Two-Photon Excited Molecular Fluorescence in Optically Dense Media M. J. Wirth and F. E. Lytle" Department of Chemistry, Purdue University, West Lafayette, Indiana 47907
Two-photon absorption is examined as a method of fluorescence excltation. The resultant emlssion Is used directly to quantitate molecular specles without correcting for the presence of a varlable matrix absorbance. Addltionally, bulk excitation Is achieved for solutions prepared with optically dense solvents. I n all cases, h e a r callbratlon curves are obtalned from submlcromolar levels up to the saturatlon concentration.
T h e application of fluorimetry as an analytical technique is limited in many cases by the spectroscopic properties of the sample environment. When a significant fraction of the incident radiation is absorbed by the matrix, the measured fluorescence intensity ceases to be a simple function of the fluorophor concentration. This problem has recently been addressed by Holland e t al. ( I ) . who have corrected the emission with a simultaneously measured value of solution absorbance. Such a scheme was shown to be valid for optical densities 5 2. Often, however, the analyte is foilnd in a more highly absorbing medium where conventional techniques are very unreliable and such correction procedures can be difficult. This paper demonstrates that the technique of two-photon excited fluorimetry can be used to quantitab molecular species in the presence of a high and/or varying matrix absorbence. Two-photon absorption occurs by a nonlinear process in which two photons simultaneously interact with a molecule to induce a transition (2). T h e energy of the resultant excited state is equal to the sum of the energies of the incident photons. This spectroscopic process obeys an absorption law that is similar t o that for one photon For small values, the fraction of light absorbed can be determined by
APjP = [6P I A ] 1C where P is the incident cpticsl power, A I ' is the change in power due to absorption, 6 is the two-photon absorptivity, 1 is the path length, C is the concentration and A is the transverse area of the incident beam. A typical value for 6 is cm4 s photon-' molecule-' ( 2 ) ,which becomes lo-'' L cm mo1-l Watt-' for molar concentrations and 333-nm radiation. T h e laser system used in this study could generate a 1-kW pulse and focus it t o a 10-Fm spot. Thus, the 6 P i A term would be lo-* L mol-' cm-' which is six or seven orders of magnitude smaller than the corresponding one-photoc absorptivity. In two-photon excited fluorescence, the emission intensity is proportional to 9.From Equation 1, it can then be seen that the signal strength is proportional to the concentration and path length as in Beer's law, while in addition, i t is proportional t o the square of the excitation power and the reciprocal of the beam area. A diagrammatic representation of two-photon excitation in optically dense media is shown in Figure 1. T o succeed as a n analytical method, several criteria must be met. First, the excitation frequency m a t be chosen to equal one-half that of an allowed transition. Second, the matrix must not absorb the excitation frequency by a one-photon process. And third, 2054
ANALYTICAL CHEMISTRY, VOL. 49, NO. 13, NOVEMBER 1977
a reasonable fraction of the resultant fluorescence should occur in transparent regicns of the sample. Under these restrictions, the intensity of the excitation beam is never significantly decreased by either one- or two-photon absorption processes. As a result one would expect to obtain linear calibration graphs from the lower limit of detection u p to a saturated solution. In this work, the excitation source is a cavity-dumped, synchronously pumped CW dye laser. Such a device combines the attractive features of tunable wave!ength; high, reproducible peak power; and low pulse energy. T h e sample solutions investigated are 2,5-diphenyl oxazole in acetone and either p-terphenyl in toluene or p-terphenyl plus bipyridine in cyclohexane.
EXPERIMENTAL Chemicals. The toluene used was Mallinckrodt AR grade; the cyclohexane was Phillips bulk grade. The ZJ-diphenyl oxazole (PPO) and the p-terphenyl were Eastman scintillation grade; the 2-2'-bipyridine was obtained from Aldrich. The bipyridine was recrystallized from acetone by changing the solvent polarity; all other chemicals were used without further purification. Single-Photon Instrumentation. All absorption measurements were made with a GCA/McPherson Model EU-700 spectrophotometer. Ail fluorescence measurements were made with a Spex Fluorolog equipped with a 150-W xenon lamp and a photon courting subsystem. Attempts to use the more common AminceBowmm in the front surface configuration were thwarted hy high levels of scattered, stray rzdiation. Two-Photon Instrumentation. The two-photon excitation of fluorophors was accomplished by the use of a synchronously pumped CW dye laser of the type described by Harris et al. (3). Using rhodamine 6G excited by a 400-mW train of mode-locked 514.5-nm radiation, the dye laser produced 15-nJ pulses at 600 nm for rates as h;gh as 500 kHz without loss of peak power. The tuning range of 56W54 nm was limited by the free spectral range of the Lyot filter. With similar excitation, sodium fluorescein produced 4-nJ pulses at 545 nm and had a tuning range of 530-573 nm. The use of other dyes has already extended this range as lcw as 515 nm (3) and as high as 668 nm ( 4 ) . Although the pulse width of the mode-locked dye laser was not determined in this study, the previously published values of 3 ps ( 5 ) ,7.5 ps ( 4 ) and 10 ps ( 6 ) yield 600-nm peak powers of 5 kW, 2 kW, and 1.5 kW, respectively. The emission intensity was optimized by focusing the laser beam into a 1-cm quartz fluorimeter cell via a 3.5-cm focal length lens. Excitation of PPO was accomplished with rhodamine 6G as the dye and the resultant emission was isolated by a Corning 7-59 filter. Excitation of p-terphenyl was accomplished with fluorescein and the emission isolated by a Corning 7-54 filter. The detector was an RCA 931A photomultiplier operating in a dc mode. The high voltage and current amplification were provided by a Pacific Photometric Model 124 photometer. Power-Squared Measurements. As previously mentioned, two-photon excited fluorescence increases with the square of the power, whereas one-photon excitation is a linear function. In most instruments utilizing pulsed lasers, the peak power of every individual excitation has to be measured and used t o correct the data. Because of the excellent pulse-to-pulse reproducibility of mode-locked lasers, a simple correction for fluctuations in average power was found tc be quite sufficient. In addition, for all of the studies a check was made t o ensure that the monitored emission had the expected quadratic relationship. This was accomplished
S , (u) s o lvent
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350
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W a v e l e n g t h (nrnl
SAg) Diagram for two-photon absorption in an optically dense solvent. Two-photon excitation accesses the S2(9) molecular state, which has twice the energy of each photon. T h e molecule internally converts to the S, (u) state, from which it subsequently emits Flgure 1.
Flgure 2. Spectral data from p-terphenyl and bipyridine. (a) Excitation and (b) emission spectrum of p-terphenyl; (c) absorption spectrum of bipyridine. The broken line indicates the UV cut-off of the solvent toluene from an absorbance of 0 to 5
by inserting a rotatable polarizer into the beam and varying the intensity incident on the sample.
RESULTS AND DISCUSSION Interfering Chromophors. A common problem in fluorimetric methods is the presence of one or more chromophores whose absorption bands coincide with those of the analyte. The observed fluorescence intensity is then described by the equation Fobxi a
Af + A ,
(1- T)
where A f and A , are the absorbances of the fluorophor and chromophore, respectively, and T is the total solution transmittance ( I ) . Thus, if the total solution absorbance is small, the species have an independent response and the emission intensity is related only to the fluorophor concentration. At high optical densities, interfering chromophores reduce the amount of radiation exciting the fluorophor by the ratio of its absorbance to the total solution value. In addition, the resulting inner filter effect alters the intensity a t different distances along the cell path length. Under these circumstances, the fluorescence is no longer simply proportional to concentration and may not even be observable at the center of the sample. Unlike pure substances, diluting the solution is not generally a practical approach to reducing the problem. Often the signal would fall below the lower limit of detection or the nature of the fluorophor could change because of a shifted chemical equilibrium. This type of interference was studied by using a model system with p-terphenyl as the fluorophor, bipyridine as the chromophore, and cyclohexane as the solvent. The pertinent spectral data are shown in Figure 2 and the interference data in Figure 3. For a fixed amount of p-terphenyl and a varying bipyridine concentration, the one-photon excited fluorescence intensity varies with the absorbance ratio as predicted. The apparent discrepancy a t low values is due to the instrumental collection efficiency changing with solution transmission. From the experimental results presented, it can be seen that with unknown levels of chromophore, the fluorophor concentration information is lost. In two-photon excited molecular fluorescence, the inner filter effect is nonexistent because the wavelength of the incident radiation is in a transparent spectral region of the solution, and the amount of power extracted by any nonlinear process is negligible. Thus, the intensity of the beam should be constant and
e-------
I 0
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4 Bipyridine
I
I ? -
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Absorban:e
Fluorescence intensity of p-terphenyl as a function of chromophor absorbance. Intensity resulting from (a) two-photon and (b) one-photon excitation. The broken line indicates the theoretically predicted intensity Figure 3.
uniform throughout the sample. The results shown in Figure 3 demonstrate that the intensity due to two-photon excited p-terphenyl is not affected by the variation in bipyridine concentration. It should be noted that the presence of chromophores absorbing the emitted radiation will interfere with the analysis regardless of the method of excitation. Reabsorption of fluorescence is a problem in the emission domain of fluorescence spectrometry and cannot be eliminated by improvements in the excitation domain as long as the same final excited state is populated. T h e self-absorption effect is simplified in the two-photon case for two reasons. First, the penetration depth of the incident beam is constant so that the relationship between concentration and fluorescence intensity is much simpler than the one-photon case. And second, the tight focus of the beam allows the sample to be excited very close to the cell wall, minimizing the path length of the emission through the solution. Optically Dense Solvents. An important special case of an interfering chromophore is the optically dense solvent. In single-photon excitation, i t is imperative that the solvent be transparent where the species of interest absorb. When this restriction precedes chemical considerations of the solvent choice, the analysis is a priori less than optimum. As a worst ANALYTICAL CHEMISTRY, VOL. 49, NO. 13, IYOVEMBER 1977
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PPO/Acetone
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Spectral data for PPO. (a) Excitation and (b) emission spectrum of PPO. The broken line indicates the UV c u t 4 for the sobent acetone from an absorbance of 0 to 5 Figure 4.
case, the fluorophor is often initially in, and cannot be removed from, an optically dense matrix. The spectral data for a model system, PPO in acetone, are shown in Figure 4. Visual inspection of solutions of this compound indicates that very little of the exciting radiation makes it to the center of the cell. As a result, a front-surface configuration is mandatory for single-photon excitation. Even with such an optical arrangement, sensitivity is decreased by the numerical value of the solvent absorption. For this system, the front surface emission intensity in acetone was 250 times less than that in ethanol, exactly as predicted. Two-photon excitation completely penetrates the sample and a right angle configuration can be utilized. In addition, the intensity is the same in both ethanol and acetone solutions. Another system, shown in Figure 2, is p-terphenyl dissolved in toluene. This is a n important model because benzenoid fluorophors are quite often found in benzenoid solvents. Compounds of this type tend to absorb in the same spectral region, hence are difficult to excite. Fortunately, most of the fluorescent benzene derivatives exhibit a large Stokes shift which displaces the emission into transparent regions of the solvent. Figure 5 shows the fluorescence calibration curves obtained for the model systems discussed above. With the laser source used in this study, the detection limit ( S I N = 1) for twophoton excitation of PPO in acetone is approximately 0.5 pM and t h a t for p-terphenyl in toluene is 0.2 pM. These results are primarily determined by the peak power of the source, which itself can certainly be improved by a t least an order of magnitude. The one-photon detection limits are approximately the same as those for the two-photon case. Although the one-photon values can conceivable be lowered, they are ultimately limited by the amount of scattered excitation entering the emission monochromator. This is an inherent problem in that the excitation and emission frequencies are usually close to each other. Naturally, the front surface arrangement maximizes the difficulty. There are no a priori reasons to assume that an increase in source power will improve the detection limit, because the background noise is increased proportionally. Again, two-photon excitation minimizes this problem since the frequencies are usually far from each other and a right angle observation can be used. Even though source fluctuations are amplified by the square of the power in two-photon cases, the precision of the method for samples well above the detection limit is still better than that for one-photon excitation. In the latter case, since the signal is measured in the presence of a large scatter background, any source fluctuation changes both the signal and 2056
ANALYTICAL CHEMISTRY, VOL. 49, NO. 13, NOVEMBER 1977
0
25 p-terphenyl / Toluene
50 ( p M )
Figure 5. Calibration graphs for fluorophorsin optically dense solvents. (a)Data for twc-photon excited fluorescence; (bj one-photon calibrations of p-terphenyl in toluene; and (c) PPO in acetone. T h e slopes for all curves were normalized to the same value at infinite dilution
the background, leading to reduced precision. This problem was exemplified in our attempts to obtain fluorescence measurements using an Amino-Bowman spectrofluorimeter. The single grating emission monochromator passed sufficient scatter to yield a background many times larger than the signal. The fluctuations in the background, minimized by a magnetic arc stabilizer, were comparable to the actual signals even a t the most favorable concentrations. One-photon measurements in optically dense media could only be made with a more sophisticated device, the Spex Fluorolog, which employs a double monochromator specifically designed to eliminate scatter. The residual amount of background was still high enough to prevent the precision from reaching that of the two-photon measurements. It is evident from Figure 5 that the one-photon curves exhibit the well-known inner filter roll-off a t high concentrations. The linearity in any fluorescence calibration fails as soon as the emitter absorbance becomes sufficiently large (1 - 7'). In as to break down the approximation that A optically dense solvents this roll-off occurs a t concentrations several hundred times higher than in transparent solvents because of the shorter effective path length. It should be noted that the linear range isn't really extended by the increased roll-off concentration because the lower limit of detection is simultaneously raised by the same factor. Fcr two-photon excitation, the total absorption is so small that there is no inner filter effect operating and, therefore, no curvature in the calibration graph. Thus, for fluorophors in optically dense matrices, this method produces the largest linear range. CONCLUSION Two-photon excited molecular fluorescence has been shown t o be a useful analytical tool for probing fluorophor concentrations in the presence of other absorbing species. Variations in chromophore concentration that dramatically alter emission intensities for one-photon excited fluorescence have no effect in the nonlinear case. In adciition, because the
-
incident radiation is not attenuated by absorbers, there is no inner-filter effect and the measured emission intensity is directly proportional to analyte concentration over an extended range. Although the sensitivity of the method is normally less than that for one-photon excitation, experience has shown that in optically dense matrices, the detection limits can quite often be better. This fact is due to the extremely large scatter blank normally present in single monochromator instruments when front-surface excitation is employed. Because of the large sepaiation of excitation and emission wavelengths and the ability to use a right angle configuration, the blank for two-photon excitation is generally dark current limited. Another advantage of two-photon excitation in absorbing matrices is the ablity to bulk excite the sample. Front surface methods risk interference from fluorophors adsorbed onto the cell wall or emission from the cuvette itself. Also, the bulk excitation capability is highly important for solid state analyses where the surface concentration may not be representative of the entire sample. The synchronously-pumped laser has not, to our knowledge, been reported as a source for two-photon spectroscopy. The possible range of wavelengths with an argon-ion pump laser extends from 420-690 nm, allowing study of a wide variety of molecular systems. Cavity-dumped operation grants additional flexibility in making the system well suited to time-resolved spectroscopy and synchronous photon counting ( 3 ) . Problems common to other pulsed dye laser sources are
substantial pulse height variations and large, potentially destructive, beam energies. The synchronously-pumped laser, however, is characterized by excellent pulse-to-pulse reproducibility via mode-locking. The picosecond duration allows peak powers sufficiently large to induce two-photon absorption, yet maintains the beam energy a t levels sufficiently low to avoid thermal effects in the sample. Finally, because of the scarcity of continuously tunable ultraviolet sources, it is advantageous that two-photon excitation accesses states in this region while minimizing problems with photolytic degradation of the sample.
LITERATURE CITED (1) J. F. Holland, h. E. Teets. P. M. Kellev. and A. Timnick, Anal. Chem.. 49, 706 (1977). (2) W. M. McClain, Acc. Chem. Res., 7, 129 (1974). (3) J. M. Harris, L. M. Gray, M. J. Pelletier, and F. E. Lytle, Mol. Photochem., 8 12). 161 11977). (4) J 'DeVries: D Bebelaar and J Langelaar, Abstracts, Ninth Biennial International Quantum Electronics Conference, Amsterdam, June 1976, Opt. Commun., 18, 24 (1976). (5) C. K . Chan and S. 0. Sari, Appl. Phys. Lett., 25, 403 (1974). (6) N. J. Frigo, T. Daly, and H. Mahr, I E E E J . Quantum. Electron., 13, 101 (1977).
RECEIVED for review June 28,1977. Accepted August 15,1977. This research was supported in part through funds provided by the National Science Foundation under Grant MPS7505907. M.J.W. gratefully acknowledges the American Association of University Women for fellowship support.
Photothermal Spectroscopy George H. Brilmyer, Akira Fujishirna, K. S. V. Santhanam, and Allen J. Bard* Department of Chemistty, The University of Texas at Austin, Austin, Texas 78712
Photothermal spectroscopy (PTS) involves the use of thermistors in contact with the sample to determine the spectral response of highly absorbing samples. The technique was evaluated with crystalline solids (CdS and Ti02)and solutions of the dyes rose bengal, methylene blue, and aniline yellow. The instrumental and cell deslgns sultable for solid and liquid samples are described and suggestions are made for further Improvements in the techniques.
There has been much recent interest in the use of thermal detection techniques for the spectroscopic examination of different types of samples ( 1 4 ) . For example, photoacoustic spectroscopy (PAS) (1-4) involves the detection with a sensitive microphone of the pressure fluctuations in a gas arising from heat produced by the absorption of radiation from a modulated light beam. This technique has the advantage of not requiring optical detection of transmitted or reflected light, and it can be applied to samples which are difficult to examine by cmventional spectroscopic methods. Thermal detection methods also have been suggested for the determination of absolute quantum yields by employing calorimevic techniques which are free from the geometrical correction problems of optical methods (5-8). These have also frequently employed microphone detectors (6, 8) or the
monitoring of volume expansion ( 7 ) . While PAS and related techniques have been used quite successfully, they require sample placement in sealed cells and utilize rather expensive, highly sensitive microphones which can be troubled by external acoustic noise. Moreover, PAS has, so far, not proved useful for spectroscopic studies of the solid/liquid interface. We thought it worthwhile to investigate the direct detection of the temperature changes resulting from radiationless processes occurring following light absorption, using a thermistor detector during irradiation of the sample with a high intensity source. Thermistors have been employed extensively for calorimetric and analytical measurements, such as thermometric titrations (9, 10). They have also found application to studies of electrode reactions (11-13) and for enzyme reactions at electrode surfaces (14). The technique we propose, photothermal spectroscopy (PTS),involves placing a thermistor in close proximity to the sample (solid or liquid) and measuring temperature changes (Le., thermistor resistance changes) during sample irradiation with high intensity monochromatic light. This technique is similar to PAS, in that the radiationless processes following light absorption are detected, but it allows a more flexible sample cell arrangement and is free of acoustic noise problems. PTS should be especially sensitive for samples of high molar absorptivity (4 such as solids or optically dense liquids, where a large fraction of the impinging radiation is absorbed. PTS ANALYTICAL CHEMISTRY, VOL. 49, NO. 13, NOVEMBER 1977
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