Wang L, Gaigalas AK, Abbasi F, Marti GE, Vogt RF, Schwartz A. Quantitating Fluorescence intensity from fluorophores: practical use of MESF Values. J Res Natl Inst Stand Technol 2002;107: