trace-level quantitation via time-resolved two-photon-excited fluorescence

Trace-Level Quantitation Via Time-Resolved Two-Photon-Excited Fluorescence

FRED E. LYTLE,* DEBORA M. DINKEL, t and WALTER G. FISHER1: Department of Chemistry, 1393 Brown Laboratories, Purdue University, West La[ayette, Indiana 47907-1393

With the use of a cavity-dumped, synchronously pumped dye laser for excitation, two-photon fluorescence cross sections are approximately eight orders of magnitude smaller than those for one-photon excitation. Thus, examination of dilute solutions has been achieved only with great dif- ficulty. Any successful instrumentation will require that the blank be essentially eliminated. To this end, time-filtered detection has been combined with two-photon excitation and spatial filtering to produce fluo- rometric detection limits of 38 pM for 9,10-diphenylanthracene and 8,6 pM for a-NPO. It is believed that this latter value is the lowest concentration yet reported for two-photon spectroscopy in fluid solution. The instrumentation and data processing are described. Additionally, a comparison is made with the performance of other recent alternative approaches involving spatial filtering and second harmonic detection. Index Headings: Fluorescence; Instrumentation, two-photon; Lasers; Time-resolved spectroscopy.

INTRODUCTION

Two-photon excitation of an electronic state is achieved via the third-order susceptibility. As such, the transition strength is very weak and displays a power-squared de- pendence2 The usual quantitative description of the re- lationship between signal strength and concentration places one of the power terms within an instrument- dependent cross section. 2 To this end, the cavity-dumped, synchronously pumped dye laser is an unusually good source for two-photon excitation. Dumping at 2 MHz can easily generate a 160-mW beam of 17-ps pulses. These parameters yield a peak power of ~4.7 kW and a pulse energy of ~80 nJ. The peak power contributes to the magnitude of the cross-section/pathlength product, producing values near 4 × 10 -25 cm 3 (typical one-photon values are near 10 -17 cm 3 for a 1-cm cell). The average power contributes to the magnitude of the signal, in the usual manner, via the sample absorptance, (1 - T). Ad- ditionally, the temporal delay between pulses is sufficiently long to ensure that the majority of the excited- state energy decays before the next excitation event.

In the absence of unwanted nonlinear processes, it would appear that the blank for two-photon spectroscopy should be near zero. This expectation is due to the ex- treme anti-Stokes nature of the process; i.e., the sample is excited in the red, and the fluorescence observed in the violet to blue, spectral region. In actuality the blank is significant, being composed primarily of Rayleigh and/ or Mie scatter. Such interference is of sufficient intensity to survive virtually all attempts at wavelength discrim-

Received 23 June 1993. * Author to whom correspondence should be sent. t Current address: Conoco Inc., Ponca City, OK 74603. :~ Current address: Oak Ridge National Laboratory, Oak Ridge, TN

37831-6113.

ination, including multiple filters and monochromators. In contrast, none of our studies have ever indicated that one-photon-excited fluorescence is a significant component of the blank. This observation appears to be due simply to the large magnitude of elastic scatter cross sections compared to those for fluorescence.

Within the last three years, instruments employing spatial isolation, 3 second-harmonic de tec t ion /and spatial filtering combined with second-harmonic detection 5 have pushed chemical detection limits into the hundred picomolar domain. Such achievements were made pos- sible by combining spectral and nonspectral methods of rejecting scattered laser radiation. In all three approaches, the synchronously pumped dye lasers were operated in a "cw" mode, producing an 82-MHz train of pulses. The high repetition rate had the undesired effect of producing lower peak power, which decreases the cross section. As a result, the source contribution to the sensitivity of the instrumentation was about a factor of eight below that predicted above for the cavity-dumped variant op- erating at 2 MHz (down 16 due to decreased peak power, up 2 due to increased average power). The above discussion indicates that the best results should have been obtained with the cavity-dumped laser. Unfortunately, second-harmonic detection was, at that time, not com- patible with cavity-dumping at 2 MHz, due to the lack of a suitable extra-cavity modulator. In a procedure to test the hypothesis that increased peak power would ira- prove sensitivity, the entire instrument was, instead, re- configured to employ t ime-correlated single-photon counting combined with software temporal filtering of the decaysY The performance of this scheme for de- tecting two-photon excited fluorescence is described, along with a comparison to that obtained by second- harmonic detection. Data will be shown that demonstrate detection limits of 38 pM for 9,10-diphenylanthracene (DPA) and 8.6 pM for 2-(l-naphthyl)-5-phenyloxazole (a-NP0). It is believed that this latter value is the lowest concentration yet reported for two-photon spectroscopy in fluid solution. Variations of the experiment will also be discussed that should provide improved detection limits.

EXPERIMENTAL

Instrumentation. A detailed description of the time- correlated, single-photon counting instrument can be found in Ref. 7. A cavity-dumped dye laser system (Spec- tra-Physics Model 375B dye laser and Model 344 cavity dumper) was synchronously pumped with a Spectra- Physics Nd:YAG laser, mode-locked at 82 MHz. In this experiment, the detector was a thermoelectrically cooled (Products For Research, Inc.) RCA 8850 photomultiplier

2002 Volume 47, Number 12, 1993 0003-7028/93/4712-200252.00/0 APPLIED SPECTROSCOPY © 1993 Society for Applied Spectroscopy

biased at -2150 V and operated in the single-photon- counting mode. The output of the photomultiplier was connected to a constant-fraction discriminator (Tenne- lec 455). A fast photodiode (Texas Instruments TIED 56) biased at a potential of -125 V and wired for a risetime of about 200 ps 8 was employed to provide a trigger coincident with the excitation of the sample. The photodiode was irradiated with part of the excitation beam.

Optics. A detailed description of the sample chamber optics can be found in Ref. 5. Briefly, a 10 × microscope objective (NA = 0.30) was used to focus the laser to a diffraction-limited beam waist inside the sample cell, while a second 10× microscope objective (NA = 0.25) collected the two-photon-induced fluorescence. The blue fluorescence was isolated from the red laser scatter by a filter combination comprised of a Corning 7-59 glass filter and a Corion LS-500 cut-off interference filter. A red- absorbing glass filter, tilted at Brewster's angle, was used as a beam stop. The residual beam reflecting off the filter was allowed to exit completely from the sample chamber. Without this feature, scattered laser radiation so dominated the measurement that sub-nanomolar concentra- tions could not be quantified.

Signal Processing. In the reverse-noninteractive configuration, the start input of the time-to-amplitude con- verter (TAC) comes from the constant-fraction discriminator, while the stop is provided by the leading-edge discriminator, which is triggered by the photodiode. 9 The repetition rate of the cavity dumper was adjusted to 2 MHz, which corresponds to the maximum signal-to-blank ratio (see Appendix). This configuration routinely provides 160 mW of power in 17-ps pulses. The TAC output pulse possesses an amplitude proportional to the time between input events. This signal is analog-to-digital (A/ D) converted and stored in the channel corresponding to its amplitude value by a 1024-channel multichannel analyzer (MCA) operated in the pulse height analysis mode (Nuclear Data ND62 Multichannel Analyzer). Af- ter collection of the decay obtained from the blank or the sample, the contents of the MCA were transferred to an IBM PS/2 Model 50 with the use of the serial port.

The signal-to-noise ratio was optimized by use of a software algorithm to implement time-filtered detection. The quantitative advantage of time-filtered detection arises from the ability to distinguish between blank and analyte signals, thereby improving the signal-to-noise ratio and decreasing the detection limits of fluorescence. This strategy has been described in detail elsewhere. 6,1° The placement and width of the signal acceptance win- dow (temporal filter) are constructed so as to maximize the number of signal counts while concomitantly reduc- ing the blank below a level where its photons dominate the variance. This optimum occurs very near the temporal position where the percentage rate of change in the blank decay equals that of the signal. The exact placement is determined by an exhaustive computation uti- lizing trial windows having every physically significant placement and width. The signal-to-noise (S/N) ratio is computed by the propagation of counting error due to subtracting the blank from the total.

Chemicals. c~-NPO was obtained from Eastman Ko- dak; DPA was from Aldrich. Both compounds were used

120

oo. t/ -~ 8 0 a

60- c o 0 40- b

2O

0 . . d , . . . . . . . . , . ,.,.. _ . . , . , . L , , , , , , , , _ _ ~ , , . . . . . , L t , . u .~, - - . L . .

0 5 10 15 20 25 30 35 40

Time (ns)

FIG. 1. Fluorescence decays of e-NPO (103 pM, S/N = 36) and cyclohexane blank: (a) signal; (b) blank. Integration time, 1 min; TAC range, 100 ns; repetition rate, 2 MHz; excitation wavelength, 590 nm; filters, Corion LS-500 and Corning 7-59.

as purchased. C yclohexane was from Burdick and Jack- son. The laser dye, Rhodamine 6G (565-615 nm), was purchased from Exciton.

RESULTS AND DISCUSSION

Limits of Detection. In order to evaluate quantitative, time-resolved two-photon spectroscopy, calibration curves were obtained for a-NPO and DPA. These two compounds were chosen on the basis of disparate lifetimes, a-NPO has a measured value of 1.8 ns, which is too short for temporal resolution to contribute signifi- cantly to the detection limit with the use of the current instrumentation. In contrast, DPA has a lifetime of 5.6 ns, which permits modest separation from scatter. Op- timal performance of the temporal filter would be obtained with lifetimes larger than 10 ns. 6 At the wavelength of excitation, 590 nm, DPA appears to have a two- photon cross section which is 6-7 times smaller than that of c~-NPO. This observation is based on detection limits obtained by the second-harmonic technique. 5 Since the detection optics were fixed and the two fluorescence spectra have maxima separated by 30 nm, a more precise statement is impossible.

A log-log plot of the c~-NPO calibration data yields a slope of 0.92 ± 0.01 with a correlation coefficient of 0.9993 for seven points extending from i × 10 -l° M to 5 x 10 -8 M. The extrapolated detection limit (S/N = 3) is 8.6 pM. Each data point was obtained by a 1-min integration time. The quality of the data at low analyte concentration is demonstrated by the decay obtained for a 103-pM solution. This signal, along with the cyclohexane blank, is displayed in Fig. 1. After processing with the temporal filtering software, these data yield an S/N of 36 (including blank subtraction). It should be noted that the leading edge of the filter did not move off the peak of the decay. This observation indicates that the computation yielded results equivalent to a steady-state measurement. 6 In an effort to lend confidence to the assumption that the data were indeed being produced by a-NPO, a lifetime of 1.1 ns was computed from the decay. Although the value is shorter than 1.8 ns, this result is expected because of bias due to scattered light.

A P P L I E D S P E C T R O S C O P Y 2 0 0 3

240

200 a)

160

E 120

O

80 b) signal

4o ¢ 212 0 ~ - i-- , -~ -, . . . . . . , " ; " ~ ' . ~ , - . . . .

15 17 19 21 23 25 27 29 31 ' 33 35

T i m e (ns)

FIG. 2. Fluorescence decays of 9,10-diphenylanthracene (110 pM) and cyclohexane blank: (a) 3-min integration time, S/N = 19; (b) l-rain integration time, S/N = 8.6. TAC range, 100 ns; repetition rate, 2 MHz; excitation wavelength, 590 nm; filters, Corion LS-500 and Coming 7-59.

A log-log plot of the DPA calibration data yields a slope of 0.98 ± 0.01 with a correlation coefficient of 0.9995 for seven points extending from 1.1 × 10 -t° M to 1 × 10 -7 M. The extrapolated detection limit (S/N = 3) is 38 pM. Each point was obtained by a 1-min integration time. The quality of the data at low analyte concentration is demonstrated by the decay obtained for a l l0 -pM solution with both 1-min and 3-min integration times. These signals, along with the cyclohexane blanks, are displayed in Fig. 2. After processing with the temporal filtering software, these data yield an S/N of 8.6 for the 1-min integration and 19 for the 3-min integration. For this fluorophore, the leading edge of the filter moved 0.32 ns toward later times in order to maximize the S/N. Optimum filter placement resulted in a 25% improve- ment in S/N with 5.5% of the signal and 17.5% of the blank being dropped from the calculation. Neither of these decays could be used to compute a reliable lifetime due to the low number of counts. However, a 500-pM solution produced a value of 4.5 ns. Again the shorter value is due to bias from scattered light.

Comparison to Second-Harmonic Detection. As stated in the Introduction, the primary objective of this study was to test the hypothesis that cavity dumping would increase the sensitivity, and thus lower the limit of detection, for two-photon-excited fluorometry. The second- harmonic instrument used a laser producing an average power of 150 mW and an 82-MHz modulated train of pulses with an average width of 12 ps. 5 The average power divided by the repetition rate gives an average pulse energy of 1.8 nJ (remember, the source is modulated). The pulse energy divided by the pulse width gives an average peak power of 152 W. Thus the peak power/ average power product is 23 W 2. The laser system used in this study produced an average power of 160 mW and a 2-MHz train of pulses with a width of 17 ps. With an identical calculation, the peak power/average power product becomes 753 W 2. Thus cavity dumping (without internal modulation) should increase the sensitivity by a factor of 33. Comparison of the data in this report with those obtained by second-harmonic detection produces the following results: 5 DPA has a second harmonic-to-

temporal resolution detection limit ratio of 5.98 nM/38 pM = 157, while c~-NPO has a ratio of 900 pM/8.6 pM = 105. Both of these enhancements exceed the above predicted factor of 33.

At this point it is necessary to consider the mathe- matics of second-harmonic detection. The laser, when modulated, has the functional form of an offset cosine. One-photon processes then produce dc and first-harmonic components. On the other hand, two-photon processes square the offset cosine producing dc, first-harmonic, and second-harmonic components. A detailed trigonometric derivation demonstrates that the desired second-harmonic component is, at best, 12.5% of the total signal. 4,~ Thus, detection by photon counting should increase the sensitivity by another factor of eight. This approach predicts a total enhancement of 264. Although difficult to compare, the differences in extent of signal averaging between the two techniques appear to be sim- ilar and are not included in this performance analysis.

For both chemical systems studied, time-correlated single-photon counting provides enhancements which are lower than estimated. This result must be due to the inability of time-filtered detection to provide adequate rejection of scattered light for short fluorescence lifetimes. That is, DPA (5.6 ns), which is amenable to some degree of temporal filtering, is off by a factor of 1.7. On the other hand, a-NPO (1.8 ns), which is not amenable to temporal filtering, is off by a factor of 2.5.

Instrumental Improvements. The incredible increase in performance produced by time-resolved detection of two- photon excitation came as a pleasant surprise. However, this generation of the instrument is hampered by our inability to find compounds that possess both long fluorescence lifetimes and large two-photon cross sections. To this end, we are in the process of replacing the RCA 8850 photomultiplier with a channel plate detector. The result should be a reduction in the impulse response of the instrument from ~960 ps to ~90 ps. This improve- ment will facilitate time-filtered detection with short- lived fluorophores, and increase the number of compounds that can be quantified at these low levels. The predicted lower limits of detection would then be 22 pM for DPA and 3.4 pM for c~-NPO.

A longer-term modification would involve replacing the Nd:YAG pump with a frequency-doubled, femtosec- ond, Ti:sapphire laser. Unpublished studies have shown that dye lasers synchronously pumped in this manner can produce 1-ps pulses even when combined with cavity dumping. 11 This change will increase the peak power, and thus the sensitivity, by a factor of 17 above that of the laser used in these studies. With both modifications, it appears that two-photon-excited fluorescence may ac- tually be capable of producing fluorescence detection limits comparable to those achieved by one-photon excitation. As an example, a-NPO should have a value near 0.2 pM. This value is identical to the lowest concentra- t ion-0 .18 pM (S/N = 2) for rubrene in a 1-cm cell-- reported by any member of this research group. 1°

APPENDIX

Optimizing Sensitivity. The peak power, pulse energy, and average power of a cavity-dumped, synchronously

2004 V o l u m e 4 7 , N u m b e r 1 2 , 1 9 9 3

20 250

200 ~" E

150 o 13.

100

50 <

18

~ " 16 o..

"~ 14

~ 12

a. 1o

& & 1'o Laser Repetition Rate (MHz)

0 0 12

FIo. 3. Performance of the cavity-dumped dye laser. Pulse width (O) and average power (D) vs. laser repetition rate.

7OO

"~ 6 0 0

~_ 500- g

4 0 0

< 300- x

200- g_

I 00 "

(3_

FIG. 4. Fig. 3.

i i i i i 2 4 6 8 10 12

Laser Repetition Rate (MHz)

Peak power/average power product derived from the data in

pumped dye laser are all a function of the repetition rate. Figure 3 shows this dependency for the average power. It initially increases linearly because the cavity energy reaches a steady state in a time shorter than the recip- rocal of the repetition rate. Eventually, extracting a pulse from the cavity removes more energy than the optical amplifier can restore before the next pulse is to be dumped. At this point, ~1 MHz, the average power be- gins to flatten. The exact shape of the curve depends upon the nature of the intracavity gains and losses, and thus needs to be determined for each type of laser. As an example, the recovery time is near a microsecond for an argon-ion laser, and the average power maximizes at repetition rates ~1 MHz. Dye laser recovery rates are near 100 ns and produce an optimum average power ~ 10 MHz. Since the major loss mechanism in dye lasers is probably due to triplet-triplet absorption, the recovery time will also show a strong dependence on the dye and the desired wavelength.

Also shown in Fig. 3 is pulse width vs. the cavity- dumping repetition rate. In a synchronously pumped dye laser, the short pulses are created by mode locking. At repetition rates below ~ 1 MHz, the gain of the dye is so high that it is difficult to lock all the modes of the cavity. This factor produces a broad pulse, as predicted by the Uncertainty Principle. As the repetition rate increases, the single-pass gain drops, and an increasing number of modes are locked in phase. This pattern produces narrow pulses. As can be seen in the figure, the change in pulse width is dramatic at first, but then levels out as the gain of the cavity is reduced to a value where synchronous pumping is locking most of the modes.

The repetition rate that should produce the maximum two-photon signal is that having the largest peak power/ average power product. As stated earlier, the peak power can be mathematically considered part of the "cross section," while the average power contributes to the signal, in the usual manner, via the sample absorptance (1 - T). The data of Fig. 3 were used to compute the peak power/average power product vs. repetition rate. This predictor of performance, shown in Fig. 4, has a broad maximum near 4 MHz.

The instrument based on second-harmonic detection, with which this study is compared, used the cavity dump-

er to both create and modulate an 82-MHz pulse train) The result was 150 mW of modulated average power and 12 ps pulses. The fact that the average power was lower than the plateau in Fig. 3 is due to electronic and acous- tooptic limitations that occur when the Bragg cell is used to both dump and modulate. Because the pulse train is intracavity modulated, the laser gain oscillates between a low and high value. The measured 12 ps is produced by the autocorrelator averaging over the modulation- induced variation in width.

Optimizing the Signal-to-Blank Ratio. The above analysis leads to a suggested repetition rate solely on the basis of maximizing sensitivity. However, what is of greater importance is optimizing the signal-to-blank ratio. As stated earlier, the blank for any two-photon detection scheme is dominated by laser scatter leaking through the spectral and spatial filtering used to isolate the signal. Photon-counting detection produces a second contribution to the blank, dark count, that is not observed in the second-harmonic experiment (due to frequency discrimination).

To achieve the optimum excitation rate predicted by the peak power/average power product shown in Fig. 4, one must operate the TAC in the reverse configuration2 This requirement exacerbates both interferences. To re- duce laser scatter in the forward TAC configuration, one can delay the trigger just enough to prevent this interference from starting the TAC. With the reverse configuration, all photons have an equal chance to trigger the TAC, and sufficiently intense scatter can prevent the fluorescence from being observed. Likewise, dark counts are seldom a major source of the blank when the TAC is operated in the forward configuration. This is because the repetition rate of cavity dumping must be kept at or below the maximum TAC trigger rate of 160 kHz. In the reverse configuration, the duty cycle increases linearly with repetition rate, and reaches 100% when the free temporal range of the TAC is larger than the laser pulse spacing.

Scatter is proportional to the average laser power. Thus, an experiment having a scatter-dominated blank will have a signal-to-blank ratio proportional to the curve of Fig. 4 divided by the average power, i.e., the peak power. The dark count is proportional to the repetition rate. Thus,

APPLIED SPECTROSCOPY 2005

1.1 -

1.0-

~ o.9-

ca o.8-

E ~ 0.7-

0.6-

'~ 0.5 N o.4 E 0.3

0.2

0.1

0.0 i i i i

0 4 6 8 10 12

Laser Repetition Rate (MHz)

FIG. 5. Normalized signal-to-blank ratio as a function of laser repetition rate. Scatter (O) and dark count (D) dominated blanks. Calcu- lated ratios derived from data in Figs. 3 and 4.

an experiment having a dark-count-dominated blank will have a signal-to-blank ratio proportional to the curve of Fig. 4 divided by the repetition rate. Both of these cases are plotted in Fig. 5, which indicates that the optimum signal-to-blank ratio will occur at a repetition rate slight-

ly below that producing the maximum sensitivity. The data in this report were taken at the compromise value of 2 MHz.

ACKNOWLEDGMENT

This research was supported in part by the National Science Foun- dation Grant CHE-8822878.

1. M. J. Wirth and F. E. Lytle, in New Applications of Lasers to Chemistry, ACS Symposium Series No. 85, G. M. Hieftje, Ed. (American Chemical Society, Washington, D.C., 1978), pp. 24-49.

2. S. M. Kennedy and F. E. Lytle, Anal. Chem. 58, 2643 (1986). 3. M. J. Wirth and H. O. Fatunmbi, Anal. Chem. 62, 973 (1990). 4. R. G. Freeman, D. L. Gilliland, and F. E. Lytle, Anal. Chem. 62,

2216 (1990). 5. W. G. Fisher and F. E. Lytle, Anal. Chem. 65, 631 (1993). 6. N. K. Seitzinger, K. D. Hughes, and F. E. Lytle, Anal. Chem. 61,

2611 (1989). 7. S. A. Nowak and F. E. Lytle, Appl. Spectrosc. 45, 728 (1991). 8. J. M. Harris, W. T. Barnes, T. H. Gustafson, T. H. Bushaw, and

F. E. Lytle, Rev. Sci. Instrum. 51, 988 (1980). 9. G. R. Haugen, B. W. Walling, and F. E. Lytle, Rev. Sci. Instrum.

50, 64 (1979). 10. G. R. Haugen and F. E. Lytle, Anal. Chem. 53, 1554 (1981). 11. W. Fisher and J. Gord, personal communication (1993).

2006 Volume 47, Number 12, 1993

trace-level quantitation via time-resolved two-photon-excited fluorescence

Documents