fluorescence lifetime measurements

Time Correlated Single Photon Techniaue:

'

The time correlated single photon (TCSP) technique is an instrumental method for measuring changes in light intensity in the nanosecond time range ( 1 - 5 ) . Many chemical and physical properties of molecules can be studied through this type of measurement. It has been used successfully to follow the luminescent intensity decay curves of molecules with extremely short excited state lifetimes. A typical fluorescence decay curve obtained by the TCSP technique is shown in Figure 1. The time between data points on the abscissa is approximately 0.44 ns. From this decay curve the lifetime of the excited state can be derived. For this type of application the lifetime is usually defined as the time needed for the light intensity t o decrease by the fraction l / e , where e is the base of the Napierian logarithm system. This report will deal only with TCSP measurements of molecular fluorescence decay curves. Molecular fluorescence generally occurs within 10-6-10-12 s following activation. Conventional fluorometers incorporating oscilloscopic detection are capable of measuring microsecond decay characteristics directly. The TCSP technique allows fluorometric studies in the nanosecond range so that lifetimes down t o approximately s can be reliably obtained. In fluorescence decay curves the light intensity decreases with time after the excitation light is removed. The probability of detecting a n emitted photon is greatest when the emission intensity is greatest. By use of the TCSP technique the decay curve is built up in a time correlated manner according to this probability. T o measure a decay curve, the sample is excited by a pulsed light source. During each excitation pulse a single photon is detected, and the time of arrival, 364A

relative to the primary pulse, is electronically measured. Because the probability of detecting a single photon is directly proportional to the emission intensity, the detected events will be distributed in a manner that correlates with emission intensity along the time axis. T o obtain undistorted data, it is essential that only a single photon is received by the detector per excitation cycle. This contrasts sharply with most other spectroscopic techniques.

concerning the excited state. This information is helpful in understanding the mechanism of excitation and deactivation processes. A molecule which has been activated to some excited state M* by absorption of radiant energy will remain in this high energy state for a variable length of time. Deactivation can proceed by numerous mechanisms such as chemical reaction, radiationless transitions, fluorescence, and phosphorescence. The principal pathways of deactivation for a molecule in the first excited singlet state are given in Figure 2. Radiative deactivation by fluorescence or phosphorescence is characterized by the decay constants hl and h4, respectively. The decay constant for radiationless transitions to the ground singlet state, So, is h z , and to the metastable triplet state, 2'1, is h ~Of. particular interest to photochemists is the reaction rate constant, h ~governing , the formation of products.

kj

M*

o...:

I

I

I

.e...

Products

The particular mode(s) of deactivation can affect the value of the experimentally determined excited state lifetime of the species. The experimental lifetime is given by the reciprocal of all decay constants operative in the system. 1

T =

The measurement of fast photophysical and photochemical phenomena can provide information regarding the nature of intra- and intermolecular and atomic interactions. The fluorescence lifetime, T , can be used mathematically in conjunction with other experimentally determined parameters such as the quantum yield, a, t o derive energy considerations

ANALYTICAL CHEMISTRY, VOL. 48, NO. 4, APRIL 1976

(1)

h i + hz

+ h3 + h4 + h ;

(2)

In situations of analytical interest a t room temperature, h4 and h j are negligible and the lifetime depends only on the rate constants h l , hp, and h3 for radiationless transitions and fluorescence. There is an intrinsic natural lifetime, T O , characteristic of the particular molecular species or element which

Report

L. J. Cline Love and L. A. Shaver Department of Chemistry Seton Hall University South Orange, N.J. 07079

potentially could serve to identify the species. This natural radiative lifetime is obtained when deactivation occurs solely via fluorescence, with all rate constants except k 1 negligible. Since this is seldom the case, T O is calculated from the relationship

(3) where T is the experimental lifetime, and is the quantum yield given by the ratio of the number of fluorescent photons to the number of absorbed photons.

The time correlated single photon technique has been referred to as the "single photon counting" method, a term which has led to confusion with conventional photon counting detection. Both are spectroscopic detection methods, but they differ greatly in principle, instrumentation, and applications. Photon counting has been used primarily to measure the magnitude of low light levels in direct digital form using relatively simple, inexpensive instrumentation (7-9). With the time correlated single photon method, both intensity levels and time dependent variations are recorded simulta-

neously with highly specialized instrument modules. Instrumentation

The time correlated single photon technique was first used by Bollinger and Thomas in 1961 ( 1 0 ) to measure the luminescent decay characteristics of scintillators excited by alpha, beta, and gamma radiation. In the same year Koechlin (11)reported the use of TCSP measurements to determine the shapes of scintillator emission curves with resolution in the nanosecond range. Since that time, significant improvements in scientific instrumenta-

Figure 2. Energy level diagram of some possible modes of deactivation of first excited singlet state SI to ground singlet state So. TI is first excited triplet state

If the chemical environment of the fluorophor is carefully controlled, the experimental excited state lifetimes can be used for qualitative analysis. This is possible only if conditions are rigidly maintained so as to render all of the modes of deactivation constant. Because of the similarity in lifetimes of some compounds, it is often necessary to use supplemental information such as luminescence spectra to positively identify them. Aaron and coworkers have developed an analysis scheme for several drugs using lifetime and spectral information (6).

Figure 3. Block diagram of time correlated single photon fluorometer Photomultiplier (PMT) 1 provides "START" signal to time-to-amplitude convertor (TAC) when pulsed light source fires. Single emission photon detected by PMT 2 provides "STOP" signal to TAC. TAC sends voltage pulse with amplitude proportional to elapsed time between "START" and "STOP" signals to multichannel analyzer (MCA) ANALYTICAL CHEMISTRY, VOL. 48, NO. 4, APRIL 1976

365A

tion have been made which allow accurate, precise measurements of lifetimes in the subnanosecond range. In particular, electronic measuring devices and photomultiplier tubes specifically designed for pulse signals are now readily available. Advances in pulsed light source technology have increased the utility of the TCSP method (1-5). The complete instrument is available commercially in modular form (ORTEC, Inc., 100 Midland Road, Oak Ridge, Tenn. 37830). Although much of the instrumentation, such as monochromators, sample cell, and power supplies, is common to most standard fluorometers, some additional specialized equipment is required to implement the technique. This specific equipment includes a pulsed (typically 1-30 kHz) light source such as a laser or spark discharge, a fast response photomultiplier tube (PMT), and a time-to-amplitude convertor (TAC). Several additional instrumental refinements are necessary but of secondary importance to this discussion. The TAC is the critical device employed in the time correlation process. Its function is to measure the elapsed time between the initial rise in intensity of the pulsed light source and the detection of an emitted photon. The principle of operation of the TCSP technique is illustrated in Figure 3. A simplified schematic drawing of a “nanosecond fluorometer” designed to obtain fluorescence decay curves is shown. One P M T is used in the START channel to observe the occurrence of a pulse from the excitation source. The second P M T is employed in the STOP channel to detect the arrival of an emission photon from the sample. Upon the initial rise in intensity of the excitation source, the START P M T sends a pulse to the TAC which triggers the voltage ramp shown in Figure 4.Simultaneously, molecules in the sample are excited by the light pulse and undergo radiative decay. This luminescent emission is observed by the STOP PMT, and a voltage pulse is sent to the STOP channel of the TAC. This trigger pulse halts the voltage ramp in the TAC, and an output pulse is generated with a voltage level directly proportional to the elapsed time between trigger pulses. The output pulses are sent to a multichannel analyzer (MCA) or computer where they are sorted according to amplitude and stored as counts in the memory. Because the amplitudes of the TAC output pulses are directly proportional to elapsed time, the channels of the MCA correspond to increments of time. The process of excitation is carried out a few thousand times per second, the exact rate being determined by the pulse repetition 366A

Time

*

Figure 4. Voltage ramp generation by time-to-amplitude convertor (TAC)

rate of the excitation light source. Following each excitation cycle the TAC is reset, and the process is continued until sufficient counts have been collected in the memory to define the decay curve. The number of counts in the memory channels are directly proportional to the luminescent intensity. Each channel is a unit division on the time axis, and the time increment between channels can be varied from approximately 0.2 ns up to a few microseconds. I t has proved very helpful to follow visually the buildup of the decay curve on an oscilloscope display. Oftentimes, decisions can be made based on this preliminary observation and the experimental conditions changed without waiting for the data reduction. A typical experimental decay curve taken from a MCA oscilloscope display was shown in Figure 1. Computer data reduction is often imperative to analyze the complex decay curves (12, 13).This is nearly always the case when more then one decay function is observed during the measurement time interval. When multiple, overlapping decay curves are measured, a computer is used to deconvolute the data and calculate the characteristic lifetimes. If the choice is available, a digital computer programmed to simulate a multichannel pulse height analyzer and interfaced directly to the time-to-amplitude convertor is preferable to an actual MCA. If direct access to a computer is not available, the MCA should have the capability of computer-compatible data output such as punched paper tape. A listing of channel contents from the MCA is sufficient for subsequent graphical analysis of single exponential long-lived decay curves. Three other techniques used to measure luminescence decay deserve mention. The simplest technique uses a photomultiplier tube, fast oscilloscope, and either a camera or printout device to record the decay curves (14,


15). Another technique uses a modified image convertor tube to follow the luminescence intensity as a function of time ( 5 ) .A third technique uses a modulated excitation source to permit measurement of the phase shift of the luminescence. The phase shift method does not record the decay curve, but the information can be used to calculate the luminescent lifetime ( I ) . The time correlated single photon technique has several advantages over these methods. It has a time resolution of about 0.2 ns, and the signalto-noise (S/N) ratio can be improved by counting longer. Sensitivity does not present a problem because only one photon per excitation pulse is required for detection, andtthe time jitter of the pulsed source is averaged over many cycles. The technique appears to be the least limited in overall applicability when pulsed excitation sources with a width a t half maximum of a few nanoseconds are employed. The precision and accuracy of the lifetime information decrease with decreasing lifetimes. For lifetime values s range, the relative in the to standard deviation is between 2 and 5% ( 4 ) . The accuracy of the TCSP technique depends to a large extent on the experimental conditions and on the sample material itself. Lack of agreement with published lifetime values is not uncommon because of the large number of factors which can actually change the lifetime and instrument calibration errors. For example, the lifetimes reported in the scientific literature for quinine averaged 18.91 & 0.56 ns ( 1 6 ) . The sensitivity of the technique is more difficult to assess. Only one luminescent photon per hundred excitation flashes is required to reach the detector to build up the decay curve. If the detected photon rate is less than this, the sensitivity can be improved through the use of more powerful excitation sources such as high intensity pulsed lasers, although the scattered

light interference becomes more severe ( 4 ) .Normally, decay curves of even faintly luminescent molecules can be obtained using conventional spark discharge excitation ( I 7). Lewis and coworkers (18) have published a paper containing many helpful ideas on the setting up and operation of a time correlated single photon apparatus. The discussion includes construction and operation of nanosecond flashlamps, installation and maintenance of photomultiplier tubes, and the general operation of the apparatus.

Probability Considerations Accurate time correlated single photon data are obtained only if no more than one emission photon is detected per excitation cycle. If multiple photon events do occur, error can result due to pulse pileup. The counts in the multichannel analyzer or computer memory locations accumulate disproportionately faster in the early portion of the decay curve resulting in badly distorted data. Pulse pileup occurs because of the inherent operating characteristics of the time-to-amplitude convertor module. The TAC voltage ramp is initiated by the start signal and is halted when the stop signal is received. The TAC then recycles after a preset time interval and is cleared for another start pulse. If a second emission photon is detected later in time and before the next excitation cycle, it will be observed by the stop photomultiplier tube but will not be counted because the TAC is disabled. Consequently, the decay curve is biased toward early arriving photons. The emission beam of photons from the sample must be attenuated so that the probability of detecting more than one photon per excitation pulse is small. Thus, the detection of a photon is a rare event, and the probability is calculated from Poisson’s equation (19)

mn P, = (4) e%! where P, is the probability of detecting n photons per excitation pulse, and m is the average number of photons detected per excitation pulse. The Poisson probability polygons shown in Figure 5 may be used to predict the frequency of occurrence of multiple photon events and the appropriate experimental conditions for minimum pileup error. When the average counting rate is one photon per excitation pulse, the probability of detecting two photons is 0.184. This is large enough to produce severe pileup error. Normally, an average count rate of 0.01-0.05 photons per excitation pulse is used, and the probability of multiple photon events is very small.

ly short-lived fluorophor. The TCSP instrument was operated a t an average count rate of 0.05 photons per excitation pulse and collected 2000 counts in the peak channel in a 5-min experiment. The choice of optimum photon count rate, and to a much lesser extent the excitation pulse repetition rate, will be influenced by properties of the sample such as fluorescence quantum yield, photochemical and thermal stability, and sample concentration. Signal-to-Noise Ratio Requirements

1. o l

Photons Detected Figure 5. Poisson frequency polygons for various average single photon count rates Curves show probability of detecting zero, one, or two photons resulting from any given excitation pulse at four different mean data collection rates. Average detected emission photons per excitation pulse ( 0 )1.0, (M) 0.2, (A)0.1, and ( 0 )0.05

Several mathematical methods for correction of pileup have been reported (20-22), but they are not needed if an average count rate of C0.05 is maintained. The major disadvantage of the time correlated single photon method is the low average emission photon count rate required. An average count rate of 0.05 means only five out of 100 excitation pulses will produce a detected emission photon. This low data collection efficiency can be a serious drawback. Ordinarily, between lo3 and lo6 counts are accumulated in the peak channel. Several hours may be required to obtain a decay curve if the pulse repetition rate of the excitation light source is low. Undistorted data can be accumulated faster by using higher average count rates in conjunction with a pileup detector module (23). This device will reject the TAC output for a detected emission photon if a second photon arrives within the TAC sweep range. Use of a pileup detector becomes detrimental a t average count rates greater than one photon per excitation cycle because as multiple photon events increase, more TAC outputs are rejected and data accumulation time is actually longer than that required a t lower average count rates. The problems of serious pulse pileup distortion and low data collection efficiency could be eliminated if an excitation light source with an extremely high pulse repetition rate is available. According to one report ( I 7), a flashlamp operating a t 20 kHz with a width a t half maximum of 3.5 ns was used to measure the decay curve of a relative-

Normally, the decay curve is divided into 100-500 increments, corresponding to the number of channels used in the multichannel analyzer or storage device. Data collection is continued until a relatively smooth decay curve is obtained. The relative intensit y of the decay curve varies over three or four orders of magnitude from less than 100 counts a t each extreme to between 103-106 counts in the peak channel. The variance in each channel, according to Poisson statistics, is equal to the number of counts in that channel. Therefore, the best estimate of the standard deviation or noise in each channel is simply the square root of the counts in the respective channels. I t is clearly advantageous to accumulate a large number of counts if high precision data are desired. S/N ratio will improve and a smoother decay curve will result when more counts are allowed to accumulate in the peak channel. The minimum acceptable S/N ratio will be determined to a large extent by the particular data reduction or deconvolution scheme employed to extract the lifetime from the decay curves. The accuracy of the computer algorithms used in the deconvolution step is sensitive to noise on the decay curve. Using the reiterative convolution method, a single exponential decay curve was extracted from an overlapping flashlamp decay curve, and an accurate lifetime calculated when lo4 counts were accumulated in the peak channel (12). When deconvolution was carried out on similar data with only lo2 counts in the peak channel, no fit was obtained and the overlapping curves could not be separated accurately. S/N ratio requirements are more stringent if the experiment involves deconvolution of a mixture of overlapping fluorophors, fluorophors with lifetimes less than the excitation source lifetime or calculation of nonexponential decays. S/N ratio enhancement is far less critical for accurate analysis of relatively long-lived single exponential decays needing only a graphical slope evaluation of the semilogarithmic plot.


367A

n is the number of fluorophors emitting in the sample. The convolution integral breaks up the excitation light source pulse into several subpulses and sums up all the contributions of these subpulses to the observed luminescence. Deconvolution is a powerful mathematical tool used for a number of different applications. Several computer deconvolution methods have been developed, any of which is applicable to analysis of molecular fluorescence decay curves. These include the method of moments ( 2 5 ) ,Laplace (26) and Fourier (27) transforms, modulating functions (28),least squares (29), and reiterative convolution (30). The validity of the deconvolution method can be evaluated by estimating the goodness of fit. This can be done by comparing the statistical variance of the data to the mean variance of the f i t (31). Ideally, the ratio of the two should equal one. Deconvolution of experimental data shows the variance of the fit to be about three times the variance of the data using the reiterative deconvolution method (30).

I

I

Time, ns Figure 6. Computer simulated decay curves of (A)fluorescence emission, (0) pulsed excitation light source, and (M) exponential decay law of fluorophor

The improved S/N ratio resulting from the larger number of counts accumulated is obtained by increasing the data collection time. The trade-off between S/N ratio and experimental time should be critically evaluated and a minimum S/N ratio specified for each instrument and data processing scheme in use. Simulations have shown that approximately lo4 counts in the peak channel are necessary for reliable reiterative deconvolution of two overlapping exponential decay curves having similar lifetimes (24). The experimental time required to obtain this count depends on the excitation pulse repetition rate and may vary widely. Data Processing Excited state lifetimes are calculated from the experimental decay curve data. For single component decay curves which are exponential, the lifetime is simply the reciprocal slope of the plot of the logarithm of intensity vs. time. Samples containing two or more fluorophors which emit a t the measurement wavelength will produce a multiple exponential decay curve. This composite decay curve must be mathematically separated into the different components before calculation of the lifetimes is possible (24). Also, a single exponential luminescent decay must be corrected for distortion if its decay characteristics are similar to those of the excitation pulse. In general, if the two lifetimes differ by 2 or less ns, then the curves must be resolved by a suitable deconvolution procedure (13). The effect of deconvolution on a 368A

composite excitation light pulse and emission light profile is illustrated in Figure 6. These curves were simulated by a digital computer and deconvoluted by a reiterative convolution technique. The lamp flash decay law was approximated by a Gaussian function, and the luminescent decay law was assumed to be Gaussian prior to the peak and exponential after the peak. Deconvolution was carried out on the curve obtained by adding the lamp flash and emission curves. The luminescent decay law which results after correction for the lamp flash decay curve is used to calculate the excited state lifetime. In this example the decay follows a single exponential decay law

E ( t ) = E ( 0 )exp ( - k T )

(5) where E ( t ) and E ( 0 ) are the emission intensities a t time, t , and time, zero, respectively; T is the elapsed time; and k is the decay constant. The reciprocal of k is defined as the lifetime. The computer algorithm corrects for the temporal characteristics of the excitation light source, nonexponential emission, and exponential decay from luminescent contaminants in the sample. The basic equation to be solved by the algorithm is the convolution integral

E ( t ) = l t I j D ( t - j)dj 0

(6)

where I , is the light intensity a t time j , and D is the decay function. The decay function can take the form n

D =

1 ai exp ( - k i T ) i= 1

(7)

where ai is an amplitude constant, and


Applications Numerous applications of the TCSP technique to nanosecond fluorometry can be found in the scientific literature. A discussion of the significance of lifetime information derived from these types of experiments is appropriate a t this point. The selected examples given here are representative of the kinds of phenomena studied and are not meant to be a complete listing of all existing uses. Many references to specific applications of nanosecond fluorometry are found in reviews by Weissler (32) and Cundall and Palmer (33). As mentioned previously, deactivation of an excited molecule M* can occur via several mechanisms. The most useful ones for analytical purposes are direct fluorescence and radiationless transitions (see Equation 2). The competition between these two modes is reflected in the fluorescence lifetime, 7.The greater the rate of radiationless deactivation, the shorter the fluorescence lifetime. Consequently, fluorescence lifetime measurements are an indirect method of obtaining information on the radiationless processes of molecular rotation and diffusion, photochemical reactions, quenching mechanisms, solvent interactions, energy transfer, and excited state complex formation. Oftentimes, the fluorescence lifetime value alone is not sufficient to characterize the process and must be combined with other experimentally determined parameters to obtain the desired result.

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Fluorescence Polarization Studies. The structure and conformation of macromolecules such as proteins and enzymes can be determined by attaching fluorescent molecules to selected macromolecular sites. These attached fluorescent probes are then excited by polarized light, and the resulting luminescence will be polarized to some extent as a result of the rotation due to Brownian motion of the macromolecules. Measurements of the amount of fluorescence polarization, P , and the fluorescence lifetime can be used to calculate the amount of Brownian rotation using the Perrin equation

+

1/P - 1/3 = (l/Po- 1/3)(1 3 ~ / p ) (8)

Po is the polarization observed in rigid media, and p is the rotational relaxation, a measure of the Brownian rotational mobility ( 3 4 ) . The quantity p is also a measure of the elongation of the macromolecule. A larger p value, compared to the relaxation value for a sphere of the same molecular weight, indicates a proportionately larger elongation in the molecule. Fluorescent probe lifetimes will vary depending on the substrate and degree of labeling, thus necessitating careful experimental control and accurate, reliable lifetime measurements before valid interpretations are possible. Conformational transitions of allosteric enzymes may also be followed by fluorescence polarization changes (35).The fluorescent probe, l-anilinonapthalene-8-sulfonate (ANS), was bound to dimer phosphorylase b, and two lifetimes of 8.7 and 19.2 ns were obtained by deconvolution of the experimental decay curve (36).After conversion to phosphorylase a, the 19.2-ns lifetime decreased significantly, indicating dissociation of ANS from the corresponding site due to a change in conformation. Fluorescent probes have also been used to follow the unwinding of the helix structure of DNA caused by complex formation with dye molecules (37). Photochemical Studies. Lifetimes of excited singlet states are useful in the elucidation of the mechanisms of organic reactions. For example, the TCSP technique was used to measure the rates of photochemical rearrangement of a series of acyclic di-.lr-methane compounds (30). Because the rearrangement proceeds through an excited intermediate, the fluorescence lifetimes are a direct measure of the rates of rearrangement. The rates of di-a-methane rearrangements are generally very rapid, on the order of 1Olo s-l. Some of the acyclic compounds had fluorescence lifetimes too short to measure reliably 370A

by the TCSP method. These picosecond decay rates were obtained indirectly by use of a “magic multiplier”,

M.

M=%

(9)

+rt

The quantum yields a t 77 OK, a.77,and a t room temperature, + r t , are measured, along with the slower low temperature fluorescence lifetime, T77. The lifetime a t room temperature can be calculated from M and 777. This approach extends the range of rate measurements down to 10l2 s-l, Quenching Studies. Fluorescence emission can be attenuated or completely eliminated by collisions of the excited molecules with other substances. The rate of attenuation or quenching can be calculated if the fluorescence lifetime and quantum yield are measured in the absence of quencher, Q , and the change in quantum yield with concentration of quencher is determined. Quenching efficiencies for different compounds can be compared by examining their respective quenching rates. This type of information can be helpful in arranging solution conditions to minimize quenching and maximize the intensity of fluorescence. When a quencher is added to a sample, the quantum yield, W, is given by

where k l is the rate of emission, kr is the total rate of radiationless processes and corresponds to k2 and 123 in Equation 2 , k , is the rate of quenching, and [ Q ] is the concentration of quencher. In the absence of quencher, the quantum yield is obtained from the following relationship.

a=-

kl (11) k l + kr By taking the ratio of Equations 10 and 11, it follows that

and a plot of a/+‘ vs. [ Q ] will be linear with a slope equal to k , / ( k l + k r ) . This slope is the Stern-Volmer constant, K,,, which is equal to

Thus, if the Stern-Volmer constant and decay lifetime 7 in the absence of quencher are determined, the rate of quenching, k,, can be calculated ( I ). Chen (16) used Stern-Volmer plots and lifetime measurements to obtain quenching rates for quinine and y pyrenebutyrate when quenched by NaCl and KI, respectively. By use of these data, a table of fluorescence lifetimes of standard solutions of known


fluorophor/quencher ratios was generated for use as lifetime standards to calibrate TCSP instrumentation. Energy Transfer. Under some conditions energy can be transferred from an excited molecule M* to a ground state molecule M’:

M*

+ M’

M

+ M’*

(14) Singlet-singlet energy transfer is important in many biochemical systems. This has been called radiationless transfer, dipole-dipole resonance transfer, Forster-type energy transfer, and long-range nonradiative transfer. Of particular interest for analytical purposes is the case where M’* fluoresces. This case is known as sensitized fluorescence. The critical distance a t which transfer occurs is partly a function of the fluorescence lifetime ( 3 4 ) . A recent application of nanosecond fluorometry in energy transfer studies is the work by Loper and Lee (38)on the steric hindrance of gas phase singlet energy transfer from napthalene to the trans-azobutane isomers. Possible energy transfer has also been reported between carotenoids and the energy receptor, chlorophyll a (39). +

Summary Fluorescence lifetimes provide additional information to aid in the characterization of molecules and molecular interactions. Because lifetimes are very sensitive to the molecular electronic configuration and to the molecular environment, they are good indicators of subtle changes in these properties. Any process which competes with the natural radiative decay will change the value of the experimental lifetime. Lifetime information is particularly helpful in the study of the structure and functions of molecules of biological interest. The excited state lifetime is potentially useful to the analyst for identification of molecular species. If a common matrix is maintained for the sample and standards, it is possible to use the experimental lifetime to aid in qualitative analysis. Valid interpretation of lifetime measurements depends on the accuracy of the measurement technique. The time correlated single photon method permits accurate observation of nanosecond and subnanosecond phenomena if the chemical and instrumental conditions are very carefully controlled. The use of the TCSP technique should increase as investigators probe the properties of fast phenomena. Further refinement of lifetime measurement techniques and use of mode-locked lasers capable of generating intense light pulses of picosecond duration will allow studies of more elusive chemical phenomena.

References (1) J.B. Birks and I. H. Munro, "Progress

in Reaction Kinetics", Vol4, Chap. 7, G. Porter, Ed., Pergamon Press, New York, N..V . .., 1W7. . ( 2 ) W. R. Ware, "Creation and Detection of the Excited State", Vol 1, Part A, Chap. 5, A. A. Lamola, Ed., Marcel Dekker. New York. N.Y.. 1971. (3) ORTEC, Ine., Application Note AN 35, Oak Ridge, Tenn. (4) A.E.W. Knight and B. K. Selinger, Rust. J. Clmm., 26, l(1973). (5) J. Yguerahide, "Methods in Enzymology", Vol26, Part C, S. P. Colowiek and N. D. Kaplan, Eds., Academic Press, New York, N.Y., 1972. ( 6 ) J. J. Aaron, L.B. Saunders, and J. D. Winefordner, Clin. Chim. Acta, 45,375 (1973). (7) M. L. Franklin, G. Horliek, andH. V. Malmstadt, Anal. Chem., 41,2 (1969). ( 8 ) H. V. Malmstadt, M. L. Franklin, and G. Horliek, ibid., 44 ( E ) , 63A (1972). (9) G. A. Morton, Appl. Opt., 7, I (1968). (10) L. M. Bollinger and G. E. Thomas, Rev. Sci. Instrum., 32,1044 (1961). (11) Y. Kaechlin, CR Aeod. Sei. (Paris), 252,391 (1961). (12) L. A. Shaver and L. J. Cline Love, presented in part at the Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, No. 517, Cleveland, Ohio, March 1975. (13) L. A. Shaver and L. J. Cline Love, Appl. Speetmsc., 29,485 (1975). (14) J. N. Demas and G. A. Croshy, Anal. Chem., 42,1010 (1970). (15) L. Hundley, T. Coburn, E. Garwin, and L. Stryer, Re". Sei. Instrum., 38,488 -I

il'X7I

(16) R. F. Chen, Anal. Bioehem., 57,593

(1974).

(17) H. E. Zimmerman, K. S. Kamrn, and D. P. Wertheman, J . Am. Chem. Sac., 97, 3718 (1975). (18) C. Lewis, W. R. Ware, L. J. Daemeny, and T. L. Nemzek, Re". Sei.Instrum., 44,107 (1973). (19) R. R. Sokal and F. J. Rohlf, "Biome-

try'', Chap. 5, Freeman, San Francisco, Calif., 1969. (20) P. B. Coates, J. Phys. E., 5,148 (1972).

(21) C. C. Davis and T. A. King, Re". Sei. Instrum., 41,407 (1970). (22) D. E. Donohue and R. C. Stern, ibid., 43,791 (1972). (23) 0. M. Williams and W. J. Sandle, J. Sei.Instrum., 3,741 (1970). (24) L. J. Cl@eLove and L. A. Shaver, presented m part at the Eastern Analytcal Symposium, New York, N.Y., Na"ember 1975. (25) I. Isenberg, J. Chem. Phys., 59,5708 114711 j_l.l,.

(26) A. Gafni, R. L. Modlin, and L. Brand,

Biophys. J., 15,263 (1975).

(27) J. Schlesinger, Nuel. Instrum. Methods, 106,503 (1973). (28) B. Valeur and J. Mairez, J. Chim. Phys., 70,500 (1973). (29) W. R. Ware, L. J. Doemeny, and T. L. Nemzek, J.Phys. Chem., 77,2038 il_".l,. iami

(30) H. E. Zimmerman, D. P. Wertherman", and K. S. Kamm, J. Am. Chem. Soe., 96,439 (1974). (31) A.E.W. Knight and B. K. Selinger, Speetroehim. Acta, 27A, 1223 (1971).

(32) W. Weissler, Anal. Chem., 46 (9, 500R (1974). (33) R. B. Cundall and T. F. Palmer. "An-

1973. (34) R. F. Chen, Fluaresc.News,8,29 1,074~ l_".-,.

(35) S.Matsumoto and G. G. Hammes, Rioehem., 14,214 (1975). (36) M. S. Tune and R. F. Steiner. Biopolymers, 14;1933 (1975). (37) J. L. Tichadou, D. Genest, Ph. Wahl, and G. Aubel-Sadron, Bmphys. Chem , 3 , I1"" Ilnncl . f L ,'".'U,. 1 G. L.Loner and E.K.C. Lee. J. Chem.

L. J. Cline Love is assistant professor of chemistry at Seton Hall University. After receiving her doctorate from the University of Illinois, Urbana, in 1969, she spent one year working with J. D. Winefordner's group at the University of Florida. Following this, she was visiting assistant professor of analytical chemistry at Michigan State University for two years and moved to her present position in 1972. Her research interests include development of molecular luminescence methods, particularly for pharmaceutical applications, extension of automated atomic spectroscopic techniques, and surface studies in the area of forensic art. L. A. Shaver obtained his bachelor's and master's degrees from Northern Arizona University and Arizona State University, respectively. He is currently m m pleting the program of study for a PhD degree at Seton Hall University. H e is on a leave of absence from FMC Corp., R & I),Princeton, N.J. Current research interests include fluorescence lifetime measurements by the time correlated single photon technique and digital computer data reduction techniques. CIRCLE 1 1 2 O N READER SERVICE CARD


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fluorescence lifetime measurements

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