Anal. Chem. 1984, 56,2957-2960
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Fluorescence Suppression by Phase-Resolved Modulation Raman Scattering A. Z. Genack Exxon Research and Engineering Company, Clinton Township, Route 22 East, Annandale, New Jersey 08801 Electrooptlc (EO) phase-sensltlve detectlon of emlsslon excited by an Intensity-modulated laser Is used to obtaln fluorescence-free Raman spectra. Phase resolutlon is utlllred to separate Raman scatterlng (RS), whlch Is In phase wlth the modulated laser, from fluorescence whlch lags the excitatlon In phase. The fluorescence background Is nulled by subtractlng spectra detected wlth the fluorescence f90° out of phase with the modulated transmission through the EO modulator. Since the light is demodulated before strlklng the photodetector, an optical multichannel analyzer can be utlk e d . When 30-MHr modulatlon Is used, RS from a cyciohexane-ethanol mixture Is measured in the presence of dlssolved (2,2‘-blpyridlne)ruthenlum dichloride hexahydrate whlch emlts fluorescence wlth an Intensity 30 tlmes larger than the RS. The fluorescence Is completely suppressed, and a dgnal-to-noise ratlo of 20 Is obtained for RS in 50 s wlth 25 mW of 4880-A excltatlon.
Raman scattering (RS) is often swamped by fluorescence from resonantly excited molecules under study or from other sample constituents including impurities. In particular cases, fluorescence may be reduced by judicious choice of exciting frequency, careful sample preparation, or the introduction of fluorescence quenching molecules or surfaces. However, in order to fully exploit resonant RS and to study inhomogeneous systems such as, for example, in vivo biological samples or molecules on dispersed supports, a general means of fluorescence suppression is required. Numerous approaches using both stimulated (1-6) and spontaneous RS (7-13) have been developed to increase the contrast between RS and fluorescence for particular samples. We demonstrate here the use of electrooptic (EO) phase-sensitive detection of emission induced by an intensity modulated laser to null fluorescence. Phase-sensitive detection can separate fluorescence from RS since fluorescence lags the excitation in phase while RS, which is a prompt scattering process, is in phase with the excitation. When extended to higher frequencies, the method of phaseresolved modulation RS can be used to suppress fluorescence even in samples with subnanosecond decay times and to measure ultrashort optical phenomena. Since fluorescence is suppressed by subtracting spectra in which fluorescence is present rather than by enhancing RS or rejecting fluorescence, statistical noise arising from the fluorescence backgrounds needs to be reduced by signal averaging. Long integration times are required in the presence of intense fluorescence. Nonlinear optical techniques such as coherent anti-Stokes RS (CARS) (1,2) or Raman gain spectroscopy (3,4), in which the frequency difference between the exciting lasers is scanned, have yielded fluorescence-free Raman spectra of transparent samples. Interference due to nonresonant terms often complicates the CARS spectrum, but these can be suppressed by using Raman-induced Kerr effect spectroscopy (RIKES) (5, 6). A host of linear methods have also been developed which separate RS from fluorescence on the basis of their different characteristics in the frequency or time domain or in their polarization properties and are applicable even to opaque 0003-2700/84/0356-2957$01.50/0
samples. Modulation of the detected wavelength can separate broad fluorescence from sharper RS features (7). The more effect method of excitation frequency modulation (8)distinguishes between fluorescence and RS in cases where the fluorescence spectrum is broad and independent of excitation frequency since the frequency of RS tracks the excitation frequency. However, the method fails when the absorption varies rapidly with laser frequency or when the fluorescence also tracks the laser frequency. This occurs in fluorescence from small molecules and in “prompt fluorescence” from an unrelaxed excited state of larger molecules as well as in “hot luminescence” (9) from unthermalized electrons in excited bands of solids. A polarization modulation approach based on the differing degree of polarization of fluorescence and RS has proven to be useful in cases where a significant difference in polarization exists (IO). However, since the detection sensitivity depends upon the RS polarization ratio, it varies for each of the RS lines. A number of methods which exploit the different temporal response of RS and fluorescence to pulsed excitation have been developed to suppress fluorescence (11-23). Since RS is prompt, it is detected in a time which is the convolution of the laser pulse width and the detector response time. Fluorescence can be discriminated against if the detection window overlapping the laser pulse is much shorter than the fluorescence decay time. Pulsed laser techniques are most effective in discriminating against fluorescence with lifetimes considerably greater than the response time of photomultiplier tubes of 20.2 ns. When long-lived fluorescence is discriminated against, pulsed methods have an advantage over modulation techniques in that the fluorescence detected is drastically reduced and does not contribute statistical noise of the RS spectrum. A related technique, developed by Nemanich, Solin, and Doehler (14), detects the modulated component of emission from a mechanically chopped laser beam using a lock-in detector following a photomultiplier tube. This method has been used to detect RS from ruby while suppressing fluorescence with a lifetime of 7 = 5 ms. RS only occurs when the beam is chopped on, while the depth of modulation of fluorescence is reduced when the period of chopping is less than 7. The shortest luminescence lifetime that can be suppressed to some degree by this technique is limited by the chopping period of the laser beam and the response time of the photomultiplier tube and lock-in detector. In this work we demonstrated an optical modulation approach in conjunction with EO phase-sensitive demodulation to observe RS in the presence of much more intense fluorescence. An extention of this approach to microwave frequencies will make it possible to suppress fluorescence with lifetimes as short as 50 ps. Shorter lived species have low quantum yields and generally do not contribute significantly to fluorescence.
EXPERIMENTAL SECTION The experimental apparatus is shown schematically in Figure 1. The intensity of a CW laser is modulated by a Pockels cell and polarization analyzer, and the backscattered light is demodulated by a Sears-Debye acoustooptic modulator before entering the spectrometer. The light may be detected in a single 0 1984 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 14, DECEMBER 1984 Sample
Laser EO Modulator
L
P
Emission EO Modulator
Oscillator
w
‘4 Spectrometer
Flgure 1. Schematic illustration of the apparatus for EO phase-sensitive detection. The EO modulator may be a Pockels cell and polarization analyzer or a Sears-Debye acoustooptic modulator followed by a slit. The demodulated light is dispersed in a spectrometer and detector. In our experiment a Spex Triplemate spectrometer and EG&G PAR intensified photodiode array detector are used. The phase shift between the modulators is introduced electronically.
or multichannel detector. Phase-sensitive detection is achieved by varying the phase of the emission modulator relative to the laser excitation. This can be done electronically as shown in Figure 1 or optically by using an optical delay line in the case of highfrequency modulation. The time resolution of this approach is limited by the frequency of the EO modulation and the phaseresolution capability of the instrument and not by the detector rise time. A similar configuration using two microwave Pockels cells could be operated to observe RS in the presence of very short-lived fluorescence. When the angular frequency of modulation of the excitating laser, w, is much less than the off-resonance frequency of the laser or the homogeneous line width, for resonant excitation, the RS intensity is in phase with the laser. On the other hand, a phase lag, 4, given by
tan 4 = U T
(1)
develops between the fluorescence and excitation, and the depth of modulation, rn,given by m = (1 + u
(2) is reduced in analogy with a series integrating circuit with RC = T . The relationship between the phases of the laser modulation, RS, and fluorescence as well as that of the emission modulator used in suppressing fluorescence is shown in Figure 2. To simplify the figure we have illustrated the case for which 4 approaches i ~ / 2rad, the phase shift which is approached in the limit UT a. Spectra are accumulated in two sequences, corresponding to transmission through the emission modulator being in and out of phase with the laser, Ti, and T,,, respectively, in Figure 2. Both the modulated and DC component of the fluorescence are detected equally in the two spectra and do not appear in the difference spectrum. However, the modulated component of RS, which is detected more efficiently with the emission modulator set in phase, is detected. For a sample with a single fluorescence decay time, independent of optical emission frequency, 4 is well-defined and the modulated component of fluorescence is the same in the two sequences for any symmetrical choice of phase shifts about 4. However, the RS is detected differently, and the difference spectrum contains only RS without fluorescence. In a related development, phase-sensitive detection using a photomultiplier tube with modulated gain has been used to resolve individual emission spectra in a mixture of two fluorescent compounds (15). For samples exhibiting multicomponent fluorescent decay, fluorescence may be effectively suppressed by modulating at high frequency such that UT^ >> 1 for each of the fluorescent compo~ T ~ ) - ~ / ~
-
I
t-
Flgure 2. Schematic iliustratlon of the modulated intensity of the laser, I,, RS, I,,, and fluorescence, I,, as well as the transmission function of the emission modulator set in phase, TI,, and out of phase, Tea, with the modulator laser. To simplify the figure the phase shlft of I, relative to I, is taken to approach 6 = d 2 , and sinusoidal modulation is shown. Note that the time average of T,I,, is equal to that of T,In
and that fluorescence is, therefore, nulled when the in- and out-ofphase spectra are subtracted. nents with a lifetime q. In this case the modulated components are reduced by U UT^, and and the phase of fluorescence for all the components approaches 90’. The phase of all the components is nearly in quadrature with the RS and may, therefore, be canceled together. Since light is demodulated by the emission modulator, the time response of the detector is not a factor and slow detection may be utilized. In addition an optical multichannel analyzer (OMA) can be used as well as a single-channel detector. In this experiment the emitted light is dispersed in a Spex Triplemate spectrometer and detected by an EG&G PAR 1420 intensified silicon photoiodide array. The laser modulator is a Lasermetrics 3030 ammonium dihydrogen phosphate Pockels cell. The half-wave voltage for the Pockels cell, which imparts a rotation of 90’ to the polarization of the light wave a t 4880 A, is 96 V. A variable bias is applied to the Pockels cell so that the optical transmission is nulled when the applied modulated voltage passes through zero. The light intensity transmitted through the analyzer is, thus, a maximum when the modulated voltage intantaneously equals the half-wave voltage. In this experiment, the Pockels cell is driven with 1W which was sufficient to produce a peak transmission which is 80% of that achieved by applying the half-wave voltage. The ratio of peak to minimum transmission of incident CW light, when modulated with an rf of 15 MHz, is 40. The bias applied to the Pockels cells is set by varying the applied dc voltage until the negative and positive going parts of the applied rf produced identical modulated transmission. The fundamental modulation frequency of the light wave is, thereby, doubled to 30 MHz. The laser light is deflected by a 90° prism (P in Figure 1) and focused to a 100-pm spot in the sample. The backscattered light is collected and collimated by the same 55-mm focal length, f/1.2 lens which focused the laser beam. The emission modulator is an SLM LM200 Sears-Debye acoustooptic modulator. The scattered light is demagnified by a factor of 4 with a pair of lenses to produce a 1-cm-diameter collimated beam in the Sears-Debye
ANALYTICAL CHEMISTRY, VOL. 56, NO. 14, DECEMBER 1984 Wavetek 178 Synthesizer Svnch.
Function 15 MHz
Doubler
Amplifier
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Matching Network
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FG.504
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Figure 3. Electronic apparatus for modulating laser and emission at 30 MHz. Function of apparatus is described in text.
modulator. In this modulator a standing electrical wave in a water-ethanol mixture produces modulated Bragg scattering out of the main beam. The undeflected central lobe of light traversing the modulator, whose intensity is modulated, is focused into the 200-pm entrace slit of the Spex Triplemate spectrometer. When driven with 10 W at 15 MHz, the ratio of peak to minimum transmission through the Sears-Debye modulator and 200-pm spectrometer slit is 5. The time-averaged transmission of light for the two modulators set in phase is 30% higher than for the modulators set out of phase. The electronic arrangement is shown in Figure 3. The phase delay between the modulators is set by the variable delay of a Hewlett-Packard 8082 pulse generator with a square-wave output. A 180' phase shift between the two sequences is obtained by manually switching into a mode which logically inverts the output of the pulse generator running at 30 MHz. The rf frequency is doubled to 30 MHz to facilitate switching 180' in phase. It is then divided in half in order to drive the Pockels which then produces light modulated at a fundamental frequency of 30 MHz. The frequency division is accomplished by setting a HewlettPackard 1915 pulse generator to produce a pulse slightly longer than the period of the 30-MHz modulation. The rf is phase-locked to the pulse generator output using a Tektronix FG504 frequency generator. The phase delay is adjusted to give nearly equal fluorescence intensity in the two sequences which differ in phase by 180' at the fundamental modulation frequency. For the large depth of modulation utilized in these experiments, harmonics of the fundamental are present in the optical modulation. For single exponential decay, the fluorescence may be nulled by subtracting two spectra with symmetrical shifts of the emission modulator phase about 4. In a subsequent publication we will show that for the case of multiple exponential decay the Raman component can still be isolated when U T >> 1.
RESULTS AND DISCUSSION The use of phase-resolved modulation Raman scattering to reveal a solvent Raman spectrum which is masked by fluorescence from dissolved dye molecules is shown in Figure 4. Figure 4a shows the fluorescence from 3 X M (23bipyridinehthenium dichloride hexahydrate in a 70-30 mixture of cyclohexene and ethanol with the two modulators set nearly in phase. The sample is irradiated with 25 mW of laser light at 4880 A. The slits of the Triplemate spectrometer were set at 200 pm,corresponding to a resolution of 22 cm-l. The decay time is measured to be 150 ns by using a first
M 2000
2400 2800 3200 Frequency ShiR (cm-1)
Figure 4. (a) Fluorescence from 3 X M (2,2'-bipyrMine)ruthenium dichloride hexahydrate in a 70-30 mixture of cyclohexane and ethanol with modulators set in phase. Sample irradiated with 25 mW of 4880-A laser light. (b) Difference between in-phase spectrum shown in (a)and out-of-phase spectrum. Fluorescence is suppressed while RS from CH modes is displayed. (c) In-phase spectrum of neat cyclohexane. (d) Difference between in- and out-of-phase spectra of neat cyclohexane.
photon of arrival fluorescence decay technique (16). Subtracting the out-of-phase spectrum from the in-phase spectrum shown in Figure 4a reveals the CH stretching region of the spectrum as shown in Figure 4b. The effect of laser power fluctuations and inexact-phase setting was corrected for by multiplying the out-of-phase spectrum by a numerical factor near unity which is the ratio of the integrated intensity of the two spectra over a range of frequencies. This results in an offset from zero in the difference spectrum shown in Figure 4b. The in-phase spectrum of neat cyclohexane is shown in Figure 4c. The difference between in- and out-of-phase spectra of cyclohexane is shown in Figure 4d. Only the modulated component of the RS contributes to this spectrum. The noise level in the difference spectrum shown in Figure 4d is smaller than in the in-phase signal shown in Figure 4c since the pixel-to-pixel variation in dark current of the OMA is eliminated in the difference spectrum. In a related experiment, the recovery of RS from toluene solvent in the presence of dissolved dye molecules with a lifetime of 8 ns has also been demonstrated with comparable signal to noise. In the case that the fluorescence intensity is much greater than that of RS, y Ifl/IRs >> 1,the statistical noise of the fluorescencesignal is the primary source of noise in the Raman spectrum. The presence of statistical noise due to fluorescence can be seen by comparing parts b and d of Figure 4. The signal detection in phase-resolved modulation RS is
(3)
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where a1and cyz are the time averages of the in- and out-ofphase transmission factors for the emission modulator, IORs is the Raman intensity detected in the absence of the emission modulator, and At is the total time for the two sequences. The noise in the suppressed fluorescence spectrum when y >> 1 is
N = d/3PflAt
(4)
where p is the transmission coefficient of fluorescence through the modulator, which is the same in both sequences. The signal-to-noise ratio of the RS is, therefore
construction. Since modulation a t greater than the fluorescence decay rates of species that contribute substantially to fluorescence is feasible, this method should prove to be a general technique for eliminating fluorescence in light-scattering experiments. In addition, the instrument could serve as a phase fluorometer to measure subpicosecond phenomena. The method of phase-resolved modulation RS should make possible the application of RS to a range of samples and excitation wavelengths in which fluorescence dominates the spectrum.
ACKNOWLEDGMENT I acknowledge stimulating discussions with my colleagues J. M. Drake, B. N. Perry, P. Rabinowicz, and R. B. Hall. The enthusiastic, off-hour technical assistance of G. Nalavany and M. Alverez is greatly appreciated. Registry No. (2,2’-Bipyridine)rutheniumdichloride, 1574657-3; cyclohexane, 110-83-8; ethanol, 64-17-5.
where (S/N), is the signal to noise for RS in the absence of fluorescence without the interposition of a modulator between the sample and spectrometer. The value of (al - a 2 ) / 2 pfor the present experimental apparatus is 0.09.
CONCLUSIONS In this paper we have demonstrated phase-resolved modulation RS in which modulation of laser intensity and demodulation of sample emission is performed by using EO modulators. The modulators are added to a standard instrument for observing Raman scattering with CW lasers. The presence of statistical noise in the phase-resolved modulation Raman spectrum due to fluorescence may necessitate considerable signal averaging. This is faclitated by the use of an OMA which is made possible with EO demodulation. The speed of detection with EO phase-sensitive detection depends upon the frequency of modulation and not upon the response time of the photodetector. By modulating a t a frequency higher than the fluorescence decay rate of the sample, we have suppressed fluorescence from a strongly fluorescing, dilute impurity and observed solvent RS. A microwave version of the present instrument is presently under
LITERATURE CITED (1) Maker, P. D.; Terhune, R. W. fhys. Rev. 1965, 137, 801. (2) Begley, R. F.; Harvey, A. B.; Byer, R. L. Appl. fhys. Lett. 1974, 2 5 , 387. (3) Lallemand, P.; Simora, P.; Bret, G. fhys. Rev. Lett. 1966, 17, 1239. (4) Ouyoung, A.; Jones, E. D. Opt. Lett. 1977, 7 , 152. (5) Heiman, D.; Heliwarth, R. W.; Levenson, M. D.; Martin, G. fhys. Rev. Lett. 1976, 36, 189. (6) Easley, G. L.; Levenson, M. D.; Toilers, W. M. I E € € J . Quantum Electron. 1978, QE-14, 45. (7) Yacoby, Y.; Wagner, I.; Bodenheimer, J.; Low, W. fhys. Rev. Lett. 1971, 2 7 , 246. (8) Galeener, F. L. Chem. fhys. Lett. 1977, 4 8 , 7. (9) Yu, P. Y.; Smith, J. E., Jr. Phys. Rev. Lett. 1976, 3 7 , 622. (10) Arguello, C. A.; Mendes, C. F.; Lelte, R. C. C. Appl. Opt. 1974, 13, 1731. (11) Van Duyne, R. P.; Jeanmaire, D. L.; Shrlver, D. F. Anal. Lchem. 1974, 46, 213. (12) Harris, J. M.; Chrisman, R. W.; Lytle, F. E.; Tobais, R. S.Anal. Chem. 1976, 4 8 , 1937. (13) Gustafson, T. L.; Lytle, F. E. Anal. Chem. 1982, 5 4 , 634. (14) Nemanich, R. J.; Solln, S. A.; Doehler, J. Rev. Sci. Instrum. 1976, 4 7 , 741. (15) Lakowicz, J. R.; Cherek, H. J. Blochem. Methods 1981, 5 , 19. (16) Spears, K. G.; Cramer, L. E.; Hoffland, L. D. Rev. Scl. Instrum. 1976, 4 9 , 255.
RECEIVED for review April 26, 1984. Accepted July 20, 1984.
