c
1
IK Im
u u 1u uuurnrnuu
Fluorescence spectroscopy is a useful quantitative tool that combines excellent sensitivity and precision with high selectivity. The une of fluorescent molecules as labels, probes, and tracers has extended the applicability of fluorimetry to include determinations of speciesthat cannot be directly detarmined fluonmetrically because of a lack of native fluorescence or the presence of overwhelming background interferences. Examples of such indirect methods include enzymatic determinations, fluoroimmunoessays, the use of fluorescent chelating agents for the determination of inorganic ions, and many others. Selectivity in fluorimetric determinations is most commonly baaed on excitation and emission wavelengths. Techniques include measurements at one or more pairs of emission and excitation wavelengths, collection of emission andlor excitation spectra, synchronous scanning, and total emisaion-excitation matrix (EEM) acquisition. Additional selectivity parameters, such aa selective quenching, fluoreseence polarition, and fluorescence lifetime, can be incorporated into theae techniques. Phase-resolved fluorescence spectroscopy (PRFS)provides a means by 11001
which fluorescence lifetime selectivity can be implemented. The use of PRFS for multicomponent determinations, fust described by Veaelova et al. (I),is based on the phase modulation technique for the determination of fluorescence lifetimes. Discussions of the theow, instrumentation. and techniqu&~forphase modulation measurementa and of PRFS can be found in several recent articles and texts (2-6). PRFS has also been referred to aa phase-sensitive fluorescence spectroscopy (5). However, we will use the term phase s e ~ i t i u eonly in reference. to the detection in all phase modulation instruments that allow discrimination between emissions of different phase.
Theory of fluorescence llfetlme determlnatlons The mean fluorescence lietime, 7 , of a fluorescent species with a single, exponential decay is the time required for the excited-state population to be reduced to '/e of the initial population immediately following excitation with light of an appropriate wavelength. Typical values range from 10-9 to 10-6 8. I can be determined directly by using pulsed excitation and observ-
ANALYTICAL CWMISTRY, VOL. 56, NO. 13, NOM=
lee4
-
ing the exponential radiative decay following the termination of the exciting pulse. The resolution and precision of a lifetime determination are limited by the duration and reproducibility of the exciting pulses and by the response function of the detector. MdticomDonent determinations can be achievid by deconvolution of multiexponential decays obtained from mixtures. Alternatively, the longest lived species can be measured alter the fluorescence contributions of the shorter lived species have decayed to a negligible value. The contribution of the longest lived species thus determined can then be subtracted from the total,multiexoonentialcurve. Each successive longest lived species can be similarlv treated until the curve has been-completely analyzed. Limiting factors in the resolution of components are the differences between the fluorescence lifetimes of the component species and the duration and reproducibility of the exciting pulses relative both to the lifetimes of the components and to the lifetime differences among the componenta. An alternative method for the determination of fluorescence lifetimes, fust described by Gaviola in 1927 (7). involves the use of continuous, sinu~~~~~~~
~~~~~~~~~~
0003-2700/84/A351- 1400101.50/0 @ 1984 American Chemical Society
Report
Linda B. McGown Frank V. Bright
Department of Chemistry Oklahoma State University Stillwater, a l a . 74078
soidally modulated excitation combined with phase-sensitive detection. Modem instruments incorporating the phase modulation approach are capable of resolution ranging from 1 to 102 ps (8-14). In the phase modulation approach, the sample is excited with light having a time-dependent intensity E(t) of the form E(t) = A(1
+ nq. sin wt)
(1)
where A is the dc intensity component of the exciting beam, nq. is the degree of ac modulation (i.e., the ratio of the amplitude of the ac intensity to the dc intensity component), and w is the angular modulation frequency (w = 2rf where f is the linear modulation frequency in hertz). The resulting timedependent emission F(t) will be demodulated and phase shifted to an extent determined by the fluorescence lifetime of the species F(t) = A’(1
+ me. m sin(& - $))
(2)
where A’ is the dc intensity component of the fluorescence emission, $ is the phase shift of the species, and m is the demodulation factor: m=cos$
(3)
The demodulation factor can also be expressed as the ratio of the degree of ac modulation of the fluorescent species to the degree of ac modulation of a scattering solution. The phase shift and demodulation effects are shown in Figure 1. The fluorescence lifetime T can he calculated from the phase shift of the species 1 T = - h @ (4) w or from the demodulation (5)
In practice, the phase shift and demodulation of a species are measured relative to either a scatteringsolution (T = 0) or a reference fluorophore of known lifetime. Modulation of the excitation beam can be achieved in several ways. Early instruments employed Kerr cells or modulated lamps. More recently electrooptically modulated Pockels cells, which require hahly collimated sources, have been used for modulation of laser sources, and the ultrasonic Debye-Sears modulator has been used for both laser and lamp sourcea. DebyeSears modulation is limited to
a single fundamental modulation frequency plus several harmonic overtones, whereas the Pockels cell can achieve a continuous range of modulation frequencies. Phase-sensitive detection results from a comparison of the detected emission with an internal, electronic reference signal of the same frequency. A direct comparison of these highfrequency signals can be used. However, the development of crm-correlation technique8 (II,15),in which the high-frequency signals are converted to low-frequency signals, has enabled much more precise measurements because of improved S I N a t lower frequencies. Possible errors in fluorescence lifetime determinations can arise from several 80including the “color effect’’ in PMT detection due to the d e pendence of the kinetic energy of photoelectrons on the energy of the incident photons, and the effects of B m ian rotation on observed fluorescence lifetime. The former problem can be avoided by use of a reference fluorescent species measured a t the same wavelengths as the sample, instead of using a scattering solution as the fluorescence lifetime reference (16,17). Also, PMTs with negligible color ef-
ANALYTICAL CKMISTFIY, VOL. 56. NO. 13, NOVEMBER 1984
1401 A
Flgure 1. Transient response of luminescent samples to a sinusoidal excitation starting at t = 0 Modulation heq-y
= 10 MW. (a) T = 10 ns: (b) r = 100 ns. Adapted vim permissionhom Reference 3
fects can he used. The effects of Brownian rotation can be avoided by the use of "magic-angle polarization," e.g., a polarizer a t Oo in the excitation beam and one a t 55O in the emission beam. Unfortunately, each polarizer causes a decrease of -54% in beam intensity. Heterogeneity, i.e., the presence of more than a single exponential decay (resulting from more than one fluorescence lifetime species), is generally indicated by a lack of agreement between the lifetime values calculated from Equations 4 and 5. Another indication of heterogeneity is disagreement between lifetime values obtained using different modulation frequencies. For a heterogeneous (multicomponent) system, the observed emission (assuming independent, uncorrelated emitters) is the sum of the individual emissions for each of the different lifetime species. For a solution containing j independent emitters
frequencies vastly improves the average precision attainable over a wide range of fluorescence lifetime magnitudes.
Theory ot PRFS In PRFS the time-dependent, ac component of the fluorescence emission described by Equation 2 is multiplied by a periodic function P(t)
nential decays, more complex curves with both sine and cosine contributions are obtained. A commercially available phase modulation spectrofluorimeter with phase resolution capabilities has been
P(t) = 0 from Oo to the detector phase angle setting @D P(t) = 1from +D to (h+ 1 W )
+
P(t) = 0 from (@D l W ) to S O o (7) and then integrated, as shown in Figure 2. A time-independent, de signal is thereby produced that is proportional to the m i n e of the difference between the phase angles of the detector (h) and the fluorescent species (@) F ( 1 . 4 ~ )= A'm,m
CO&D
- 4) (8
Therefore, at a given wavelength (A), maximum phase-resolved intensity for a component is obtained when h = zero pbase-resolved intensity results from observation a t the ''null phase angle" (h= SO0), and intermediate intensity values will be observed at detector phase angles between the phase angle and the null phase angle of the component. For a multicomponent, heterogeneous system of j independent emitters, the phase-resolved intensities are additive
+,
Recently, a continuously variable, multiple-frequency phase modulation instrument with cross-correlation electronics capable of a resolution of several picoseconds has been described (U). In this instrument, Poekels cell modulation of an argon ion laser excitation beam is used to achieve modulation frequencies ranging from 1to 160 MHz. Such an instrument should greatly simplify the detection and deconvolution of mnltiexponential decays. In addition, since the precision of fluorescence lifetime determinations is a function of the modulation frequency relative to the fluorescence lifetime, a wide range of continuously selectable modulation 1402 A
+*
Figure 2. Depiction of the output (shaded area) from a phase-variable rectifying detector resulting from integration of the ac component of the emission signal from component A (FA(x,t))over the interval of bo to bo 180° (for which P(t) = 1) (a) h = 4~ (the dsteCmr is "in phase" with A):
+
(9)
For a single component with exponential decay, the curve of phase-resolved intensity vs. detector phase angle is a simple cosine function. For multiexpo-
ANALYTICAL CHEMISTRY, VOL 56, NO, 13, NOVEMBER 1984
(b) h = +A
t 90' = null phase angle (A is "nulled". i.e.. hasrem phaseresoived intensily): (e) h is sat at an intwmdiate value between and +A t 90'. A b t e d with wmissim horn Reference 6
I
The next step in lab a u t o m t i o ~ VAX LlMS
described by Mattheis et al. (6).The instrument uses a xenon arc lamp source with Debyesears modulation to achieve a modulation frequency of 6.18, or 30 MHz, with phase-sensitive photomultiplier tube detection. The cross-correlation technique described by Spencer and Weber ( l l ) ,in which the photomultiplier gain is modulated at frequency w Aw to produce a cross-correlation signal of Aw9which is isolated with a low-pass filter, is used to convert the high-frequency (6,18, and 30 MHz) signals to low frequencies (25 Hz).A ratiometric mode can be used for both phase modulation lifetime determinations and phaseresolved intensity measurements, in which a reference, phase-sensitive photomultiplier tube detects a portion of the modulated excitation beam to compensate for modulation drift and source output fluctuations. It is important to note that the “color” and Brownian rotation effects that may occur in the determination of fluorescence lifetimes will not affect PRFS multicomponent determinations, provided that samples and standards are prepared in similar matrices and are all measured under conditions of identical wavelengths and detector phase angles. Any other instrumental or measurement artifacts that affect observed fluorescence lifetime values but remain constant under constant measurement conditions, such as croas-sectional inhomogeneity in the modulation of the excitation beam, will similarly cancel out and have no effect on PRFS multicomponent determinations.
+
Flgure 3. Repesentatii of the selective nulling of each of two hypothetical components. A and B (a) %&iemm
~vonscenceaniasion s m of A miw),B (red). and Y. wlution m i n i n g both lblack);(b)P(otof~fiuxeacamx,intenslty(PRFi)asafvnctionofdetector~ang(e (m0) fa A (blue) and B (red) (rA 70):(e) Phawwesolvedflvaescence spact’a of B (red)md of A ( b b ) OMained bY meawing a polvtim mmainlng born at h e m111 awhs of A md 8. respec~IV.SIY. ~ a p t e d with pami~~ion ham ~ e t e n m ~ 5e
n) to generate the equations
I+,, = il,mn,c,+i2,+n,c2+....+f,,+n,
Ioo2 = il.+,cl+ I~,+,C, + ....+ r,,,,c, I*- = it,+&+ iz.mo,Cz+ .... + in,d2, (10) where I is the phase-resolved intensity of the mixture at a given detector phase angle @D, and I; is the phaseresolved intensity of component i at that phase angle. If m = n, the square
Fluorescence lifetimes..
.
at thflick of a switch, SLMIAMINCO changes your outlook on light ~
3 concentrate on the mment, not the inshllment The rapidity of -Le measurement
I
The SLM 4800' Specmfluoromm is illuminating entirely new areas of biological investigation. Investigations into cancer and enzyme research. Inquiries into molecular collisions involving quenchers, rotational and translational diffusion,or the formation of complexes with solvents and solutes. And as the measurement of fluorescence lifetimes leads to unuied pathways of bioresearch, the SLM 4800 is making these measurements more precise than ever.
v-"3.
that you finish with the same samk.you started with! Other techniques often take hours I for a statisticallv , , simificant measurement The SLM 4800 can even follow slow kinetics processes via fluorescence lifetimes!
Y
Phase modulation-precise to 50 picoseconds Unlike pulse excitation, phase modulation employs a light beam that is modulated sinusoidally-much like adimmersu'.~' n
Christmas bee lights and innovative biotechnology Unlike ahwrption spectrophotomeq, the measurement of fluorescence involves exciting hiomolecules with light, then measuring the emission of energy as the molecule returns to its ground state. So, as they're energized and returned to ground state, these molecules blink on and off, much like ~. Chrisunas lights. If you held a light meter in hont of this string of lights, you might learn the average amount of light of the entire string hut not how lrmg each light was l i t Not unless you measure their "lifetimes? But when the individual blinking ofa light lasts forjust billionths of a second, you need incredibly accurate equipment to measure that light No other insrmment can do the job as easy, as h t ,or as accurately 4800 Suhnanosecond Fluorometer.
/
/
Christmas lights, turning the light intensity up then down. Since the molecules will emit energy in the same propodon that they dsrnbed ~nergy, mo~ecu~es emit photons in a sinusoidal modulation. By comparing the light input to the output we can use that modulation or phase difference to calculate fluorescence lifetimes. Down to 50 picoseconds! i that's with no messy
~
1
our computer requirements for lifetime calculation and multiple decays is so simple, you can do it with most popular micmcomputen, or even a hand-held programmable calculator
The SLM 4800-easier to use than the pulse method Until the S1.M 4800 came along, pulse measurements were the most popular way to measure fluorescence lifetimes. But Shedding light on the future the phase technique is surpassing the of biological research pulse technique in popularity for a wide Biological systems are ofien very dimcult mriety of applications. And for a wide mriew, of r~awns. I measuremens, owing to their low levels of fluorescence, high turbidity and heter It doesn't take an electrical engineer to ogeneity. SLM has solved each problem. operate the SLM 4800. Its ease of use ~~
I
The minimum optical configurduon used in SLM monochromators resuls in impressive stray-light figures. The superior stray-light rejection of SLM monochromaton is very important when you are looking at samples with high scattering, such as lymphncytes,just as our high light throughput helps with weakly emitting specimens. Our unique cross-correlation technique allows UD to a million times more simal from the phototube than do conventional lifetime techniques, and an order of magnitude improvement in time response. With the SLM 4x00, you can directly measure the individual emission specuum of each fluomphore from a mixture of fluorophores, or determine concentrations of multiple analytes in solution that are othelwise zmraolvahle, using the phase-resolved tool. Multiple lifetimes and their weighting factors a n he determined with a precision that makes assays p&ihIe. Use polarized lifetimes to determine the onal rate, viscosity or molecular e of molecules in solution. Examine the kinetics of quenching interactions. Study energi transfer to calculate intermolecular distances. All of this information at the touch of a single button.
I
The SLM 4800 is exciting more than just molecules If you're interested in the potentials of flnorescencelifetimes and would like more information, call or write SLMI AMINCO at the number below. We've got an entire library of the \ latest information on this exciting, new technology
fluorescence lifetimes or are interested in the potentials o the SLM 4800 b q m i fluorescence lifetimes, call us for a free brochure.
SLM Inslrurnents, Inc.. 810 W. Anthony Drive, Urbana, IL 61801 US..&, (217) 384-7730 TELEX 201 CIRCLE 196 ON READER SERVICE CARD ANALYTICAL CHEMISTRY, VOL. 56. NO. 13. NOVEh
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A HALL DETECTOR”* IT’S NOT. IT’S BETTER. Selective detection of halogen, sulfur, and nitrogen compounds. Our great new Model 4420 Electrolytic Conductivity Detector represents the first major advance in ECD’s since the Hall Detector@*. The Model 4420 is in fact the latest design of Dr. Randall C. Hall, 0.1. Corporation’s senior chemist, who did pioneering work in ECD’s in the early 1970’s. The 4420 takes fullest advantage of currently available technology, and it makes important differences in overall performance, reliability, convenience and results. Engineered to fit. The 4420 has been specifically engineered to interface with the following GC’s: Hewlett Packard’s 5890,5880, and 5790 TRACOR 540,565, and 570 Perkin-Elmer Sigma Series and 8300 Varian 3700, Vista Series and 3313400 The Model 4420 can be installed in less than 30 minutes. It uses the same mounts as the manufacturers’ original equipment, and all splitters and columns fit properly. Connects to both packed and capillary columns. Matters of convenience. The model 4420’s novel flow-through conductivity cell (patents applied for) eliminates baseline perturbations from cell contamination. Reaction tubes can be installed quickly and easily. Reaction temperature is read out continuously, and the 4420 has automatic protection against temperature runaway. It’s a pushbutton detector. Easy, quick, convenient and reliable. Use it in all ECD applications - and consider using it in lieu of an electron capture detector in many applications. For complete details on the Model 4420, write for a brochure or phone 800-445-0016. In Texas use 409-693-1711, collect. *Hall is a Recirtered Trademark of TRACOR. Inc ._ Detector’ _
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 13, NOVEMBER 1984
matrix can be solved exactly for the concentration of each component. If m > n,the overdetermined matrix can be “solved” using an iterative procedure such as the Gauss-Newton method (19) or the Marquardt method (20). The direct nulling method is simply a special case of this approach in which the resulting matrix has zero elements on the main diagonal. Results are shown in Table I for the simultaneous determination of POPOP (1,4-bis(5-phenyloxazol-2yl)benzene, r = 1.35 ns) and dimethylPOPOP (1,4-bis(4-methyl-5-phenyl2-oxazolyl)benzene, T = 1.45 ns) using the direct nulling, indirect nulling, and simultaneous equation approaches (21).Both the indirect nulling 1 and the simultaneous equation 1approaches, for which the same number of measurements per solution were used as in the direct nulling determinations, show greatly increased accuracy for POPOP but not for dimethylPOPOP. The use of more phase angles and triplicate measurements (indirect nulling 2 and simultaneous equation 2) results in improved accuracy for both approaches relative to direct nulling. The choice of location of the detector phase angles to be used for a particular determination depends on the relative contributions of each component and the fluorescence lifetime (and therefore phase angle) differences between the components. A compromise must be made between maximum resolution between components (at the null phase angles of the components) and maximum precision (at regions of high total intensity, usually at the phase angles of the components). It has been shown that for two components, A and B, with essentially complete spectral overlap (A = fluorescein physically adsorbed to albumin and B = fluorescein isothiocyanate covalently bound to albumin) and very similar lifetimes ( T A - T B zs 300 ps), an effective compromise occurs at 4~ ~ J Af 45’ and ~ J D= 4~ f 45O (22).Table I1 summarizes results for this two-component system obtained by solving the 2 X 2 matrices generated using three different pairs of detector phase angles, including the pliase angles of the components, the phase angles shifted by 45’’ and the phase angles shifted by 90’ (the nulling phase angles). Fluorescence lifetime selectivity can be combined with wavelength selectivity simply by making the measurements at appropriate combinations of detector phase angle with emission and excitation wavelengths. A fivefold improvement in accuracy has been demonstrated for the determination of three components (POPOP, dimethylPOPOP, and anthracene) using
Shimadzu. Integrators wecialist. 1 n giant stel, for A
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the time of actual analysis. And with its userfriendliness, you can anjoy the benefits of data processing and automatic operation using user-defined BASIC programs. All this is standard with Shimadzu's high-performance integrator for chromatography. System-expanding options include a double track floppy disk driver, CRT display, full keyboard and digital I/O port. The C-R3A is compatible with all commercial GC and LC instrumentsno problem here. In fact, no problem anywhere with the C-R3A. Just answers to a11 your GC needs. Circle 207
neiaiive errors for me rnr3 aeierminaiion oi fluoresceln (A) * and fluorescein isothiocyanate (B) hC Iawe 11.
- 450 +*-9O0
+A
-
+a 450 +B-900
3.0 -94.3
-2.7 -12.4
6.4 273
5.8 34.7
16 1740
-'
sically adto aibumln. raiemly bound to aibumln. = 490 M; h,= 520 nm; moduLatiw, frequency = 18 MW 4, = 237.0': Q. = 235.3°. Average relative m r s of determimtion of specles A and 0 in four soIu1Iw m i n i n g both speck. ea& at elther 50.0 u 25.0 nM in cuvene. Averspes of me ab601m value11of me relative errors we shown to Indicate average errw mag-
phase-resolved measurements with wavelength selectivity in combination with appropriate detector phase angles, relative to conventional steadystate measurements a t the same wavelengths (23).It should he noted that the two approaches involve the same experimental methodology and equal numbers of measurements, in both cases generating 3 X 3 matrices that are then solved for the concentrations of the three components using Cramer's rule. The only difference between the steady-state and the phaseresolved approaches is that, in the lat-
ter approach, measurements are made using modulated excitation and phase-resolved detection. In summary, the approach to be used for a particular determination will depend on the fluorescence spectral and lifetime charaderisties of the analyte components. For species with large lifetime differences, direct nulling may be the best approach. Direct nulling also offers the ability to acquire individual spectra of each component for two-component systems. For analysis of samples in which two or more of the components have simi-
lar lifetimes, the indirect nulling or simultaneous equation approaches may he preferable. Multiple modulation frequencies. Another approach to the acquisition of simultaneous, independent equations is the use of multiple modulation frequencies. The theory of the resolution of heterogeneous systems using multiple modulation frequencies (24) and various applications (25-28)have been described. These studies involve the measurement of phase and modulation directly, as opposed to the measurement of phase-resolved intensities, and are therefore not examples of PRFS.However, they are mentioned here because the use of various comhinations of detector phase angle with modulation frequency for PRFS measurements could provide another dimension of information for multicomponent analysis.
Analytical applications and future directions The original applications of PRFS described by Veselova and co-workers included the resolution, using the direct nulling approach, of mixtures of 3-acetylamino-N-methylphthalimide ( r = 2.25 ns) and 3,6-diacetylaminoN-methylphthalimide (T = 10.7 ns) in ethanol (1)and a study of solvent effects on the fluorescence spectrum of 3-amino-N-methylphthalimide(29). Lakowicz and Cherek used directnulling PRFS to resolve mixtures of TNS (6-@-toluidino)-Z-naphthalenesulfonic acid, r = 11.6 ns) and PRODAN (6-propionyl-2-(dimethylamino)naphthalene, T = 3.6 ns) and of dibenzo(a,h)anthracene (T = 31.0 ns) and dihenzo(cg)carhazole (r = 4.7 ns) (5). They also described the resolution of heterogeneous fluorescence from proteins and from mixtures of aromatic amino acids (30).Lakowicz, Thompson, and Cherek used direct nulling PRFS to study the relaxation of model membranes using TNS as a prohe (31). The introduction of the use of multiple, non-nulling detector phase angles has extended the applicability of PRFS to include systems with more than two components (23)and systems that have two components with very similar lifetimes (within 100 ps of each other) and extensively overlapping fluorescenceexcitation and emission spectra (22). Macromolecule-ligand binding. If a sufficiently large fluorescence lifetime difference exists between a species free in solution and hound to a macromolecule, it is possible to determine the fractions of free and bound ligand, even if the free and hound species have identical spectral characteristics. It is assumed that the hound species has either a single exponential decay or several discrete and charac-
,
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ANALYTICAL CHEMISTRY. VOL. 56. NO. 13. NOVEMBER 1984
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Flgure 4. General description of phaseresolved fiwoimmunoassay
terizable decays, corresponding to different binding sites or microenvironmenta. For example, Lakowicz and coworkers used the direct nulling approach to investigate the binding of indole derivatives to micelles, exploiting a fluorescence lifetime difference of -10 ns between the Bee and the micelle-bound species (32). An interesting application of the use of PRFS for studying macromolecule-ligand binding is the development of a homogeneous fluoroimmunoassay technique in which the fluorescence lifetime difference between the free and the antibody-bound fluorescent-labeled antigen is used to determine the fractions of the two species (Figure 4). For example, a phase-reaolved fluoroimmunoaaaay (PRFIA) for phenobarbital using the simultaneous equation approach has been described (33).Fluorescence lifetimes for the free (actually. nonspecifically adsorbed to albumin) and antibody-bound fluorescein-labeledantigen were found to be 4.04 ns and
3.94 ns, respectively. PRFIA is equally
applicable to hapten and macromolecular antigens, provided a sufficient lifetime difference exists between the free and hound labeled-antigen species. Elimination of fluorescent interferences. A single, exponentially decaying interferent can be easily eliminated using direct nulling, but sensitivity for the analytes will deteriorate as the fluorescence lifetime of the interferent approaches those of the analytes, in which case the indirect nulling approach can be used. The simultaneous equation approach is an alternative to nulling, in which interferenta are treated as separate, potentially resolvable species. This approach has been applied to the elimination of bilirubin (T 1ns) interference in the determination of fluorescein (T 4 ns) in aqueous solutions containing 1nM fluorescein in the presence of as much as 10 pM hilirubin (34). Bilirubin and fluorescein have extensively overlapping emieaion
-
-
,.gun 5. Three-dimensional dgta m y depictions of phase-resolved fluorescenceIntensity (PRFi) as a function of syncluonousl, scanned excitaticn and emission wavelengths for x,ranging from 330 to 430 nm and & = x, 50 nm. and of detector phaseangle & forthe range Oo to 3 1 5 O (a) wpop. 1.5 ,AI; (b) DlmenyWPOP. 2.0 ~IM;(0)AnUvacme, 1.0 iIM;(d) MulUccmponemmlution--wwp(0.51 @AMI dlm&~lwWp ( O M pM). mthcma
+
(0.67 P Y
1414A
ANALYTICAL CHEMISTRY, VOL. 58, NO. 13, NOVEMBER 1984
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and excitation spectra and are not readily resolvable using wavelength selectivity.
Multiparametric multicomponent determinations. Perhaps the most attractive feature of the phaseresolved approach to fluorescence lifetime selectivity is the ease with which i t is combined with other fluorescence selectivity parameters. For example, a three-dimensional data format can be envisioned in which intensity is a function of synchronously scanned wavelength on one axis and detector phase angle on the other, as shown in Figure 5. An EEM could similarly be combined with the detector phase angle variable to generate a four-dimensional format to maximize wavelength information in addition to lifetime information. Selective quenching or enhancement could easily be incorporated. Further selectivity based on polarization (planar or circular), selective binding to macromolecules or micelles, or multiple modulation frequencies could greatly extend the applicability of PFWS to multicomponent determinations.
(23) MCCown, L. B.; Bright, F. V. Anal. Chem. 1984,56,2195-99. (24) Weber, G. J. Phys. Chem. 1981,85,
_”.
9 CL”F.2 ”d_
(25) Jameson, D. M.; Weher, G. J. Phys.
Chem. 1981,85,95368.
(26) Barrow, D. A.; Lentz, B. R.,unpuh-
lished data.
(27) Gratton, E.;Lakowicz, J. R.; Maliwal,
Lauko, G.;Limkeman, B.; Cherek, H.; M. Biophys. J.,in press. (28) Lakowicz, J. R.;Gratton, E.;Laczko, G.; Cherek, H.; Limkeman, M.Biophys. J., in press. (29) Veselova, T. V.; Limareva, L. A,; Cherkasov, A. S.;Shirokov. V. I. Opt. Spectrosc. 1965.19,39-43. (30)Lakowia, J. R.; Cherek, H. J. Biol. Chem. 1981,256,6348-53. (31) Lakowia,J. R:; Thompson, R. B.; Cherek,H.Brochrm. Btophys. Acta 1983. 734,29&308. (32) Lakowia, J. R.;Keatinga, S.J.Biol. Chem. 1983,258,551%24. (33) Bright, F. V.; McCown, L. B. Talanta,
I
(a?pfght.
F. V.; McCown. L. B. Anal. Chim. Acta, in press.
b d
References (1) Veaelova, T.V.; Cherkaaov, A. S.;Shi.
rokov, V. I. Opt. Spectrosc. 1970,29. 611-18. (2) Lakowia, J. R. “Principles of Fluorespy”; Plenum Press: New
;7: .“
(3) Demaa, J. N. “Excited State Lifetime
Measurements”; Academic Press: New York, N.Y., 1983. (4) Jameson, D. M.;Gratton, E.;Hall, R. D. Appl. Spectrosc. Reu. 1984.20, 55-106. (5) Lakowice. J. R.;Cherek, H.J. Biochem. Bioph s.Meth. 1981,5,19-35. (6) Mattheis, XR;Mitchell, G. W.; Spenear, R. D. In “New Directions in Moleeular Lumineseance”; Eastwood, D., Ed.; ASTM,lB83; .50. (7) Gaviola, E. j .Phys. 1927,42,853-61. (8)Bonch-Bruevich, A. M.;M o l h o v , V. A.; Shirokov, V. I. Bull. Acad. Sci. USSR Phys. Ser. 1956,20,54144. (9) Birks. J. B.: Dvson. D. J. J. Sei. Instrum. 1961,38;282185. (10) Muller, A.; Lumry, R.;Kokubun, H. Rev. Sei. Inatrum. 1965,36.1214-26. (11) Sf ncer, .. R.D.; Weher, G. Ann. N.Y. Aca Scr 1969,158,361-76. (12) Salmeen, I.; Rimai, L. Biophys. J. 1977,20,33642. (13) Harr, H. P.;Hauser, M.Reu. Sci. Instrum. 1978.49,632-33. (14) Gratton, E.;Lopez-Delgado, R. I1 Nuow Cimnto 1980,J68,110-24. (15) Birks, J. B.; Little, W.A. Proc. Phys. Soc. ISSS, A66,921-28. (16) Lakowia, J. R; Cherek, H.; Balter, A. J. Biochem. Biophys. Meth. 1981.5,
Linda McCown receiued her BS degree from the State Unioersity ofNew York at Buffalo in 1975 and her PhD from the University of Washington in 1979. Her research interests include deuelopment of analytical techniques exploiting multiple selecliuity parameters; homogeneous immunoassay techniques and techniques for studying the interactions between small molecules and macromolecular systems; and enzymatic analysis teehniques, with emphasis on clinieal appliealions.
1 x 1 4 ___
(11) Barrow, D.A,; Lants. B.R J. Biochem Bioph s Meth. 1983,7,211-34.
(18) Gratton, kilimkeman, M. Biophys.
J. 1985,44,315-24. (19) Hartley, H. 0.Technometrics 1961, 3,2l3wN.
uardt, D. W. J . Indust. 5 0th. 1963,11, 43141. (21) hffi L. B.; Bright, F. V. Anal. Soc.
Ap 1
own,
Chim. Acta, in preaa.
(22) Mffiown,L. B. Anal. Chim. Acta
1984,157,32732.
Frank Bright is completing work for his PhD under the direction of Linda McGown. His current research interests are analytical applications of both conventional and phase-resolued fluorescence spectroscopy for multicomponent analysis as well as their use in immunoassays under both batch and flow conditions.
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