Anal. Chem. 1986, 58, 3139-3144
(Figure 7a) does not correspond to chrysene emission (Figure 7b) but originates from another unidentified species in the complex sample. Our chromatographic analyses of the SRC-I1 sample indicated that the concentration of chrysene in this was about than that Of pyrenee With the setup used in this work, the highly specific information of the FLN spectrum has required narrow spectral band-pass, leading to a subsequent decrease in sensitivity. This problem may be overcome by the use of frequency-doubled dye lasers as excitation sources.
CONCLUSION
'"-
Our show that the technique Of FLN may be cessfully applied to samples adsorbed on filter paper at liquid helium temperature. This adds further versatility to spectroscopyon which so far have been shown to be extremely useful in analytical applications of roomtemperature phosphorimetry (2)' The well-established advantages of room-temperature phosphorimetry, such as practicality, simplicity, low cost, and high sensitivity, may be fruitfully complemented by the complex FLN technique providing more specific information. The trade-off between benefit and level Of sophistication can be decided upon by the investigator. Simple and cost-effective analyses and screening of major components can be performed a t room temperatureto prioritize large numbers of samples before a more detailed analysis should be conducted on a subset of samples using cryogenic techniques: such a strategy would reduce the total cost of environmental control and health effects assessment studies. Registry No. Cellulose, 9004-34-6.
LITERATURE CITED (1) Winefordner, J. D.; Schulman, S. G.; O'Haver, T. C. Luminescence Spectrometry in Analytical Chemistry; Wlley: New York, 1972. (2) Vo-Dinh, T. Room Temperature fhosphorimerry for Chemical Analysis; Wiley: New York, 1984. (3) Vo-Dinh, T.; Suter, G. W.: Kallir, A. J.; Wild, U. P. J. fhys. Chem. 1985, 8 9 , 3026. (4) Shpolskii, E. V. SOC.fhys. Usp. 1959, 2 , 378. (5) Vo-Dinh, T.; Kreibich, U. T.; Wild, U. P. Chem. fhys. Left. 1974, 352. (6) Kirkbright, G. F.; de Lima, C. G. Analyst (London) 1974, 99, 338. (7) Yang, Y.; D'Silva, A. P.; Fassel, V. A. Anal. Chem. 1981, 53, 2107. (8) Colmsjo. A.; Stenberg, U. J. Chromatogr. 1979, 169, 205. (9) Vo-Dlnh, T.; Wild, U. P.; Lamotte, M.; Merle, A. Chem, fhys, Left. 1976, 3 9 , 118.
3139
(10) Wehry, E. L.; Mamantov, G. Anal. Chem. 1979, 51, 643A. (11) Personov, R. I.; Kharmalov, B. M. Opt. Commun. 1973, 7 , 417. (12) Eberly, J. H.; McColgin, W. C.: Kawaoka, K.; Marchetti, A. P. Nature (London) 1974, 251, 215. (13) Brown, J. C.; Edelson, M. C.; Small, G. J. Anal. Chem. 1978, 5 0 , 1394. (14) Hofstraat, J. W.; Engelsma, M.; Cofino, W. P.; Hoornweg, G. P.; jer, C.; Velthorst, N. H. Anal. Chim. Acta 1984, 159, 359. (15) Brown, J. C.; Duncanson, J. A., Jr.; Small, G. J. Anal. Chem. 1980, 52, 1711. (16) Sanders, M. J.; Cooper, R. S.;Small, G. J.; Heisig, V.; Jeffrey, A. M. Anal. Chem. 1985, 57, 1148. (17) Matthew, J. S.:Cooper, P. 5.;Jankowiak. R.; Small, G. J.; Heisig, V.; Jeffrey, A. M. Anal. Chem. 1986, 5 4 , 816. (18) Personov, R. I.; AI'Shits, E. I.; Bykovskaya, L. A. Opt. Commun. 1972, 6, 169. (19) Griesser, H. J.; Wild, U. P. J. Chem. fhys. 1979, 73, 4715. (20) Personov, R. I. I n Modern Problems in Condensed Matter Sciences;
Agranovich, V. M., Hochstrnsser, R. M., Eds.; North-Holland: Amsterdam, 1982; Vol. 4. (21) Rebane, L. A.; Gorokhovski, A. A.; Kikas, J. V. Appl. fhys. 1982, 93, 235. (22) Renn, A.; Meixner, A. J.; Wild, U. P.; Burkhalter, F. A. Chem. fhys. 1985, 93, 157. (23) Wild, U. P.; Bucher, S. E.; Burkhalter, F. A, Appl. Opt. 1985, 2 4 , 1526. (24) Friedrich, J.; Haarer, D. Angew. Chem. 1984, 23, 113. (25) SUter, G. w.; Wild, u. p.; Holzwarth, A. R . Chem. fhys. 1986, 102, 205. (26) Friedrich, J.; Haarer, D. J. Chem. fhys. 1983, 79, 1612. (27) Gorokhovski, A. A.; Kikas, J. V. Opt. Commun. 1977, 21, 272. (28) AI'Shits, E. I.; Personov, R. 1.; Kharlamov, B. M. Chem. fhys. Left. 1976, 40, 116. (29) Suter, G. W.; Wild, U. P. J. Luminesc. 1981, 2 4 / 2 5 , 497. (30) Suter, G. W.; Kallir, A. J.; Wild, U. P.; Vo-Dinh, T. J. fhys. Chem., in press. (31) Hara, K.; Ware, W. R . Chem. fhys. 1980, 51, 61. (32) Environmental Carcinogens : Polycyclic Aromatic Hydrocarbons; Grimmer, G., Ed.; CRC: Boca Raton, FL, 1983. (33) Silsbee, R. H. fhys. Rev. 1962, 128, 1726. (34) McCumber, D. E.; Sturge, M. D. J. Appl. fhys. 1983, 34, 1682. (35) Krivoglaz, M. A. Fir. Tverd. Tela. 1964, 6 , 1707. (36) Osadko, I.S.Fib. Tverd. Tela. 1975, 17, 3180. (37) May, W. E.; Brown-Thomas, J. M.; Chesler, S. N.; Guenther, F. R.; Hiipert, L. R.; Parris, R. M.; Ritchie, S.A.; Wise, S.A.; Hertz, H. S. I n Advanced Techniques in Synthetic Fuel Analysis ; National Technical Information Service, CONF-811160, Springfield, VA, 1984; Chapter 24, pp 381-404.
RECEIVED for review May 12,1986. Accepted August 15,1986. This research was jointly sponsored by the Swiss National Science Foundation and the Office of Health and Environmental Research, U.S. Department of Energy, under Contract DE-AC05-840R214200with Martin Marietta Energy Systems, Inc.
Rapid Frequency-Scanned Fiber-optic Fluorometer Capable of Subnanosecond Lifetime Determinations Frank V. Bright, Curtis A. Monnig, and Gary M. Hieftje* Department of Chemistry, Indiana University, Bloomington, Indiana 47405
'
A new fluorescence lifetlme instrument is described that allows the rapid acquldtlon of the frequency-response spectrum of a fluorescent sample. The instrument employs for excltatlon a continuous wave (CW) or mode-locked argon-ion laser and is capable of scanning and acqulrlng In less than 10 ms the complete frequency-domain spectrum between 250 and 1000 MHr. Following a simple data-analysis scheme, the fluorescence lifetime of the analyte can be easily calculated. Fluorescence lifetimes for several common nanosecond and subnanosecond fiuorophores are determlned and shown to agree with previously reported values.
One area of active research in chemical analysis is the
measurement and quantitation of brief chemical phenomena. Of such short-lived chemical events, fluorescence decay processes, which occur on a nanosecond and subnanosecond time scale, are probably the most commonly studied. Interestingly, some molecular transformations of fluorophores, i.e., rotations, energy transfers, and migrations, occur on the same time scale as does fluorescence. Consequently, the determination and measurement of fluorescence phenomena provide an internal reference time which can be used to probe indirectly the magnitude and rate of these additional processes. The decay of fluorescence is typically measured by using one of two techniques. The first is a time-domain measurement of the fluorescence decay that follows excitation with a very short pulse of light. This procedure is commonly
0003-2700/86/0358-3139$01.50/00 1986 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 14, DECEMBER 1986
termed the impulse-response approach (1-5). The popular technique of time-correlated single-photon counting is one example of such a time-domain technique (6-10). The second method involves measuring the response of the fluorescence in the frequency domain. Here, a sinosoidally modulated excitation source excites the sample and is followed by a determination of the phase shift or amplitude demodulation between the excitation and emission waveforms (11-15). The main advantages of the impulse response approach are its sensitivity and its simplicity of interprepation for complex decay kinetics. However, the most commonly used impulseresponse technique, the time-correlated single-photon-counting method, is slow and sometimes takes hours to acquire the necessary data; consequently, it requires a very stable excitation source. In contrast, the frequency-response approach is much faster and requires only that measurements be made at a series of discrete source-modulation frequencies, the required number of which depends on the complexity of the decay kinetics (11-21). The most common frequency-domain instruments are phase-modulation fluorometers. Recently, Gratton and co-workers have introduced sequentially scanned continuously variable frequency phase-modulation fluorometers, operating up to a modulation frequency of 300 MHz (16). These instruments have been shown to have wide-spread utility and have resolved contributions from synthetically prepared binary and ternary mixtures (17-19). Additionally, these instruments have precision on the order of several picoseconds (20) and have been used to resolve emission spectra for binary mixtures (21). Ramsey and Hieftje have shown that the signal-to-noise ratio of a frequency-response approach is highest if a multifrequency excitation source is employed for excitation and if all frequencies are monitored simultaneously (22). In an earlier study, Hieftje and co-workers employed the mode noise from a continuous wave (CW) argon-ion laser as the multifrequency excitation source and a radio-frequency spectrum analyzer as a frequency-selective detector (23). Unfortunately, the swept-frequency spectrum analyzer was very costly and monitored only a single frequency at a time. More recently, Bright and co-workers employed an ultra-high-frequency television tuner as a selective filter, demonstrating the applicability of laser mode noise (demodulation measurements) to two-component decay systems (24). In the studies presented EElre, a CW or mode-locked argon-ion laser is used to excite remotely the fluorescence in a sample via a fiber-optic probe. Clearly, conventional optical systems could also be used and with a resultant increase in sensitivity, but the fiber-optic probe permits lifetime measurements of fluorophores that are remotely located from the excitation source and detector. The harmonic content of the mode-locked pulse train or the naturally occurring mode noise from a CW laser serves as the multifrequency excitation source. The high-frequency information is subsequently detected by a fast-sweeping radio-frequency receiver. The total time for the collection of the frequency range of interest (250-1000 MHz) is approximately 10 ms. This time is nearly 4 orders of magnitude shorter than that for time-correlated single-photon-counting approaches (7-9) and about 2 orders of magnitude less than that for any other frequency-domain techniques (11-21). The instrument described here differs significantly from that described earlier by Hieftje and co-workers (23). First, it is much faster at acquiring the frequency information than the earlier instrument. This new instrument also employs a fiber-optic probe for remote sensing, has signal-averaging capabilities, and is capable of determining single-exponential nanosecond and subnanosecond lifetimes. Furthermore, the costly spectrum analyzer in the earlier instrument has been
replaced by a simple radio-frequency swept receiver. With this new instrument, lifetimes have been rapidly and remotely determined for rose bengal, rhodamine 6G, and rhodamine 6G quenched by KI. The instrument is shown to be useful for elimination of quencher errors and to provide excellent sensitivity.
THEORY Determination of nanosecond and subnanosecond fluorescence lifetimes by the frequency-response approach requires high-frequency (MHz-GHz) sinusoidally modulated light for sample excitation. For a sample excited with sinusoidally modulated light, the resulting fluorescence is equal in frequency but phase shifted and demodulated (decreased in amplitude) by factors which depend upon the excited-state lifetime of the fluorophore (11-21). The fluorescence lifetime ( 7 ) is related to the demodulation factor M in the following manner (11-21):
where w is the angular modulation frequency (27rf) and f is the linear modulation frequency (MHz-GHz). The demodulation factor is determined simply from the fluorophore’s modulation ratio (mf) and from the modulation factor for scattered light (mo)as (24)
where ac, and acfluorare the ac amplitudes for scatter and fluorescence, respectively, and dcScatter and dcfluorare the dc signals for the scatter and fluorescence, respectively. In certain instances it is better to use a reference fluorophore of known lifetime than a scattering solution in order to correct for “color effects” (25,26). Regardless, determination of the demodulation factor allows one to calculate the fluorescence lifetime by using information a t a single modulation frequency. Of course, measurement of the demodulation factor at a single modulation frequency does not provide enough information to resolve complex kinetics or multicomponent decays. Multifrequency modulation provides a more complete view of the frequency response of a system and permits complex kinetics to be unraveled (16-21). For a homogeneous emitting population the fluorescence will follow first-order kinetics and will decay exponentially with time. The Fourier transform of this exponential function yields a Lorentzian in the frequency domain. If this Lorentzian function can be measured, the fluorescence lifetime can be calculated from 7 = -
1
(3)
Wfwhm
where wfWb is the angular modulation frequency at which the ac amplitude level is half its dc value (27). Alternatively, the complete Lorentzian (Lf) (4) can be fitted to yield the fluorescence lifetime (27). Clearly, both of these latter methods require the acquisition of demodulation information at more than a single modulation frequency. In such a situation, complex decay kinetics can be resolved (24,28,29). In fact, Bright and co-workers have demonstrated the resolution of spectrally overlapping fluorophores with lifetime ratios of 1:4.5 by using only modulation information (24).
ANALYTICAL CHEMISTRY, VOL. 58, NO. 14, DECEMBER 1986
j L
ML -
J
l--A
A r Ion Laser
I---\
1 I
RF A t t e n
RFSR
Signal A v e r a g e r
Signal Out
Flgure 2. Block diagram of the radio frequency swept receiver: RF Atten., radio-frequency input attenuator; DBM, double-balanced mixer; VCO, voltage-controlled oscillator.
Storage Scope
--
*
DBM
In
I
3141
i
Figure 1. Schematic diagram of the new rapid frequency-scanned fluorometer used for nanosecond and subnanosecond fluorescencelifetime determinations: ML, mode-locker and driver unit: M, and M,, flat mirrors; I , iris; L,, 7-cm focal length lens; X,Y,Z. fine-adjustment fiber-optic translation stage; OFS, optical-fiber spool; FM, fiber-optic mount; L,, 35-cm focal length lens; PM, photomultiplier; RFSR, radio frequency swept receiver; MCA, multichannel averager.
EXPERIMENTAL SECTION Instrumentation. Rapid-Scanning Multifrequency Fluo-
rometer. Figure 1shows a schematic diagram of the new rapidscanning frequency-resolved fluorometer used in this work. All optical components were rigidly mounted on a 1.2-m X 3.7-m optical table (Unidex). The instrument employs a mode-locked argon-ion laser (Model 171 laser, Model 342 mode-locker, and Model 452 mode-locker driver, Spectra Physics, Inc.), operated with a mode-locking frequency of 40.9800 MHz. Mirrors MI and Mz were used to direct the output of the laser through an adjustable iris and lens (focal length = 7 cm) onto one arm of an optical fiber mounted on a fine-adjustment x,y,z translation stage. The fiber-optic probe was 16 m in length and consisted of two identical 16-m segments of 250-pm diameter cladded-multimode fiber (P/N C02-40000-05-03,Valtec, Inc.). Fluorescence radiation was collected by a second optical fiber whose output end was held rigidly in a stationary fiber-optic mount. The light from this mounted fiber was then focused by a lens (focal length = 35 cm) into a double monochromator (Model 1680 Spectramate, Spex, Inc.) with a spectral band-pass of 9.0 nm. Detection of the resulting fluorescence emission was achieved with a photomultiplier (Model 31024, RCA, inc.) operating a t a biasing voltage of -3500 V dc. At the distal end of the fiber system, both excitation and fluorescence segments were epoxied within a 5-mm 0.d. glass capillary and could then be directly immersed in the sample under study. Both the ac and dc components of the fluorescence were required for calculation of the fluorescence lifetime. These two measurements were performed sequentially. For dc detection, the output from the photomultiplier was connected to a picoammeter (Model 414S, Keithley, Inc.) and the dc level recorded by a digital voltmeter (Model 8024A, Fluke, Inc.). To record the high-frequency signal, the output was connected to a radio frequency swept receiver (Model ESR-204-1, ACL, Inc.) whose operation will be described in the next section. The output of the receiver was connected to a multichannel signal averager (Model 1080 instrument computer, Model SD-81/2 12-bit digitizer and Model SW-80 sweep controller, Nicolet, Inc.); the averaged spectrum was then sent to a digital oscilloscope (Model 3091, Nicolet, Inc.) which permitted serial transfer to an IBM-PC through an RS-232 port. Data collection, regression, and plotting were all performed with an interactive BASIC program. Radio Frequency Swept Receiuer. A simplified block diagram of the radio frequency swept receiver is shown in Figure 2. In this device, the input first passes through a variable radio-frequency attenuator, which is adjusted to prevent saturation of the later electronics. Following the attenuator, the signal goes to a double-balanced mixer, the other input to which is provided by a voltage-controlled oscillator (VCO) driven by a voltage ramp. This voltage ramp serves also as a trigger for the data-collection
electronics. The output from the voltage-controlled oscillator is a linear frequency ramp that sweeps from 250 to loo0 MHz. The averaged (filtered) output of the double-balanced mixer is the cross-correlation (30,31)of the input waveforms. In the simplest case, if a single frequency in the 250-1000 MHz region is sent into the swept receiver, a dc signal proportional to the input waveform’s amplitude will appear at the output of the filter when the voltage controlled oscillator’s frequency matches the frequency of the input waveform. In the more general situation, the spectrum of any input waveform can be “captured” at the output of the double-balanced mixer by triggering the collection electronics at the onset of the voltage ramp. For our specific system, the entire scan from 250 to 1000 MHz required only 6 ms with an approximate 2-ms delay time between the cessation of one scan and the beginning of another. When it is desired, multiple sweeps of the frequency range can be coupled with signal averaging in order to improve signal-to-noise ratios (S/N). Reagents a n d M a t e r i a l s . Rhodamine 6G and rose bengal were purchased commercially (Exciton Chemical Co. and Aldrich Chemical Co., respectively) and used without further purification. A 1.00 mM stock solution of each was prepared by dissolving the appropriate amount of each compound and diluting to 100 mL with absolute ethanol (AAPER Alcohol & Chemical, Co.). Each solution was then sonicated for 30 min to ensure complete mixing. Potassium iodide (Mallinckrodt) solutions were prepared by dissolving the appropriate quantity of potassium iodide in a 5050 (v:v, ethano1:water) mixture. For all results presented here, no additional steps were taken to degas the solutions. All measurements were carried out by using disposable polystyrene cuvettes (Fisher Scientific) as sample containers. Sample solutions were prepared fresh each day from the stock solutions using high-precision micropipets (Rainin, Inc.). General Operation. Initially, while monitoring scattered laser light, the radio frequency swept receiver was adjusted to achieve the largest signal-to-noise ratio. Specific adjustments were made on the input radio-frequency attenuator and output amplifier (Figure 2). The number of scans averaged and the bit resolution of the averager were next adjusted to maintain an adequate signal-to-noise ratio (S/N) while avoiding saturation of the averager’s finite memory. In general, moderately strong input signals (10-80 pA) needed between 64 and 256 scans to achieve S/N above 10. However, the high-frequency mode beats of longer-lived fluorophores are very weak and require more signal averaging. The averaged signal from each sample, scattering solution (water), or reference fluorophore (rhodamine 6G in 1.2 M KI) (32) was stored on the digital oscilloscope and sent to the IBM-PC for data processing. This data processing involved simply the division of the fluorescence modulation amplitude vs. frequency spectrum by that of the reference spectrum (scattered light or quenched rhodamine 6G) as described earlier.
RESULTS AND DISCUSSION The sinusoidal modulations in our system are generated either by the longitudinal mode beats from a CW laser or from the harmonic content of a mode-locked laser pulse train. Of course, in both cases, the frequency content and range of the modulations are the same. Furthermore, the data acquisition time is the same regardless of the number of modulation frequencies employed. In the simplest (CW) case, the frequency spacing between adjacent mode beats for the CW argon-ion laser used in this
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ANALYTICAL CHEMISTRY, VOL. 58,NO. 14,DECEMBER 1986
study is approximately 82 MHz. Discrete mode beats occur at integer values of (c/2L)n, where c is the speed of light, L is the laser cavity length, and n is an integer. For a rare-gas-ion laser, these mode beats extend up to approximately 4 GHz, limited by the spectral line width of the active lasing medium (33). These beats are the result of mixing among the laser's longitudinal modes. The amplitudes of the mode beats are variable and depend on the gain profile of the lasing medium and on the interactions of the various modes. The second operating configuration is with the laser being pulsed (mode-locked). In this case a train of 160-ps (full width at half maximum) pulses is generated at an interpulse spacing of 12.2 ns. These pulses are Lorentzian in shape and can be described by the following expression (20): m
L(t) =
n=-u
(b2
+ [ t - nTI2]-'
1
(5) -
~~
where T i s the spacing between adjacent pulses, b is half of the full width at half maximum of the pulse, and t is time. From the convolution theorem the frequency-domain analogue of L ( t ) becomes
In 2 =-
1000
Frequency ( M H ~ )
Flgure 3. Amplitude vs. frequency spectra for six laser lines from the argon-ion laser in CW operation. In each case the laser output power is 300 mW and the dc photomultiplier tube current is 30 b A . The gain for the laser lines increases from 501.7 to 488.0nm. Each spectrum is the average of 256 swept-frequency scans.
which is simply a set of frequencies equally spaced by f (re) convoluted with an exponential envelope. calling o = 2 ~ fand Again, in our case this frequency spacing is approximately 82 MHz. The -3 dB bandwidth (fL) of this envelope is therefore given by {I,
750
300
250
I
(7)
"fwhm
and corresponds to a frequency of 1.38-GHz, well above the 1-GHz frequency limit of the radio-frequency receiver and the photomultiplier. This 1-GHz cutoff could be overcome by eliminating several of the dynodes in the photomultiplier's dynode chain, but would decrease the S / N dramatically. One other way to alleviate this problem would be to use a microchannel plate photomultiplier tube, but because of the radio-frequency receiver's limits, no further frequency content would be gained. For both modes of laser operation (CW and mode-locked) the variabilities among modulations at different frequencies are compensated by means of a scattering suspension or a reference fluorophore. This procedure compensates also for the instrument response function, Le., the frequency response of the photomultiplier tube, coaxial connectors, and the associated detection electronics. With the present system, the highest measurable frequency is dictated by the response time of the detector and is very nearly 1 GHz. We are limited on the low-frequency end (below -250 MHz) by the frequency range of the voltage-controlled oscillator in the radio frequency swept receiver. Therefore, we can in about 6 ms detect nine mode beats between 328 and 984 MHz. Figure 3 shows the amplitude vs. frequency (mode-beat) spectra for six oscillating lines from the argon-ion laser operating with the mode-locker driver off (CW operation). These spectra were collected by scattering the input fiber's laser light from an aluminum surface into the collection fiber. The mode beats in Figure 3 are weak because of the lack of phase coherence among the oscillating modes. Because they are weak, these beats can be detected only at relatively low frequencies. In contrast, Figure 4 shows the same kinds of scans taken with the laser mode locked and under the same experimental conditions (power and photomultiplier current) used for Figure 3. These "mode beats" are much stronger and can be detected out to 984 MHz. The roll-off at high frequencies is a t least partially attributable to the response time of the photomul-
250
500
750
1000
~ ~ ~ q u e (nHcHyZ )
Figure 4. Amplitude vs. frequency spectra for six laser lines from the argon-ion laser in mode-locked operation. I n each case the laser output power is 300 mW except for the 501.7-nm line which was 240 mW; the dc photmultiplier tube current is 30 MA. The gain for the laser lines increases from 501.7 to 488.0 nm. Each spectrum is the average of 256 swept-frequency scans.
tiplier tube and could be alleviated as describe above. The discrete peaks under mode-locked operation are stronger because of the phase coherence induced by the mode-locking process. Interestingly, as the gain (laser output power/ plasma tube current) of the various laser spectral lines decreases, the mode-beat structure in Figure 4 becomes much more pronounced, probably because of the efficiency of the modelocking process for lower gain laser transitions. For example, mode locking the high-gain 488.0-nm line is very difficult and unstable above an output power of 200 mW with our laser, whereas the low-gain 501.7-nm line is very easy to mode lock a t the same power. The effects of varying the emission wavelength on the amplitude vs. frequency spectrum of a solution of rose bengal are shown in Figure 5. For this series of scans the laser was operated under mode-locked conditions. Each trace in Figure 5 is a single 6-ms scan. As expected, the mode beats become larger near the emission maximum of the sample. It appears in Figure 5 that some higher frequency mode beats are lost in the lower amplitude traces. However, these mode beats
ANALYTICAL CHEMISTRY, VOL. 58, NO. 14, DECEMBER 1986
3143
Rose Bengal
500
250
500
750
1000
Fraquancy (MHz)
Figure 7. Ampliude vs. frequency spectrum for a 0.25 p M rose bengal solution. The spectrum is the average of 1024 scans. The spectrum is unnormalized by scattering spectrum.
~
250
500
750
1000
Rhodamine 6G
I
Frequency (MHz)
Flgure 5. Ampliude vs. frequency spectra as a function of emission M rose bengal solution. Each trace represents wavelength for 2 X a single scan. Mode-locked laser excitation at 514.5 nm with the laser operating at 1.0 W average power. The spectrum is unnormalized by scattering spectrum.
250
500
750
1000
Frequency (MHz)
Figure 8. Amplitude vs. frequency spectrum for a 1 /.LMsolution of rhodamine 6G. The spectrum is the average of 4096 scans. The spectrum is unnormalized by scattering spectrum.
Frequency
(MHr)
Figure 6. Amplitude vs. frequency spectra for 2 X lo-’ M rhodamine 6G solution as a function of added KI. Each spectrum is the average of 64 scans. The spectrum is unnormalized by scattering spectrum.
are not lost but merely buried in the noise, a fact that is apparent if the traces are normalized. The fluorescence lifetime calculated for all traces in Figure 5 is 0.70 f 0.09 ns, a value which agrees well with the value of 0.9 ns reported in the literature (34). The effect of the quenching by iodide ion of rhodamine 6G fluorescence is shown in Figure 6. It is evident that the mode beats become greater and their roll-off with frequency is reduced as the concentration of quencher is increased, suggesting that the fluorescence lifetime of the fluorophore has decreased. A Stern-Volmer plot (35) of the data yields a Stern-Volmer constant of 31.0 M-l, in excellent agreement with previously reported values of 30.1 M-’ (32). Previous work has demonstrated that division of an observed fluorescence signal by the observed fluorescence lifetime can correct for errors in quantitating fluorophores in the presence of quenchers (36). Ten replicates of each of the four samples used in constructing Figure 6 were run and corrected by the lifetime-division method and agreed to within 8% of M). The relative standard deviation the true value (1 x was 7% over the quenching range studied. The detection power of the new instrument was demonstrated by the collection of the amplitude vs. frequency
spectrum for a 0.25 /.LMsolution of rose bengal (Figure 7 ) . The acquisition time for this spectrum, which is the average of 1024 individual scans, was only 70 s. The fluorescence lifetime for this sample was determined to be 0.84 f 0.06 ns, again in good agreement with the previously reported value of 0.9 ns (34). Earlier methods that employed laser mode noise have not been useful for the determination of longer-lived fluorescent species because of the limited number of low-frequency mode beats and because the high-frequency mode beats were strongly attenuated and could not be detected with sufficient sensitivity (23). This problem is less severe with the present instrument because of its sensitivity and signal-averaging capabilities. Figure 8, which shows the amplitude vs. frequency spectrum for a 1/.LMsolution of rhodamine 6G, was acquired by the averaging of 4096 scans and clearly shows three mode beats. The fluorescence lifetime determined from Figure 8 for rhodamine 6G was 3.37 ns, which is also in good agreement with a value of 3.7 ns determined from the natural lifetime value of 3.9 ns (34)and a quantum yield of 0.95 for rhodamine 6G in ethanol (37). It is readily apparent from Figures 3-8 that most of the information contained in the amplitude vs. frequency spectrum is useless except for the mode beats themselves. That is, during the sweeping process much information is acquired, but only a small fraction is pertinent. In the future we plan to modify the radio frequency swept receiver so that it can operate in a “slew-scan”fashion, hopping from one mode beat to the next. It has been suggested (22) that this way of collecting mode-beat information is best from a signal-to-noise standpoint, unless truly simultaneous monitoring of all mode beats is possible.
CONCLUSION Overall, the new rapid frequency-scanned fluorometer is simple to operate, shows good detection power, allows remote
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 14, DECEMBER 1986
sensing, and is capable of determining nanosecond and subnanosecond fluorescence lifetimes. The use of the fiber-optic probe demonstrates, for the first time, that frequency-domain measurements can be used in conjunction with optical fibers, even though the overall throughput (excitation and collection of luminescence) for the fiber-optic probe is certainly at least 100-fold less than in conventional optical systems. An initial comparison of measurement precision suggests that the continuously variable phase-modulation instruments (16-21) are more precise than the present one; however, our instrumental configuration employs optical-fiber sensors. In fact, it is not unreasonable to assume that the measurement precision would decline if fiber optics were coupled to the earlier instruments simply because of the resulting 100-fold decrease in light throughput. Unfortunately, we are aware of no earlier literature that demonstrates frequency-domain lifetime determinations through optical fibers, to which we could directly compare.
ACKNOWLEDGMENT The authors are indebted to Jerry Stout of the Indiana University electronics shop for providing the radio frequency swept receiver and to Robert Addleman and the Indiana University Chemistry Department NMR group for the loan of the signal averager used in this study. We also thank Andrew W. Steele for providing the initial digital oscilloscope program. Registry No. KI, 7681-11-0;rose bengal, 11121-48-5;rhodamine 6G, 989-38-8. LITERATURE CITED Ware, W. R. in Creation and Detection of the Excited State; Lamola, A. A., Ed.; Marcel Dekker: New York, 1971; Chapter 5. Demas, J. N. Excited State Lifetime Measurements; Academic: New York, 1983; Chapter 2. Lakowicz, J. R. Principles of Fluorescence Spectroscopy: Plenum: New York, 1983; Chapter 3. Badea, M. G.; Brand, L. Methods Enzymol. 1971, 6 1 , 378. Birks, J. B. Photophysics of Aromatic Molecules ; Wiley-Interscience: New York, 1970. Bollinger, L. M.; Thomas, G. E. Rev. Sci. Instrum. 1961, 32, 1044. Cline Love, L. J.; Upton, L. M.; Ritter, A. W. Anal. Chem. 1978, 5 0 , 2059.
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RECEIVED for review May 12, 1986. Accepted August 18,1986. Supported in part by the Office of Naval Research, by the National Science Foundation through Grant CHE 83-20053, and by the Upjohn Co.
