MOLECULE IN A HAYSTACK
Optical Detection and Spectroscopy - __ _ of Single Absorbers in I _ _ _ _ _ W. E. Moemer
IBM Research Division Almaden Research Center K95/801. 650 Harry Rd. San Jose, CA 95120-6099
L. Kador
University of Bayreuth Lehrstuhl fur Experimental Physik IV 8ayreuth. West Germany In the past few years, progress has been made in the optical detection of single absorbers confined by various means. For example, single ions confined in vacuum electromagnetic traps have yielded to the techniques of the optical spectroscopist, allowing direct measurement of quantum jumps, Doppler sidebands, and other fundamental phenomena such as ion crystallization ( I , 2). In liquid media, optical trapping and manipulation of single viruses and single live motile bacteria have been achieved through the use of radiation pressure techniques (3).By using laserinduced fluorescence and a novel hydrodynamically focused flow to confine 0003-2700/89/A361-1217/$01.50/0 @ 1989 American Chemical Society
the molecules and reduce the scattering volume, single molecules of the protein B-phycoerythrin with the equivalent of 25 rhodamine 6G chromophores have also been detected ( 4 ) . The ability to detect a single ahsorhing molecular (or ionic) impurity in a solid (called single-molecule detection, or SMD) would provide a novel prohe of the local structure and dynamics of crystalline and amorphous solids on a truly local, site-selective scale. Compared with the other single-absorber experiments, SMD poses a different set of experimental challenges. The prohlem can be likened to finding a needle in a haystack. Unlike the ion-trap experiments, for example, the molecule of interest is held in a solid containing a large number (-lO‘~-1OLp) of “nonabsorbing” host molecules within the laser focal volume. If laser-induced fluorescence excitation were selected for SMD and the host molecules had appreciable Raman (or Rayleigh) scattering cross sections, the signal from the one ahsorhing molecule could he swamped by the scatter-
ing signal of the host. Unlike the hydrodynamic focusing experiments, it is not always possible to reduce the host scattering volume. Furthermore, it would he useful to measure the absorption spectrum of a single absorber rather than detect its presence digitally at a fixed laser wavelength. Removing interfering impurities when concentrations of the molecule of interest are very small is another experimental challenge in SMD. It would
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he useful to have adetection technique that could operate at relatively large impurity concentrations (IO-: moll mol) and still identify one absorbing molecule in the presence of -106 or more identical absorbing molecules. In this article we will describe recent experiments (5,6) in the optical detection and spectroscopy of single-ahsorbing impurities in a solid, using the mod-
ANALYTICAL CHEMiSTRY. VOL. 61. NO. 21. NOVEMBER 1. 1989 * 1 2 1 7 A
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Quantumjump: When many absorbers are present. one may define the transition rate from one energy level to another. However. when only one absorber is present. discrete jumps can be Observed as the absorber jumps from one quantum state to another. These quantum jumps occur stochastically,with a rate that agrees with the normal transition rate only on average. Site: The specific local environment around an absorbing impurity center. Phonon: lattice vibration of a solid host matrix. Zero-phononiransitlons:Optical or vibrational transitions of an impurity center in which no phonons of the host matrix are created or destroyed. Such transitions are Only appreciable when the electron-phonon (or vibration-phonon) coupling is not too large. bmogeneour absorptlon h e : The absorption profile of a single impurity center. In most ses, the homogeneous lines of all of the impurity centers in the solid are identical in d t h ; hence, this width is called the homogeneous width. Inhomogeneous absorption Ilne: The type of absorption line that occurs for zerc-phonon transitions of Impurity centers in solis at low temperatures. The inhomogeneous line is almost always much wider than the homogeneous width because of the spread of center frequenciesfor the various impurities. Number Ilucluations: For an addiive physical quantity that is measured for many independent members of a random ensemble. the value of the quantity fluctuates with a relative size equal to 1 over the square root of N. the number of members sampled. Coherent translent tMhnlqueS Methods for measuring the homogeneous width using pulses shorter than all the excited-state lifetimes,such as photon echo%%stimulated photon echoes. and free-induction decays. Quantum Ilmn: In optical spectroscopy. the signal-to-noise regime where the noise is dominated by Poisson shot noise fluctuationsof the light beam itself. Quadretk Stark enecct:The shifting of the resonance frequency of a center in an external electric field in which the shift is proportional to the square of the electric field. Laser FM spectroscopy: An optical detection technique in which a sample with narrow absorption features is probed with a frequency-modulated (or phase-modulated) light beam. The signal derives from the conversion of FM Into AM by the sample absorption.
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ANALYTICAL CHEMISTRY. VOL.
el system composed of pentacene suhstitutional impurities in p-terphenyl crystals a t 1.5 K (Figure 1). A pentacene molecule can substitute for any one of the four p-terphenyl molecules in the low-temperature unit cell (7), giving rise to four SI SO0-0 optical absorption origins near 593 nm: O,, 02, 03,and 04.We will focus on the origins O1 and 02.because the homogeneous linewidths are smaller for these than for the other origins. (See Reference 8 for details of the low-temperaturecrystal structure.) In our elW-rm-thick samples a t impurity concentrations of 10-I moll mol, therefore, the background "haystack" is composed of 1013host molecules plus 106 additional pentacene molecules. Our method of selecting a single resonant impurity for optical probing relies on the phenomenon of inhomogeneousbroadening that occurs for all zero-phonon transitions a t low temperatures. Zero-phonon transitions can become very narrow as the temperature is lowered, because hroadening processes in which a host phonon scatters off the electronic excited state are quenched. In essence, the ahsorp-
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tion lines for the various guests become so narrow that the normally hidden distribution of center frequencies becomes apparent (Figure 2). The lowtemperature inhomogeneous profile (for a particular origin) is composed of many narrow homogeneous (usually Lorentzian) absorption lines witha distribution of center frequencies caused by dislocations, point defects, or random intemal electric and strain fields and field gradients in the host material. Clearly, one absorber may be selected for spectroscopyby proceeding out into the wings of the inhomogeneousline, as shown on the right side of Figure 2. It would also be possible to drastically lower the impurity concentration and work near the center of the inhomogeneous line. We chose the approach described herein because by tuning to the center of the inhomogeneous line where N is large, we can use a conveniently large alignment signal to optimize the detection system. Then, by tuning out into the wings. single-molecule absorptions can be observed. Because of the randomness associated with imperfections in the host material, inhomogeneous absorption lines
(at least near their centers) are often approximated by smooth, Gaussian profiles (9). However, because the inhomogeneous line on a microscopic scale is simply a superposition of discrete homogeneous lines with widths as much as 1000 times narrower than the overall inhomogeneousprofile, the true shape of the inhomogeneous line cannot, in reality, he a smooth function. In fact, unavoidable number fluctuations in the density of absorbers per unit wavelength interval should give rise to a "spectral noise" on the overall Gaussian background that scales as the square root of the mean number of centers in resonance. To be precise, if we define the averagenumber of centers in
the probed volume within one homogelleous width of the laser wavelength as NH, there should be a statistical fine structure (SFS)present on the abeorption profile scaling in absolute magnitude as & (in the limit of NH >> 1). Because SFS arises from the superposition of many overlapping impurity absorptions, the absolute magnitude of the SFS is clearly larger than a singlemolecule absorption signal (where NH 1).Therefore, Observations of SFS would be expected to precede true single-molecule detection. Recent observations of SFS in the pentacene in p-terphenyl system provided a crucial first step toward singlemolecule detection and spectroscopy
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uro 1. Pentacene molecules may substitute for any one of the foi
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vphenyl nmlecules in the low-temperature pterphenyl unit cell. (SeeRelwence 8 far details.)
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ANALYTICAL CHEMISTRY. VOL. 61. NO. 21. NOVEMBER 1, 1989
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I
Laser
--\
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gum 2. Schematic showing the source of a statistical fine structure on inhomogeneous lines and the principle of singlemolecule detection in solids. The lower part of Um lwe &wa how me number of impwhy molecules In reemanat m Um probed volume csn be varled by changlnp Um 1~ wavelenpn. The laser llnewldm (-3 MHz) Is negllglble.
200 pm
100 MHz
Figure 3. Statistical tine structure (SFS) versus laser spot posltlon and l a w f r e quency near the inhomogeneous line center for pentacene in pterphenyl. A sequence of 100 SFdCWa w a , obtained. moving Um 2&pm m e r Spol by 2 Icm aher each SpBCbYm. and Um resub were planed 86 B color enCming ot mS SFS slgonal to s t o w mS SFS “landrcspe.” 1220,.
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(I&I2). This was achieved by using high-resolution, high-sensitivity laser frequency modulation spectroscopy (FMS) (13)to probe the opticalahsorption in a zero-background manner sensitive only to narrow spectral features. This detection method will be described in more detail below. Because its source is a random process, the SFS spectral structure changes for different prohe volumes, and an “SFS landscape” of the inhomogeneous line can be generated by acq u i r i i SFS spectra as a function of laser spot position (Figure 3). The bumps and valleys in this figure may be regarded as the “fuzz” on the top of the “haystack” represented by the pentacene inhomogeneous line that results from the statistics of independent, additive random variables. More physical information may he derived directly from SFS measurements: By computing the autocorrelation of the measured spectra ( I O - E ) , the homogeneous width of the underlying Lorentzian profiles may he determined. Autocorrelation analysis can be used to remove the information ahout the random quantity (the center frequencies of the individual absorhers), and thus reveal that which is similar for all ahsorhers (i.e., the shape of the homogeneous profile). Autocorrelation analysis examines how two copies of the same SFS spectrum overlap aa the two copies are displaced relative to one another. S t a r t i with perfect overlap at zero displacement, the autocorrelation function decays to zero in a manner characteristic of the underlying homogeneous lineshapes contributing to the spectrum. The result of a straightforward analysis (IO-1.2) is that the expected value of the autocorrelation of the SFS signal has a spectral width at the origin equal to twice the homogeneous width. Using this scheme to analyze SFS spectra similar to thoae in Figure 3, we obtain the homogeneous width (full-width at halt-maximum) y = 7.9 f 0.8 MHz for the O1 site of pentacene in p-terphenyl at 1.4 K, which compares favorably with the reported value (14)of 7.8 0.6 MHz from coherent transient techniques. After the fvat observations of SFS, other researchers moved closer to achieving single-absorber detection in solids by observing SFS at increasingly lower impurity concentrations. Lange et al. (15) relied on fluorescence excitation of Sm2+ions in CaFz at 77 K with a fixed frequency laser in tightly focused spots. These researchers saw Poisson fluctuations in the detected fluorescence as a function of the position of the focal spot and concluded that they had reached the level NH = 5. Another
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novel approach, developed by Yen and eo-workers at the University of Georgia ( E ) used , laser fluorescence excitation in a glass fiber doped with Nd3+ ions. The fiber geometry effectively maintains a small focus and a small probing volume to reduce background signals from the host. They concluded from the mea_sured SFS that they had reached NH values on the order of a few tens of ions. In both cases, special detection geometries were necessary to avoid host background fluorescence. Our approach to SMD avoids the problem of fluorescence background by using a powerful absorption technique, laser FM spectroscopy (FMS) (12). The basic operation of FMS is illustrated in the upper part of Figure 4. A tunable single-frequency laser beam at w , is passed through an electroopticphase modulator EO to produce light that is frequency-modulated at an rf frequency w, in the MHz range. This FM light beam has the spectrum shown in the upper center of the figure: a carrier at the original frequency, plus two sidebands displaced by fw, from the carrier. If this FM light beam were sent directly to a high-speed photodiode detector (which measures the envelope of the power absorbed), no Photocurrent at W , would be detected. In other words, an FM light beam has no amplitude modulation (AM). In the frequency domain, this lack of AM can be understood by noting that one of the two sidebands is in phase and the other out of phase with respect to the carrier. Therefore, the beat signals caused by each sideband interfering with the carrier cancel each other out. When a spectral feature is present that disturbs the balance between the two sidebands, an rf photocurrent appears that is proportional to the difference in optical absorption at the upper and lower sidebands. This pbotocurrent at W , may be detected (phase sensitively) by an rf lock-in composed of a mixer M driven with a local oscillator derived from the original rf source. Thus the low-frequency or baseband signal at the I port of the mixer is the standard FMS signal, with the following properties (17): (1) it appears on a background that is derived from the laser noise at w,, which may be at the quantum limit if no excess noise is introduced by the detector; (2) the size of the signal is largest when the spectral feature is narrower in width than w,; and (3) for wide spectral features, the FMS signal is proportional to the derivative of the feature, and with increasing width the amplitude approaches zero. The first property is responsible for the quantum-limited performance of FM spectroscopy that
has been achieved with simple (nonavalanche) photodiodes (18).Properties 2 and 3 are particularly important for SMD, because any undesired optical absorption from other impurities or from thep-terphenyl host that is broad compared with wm is not detected with appreciable amplitude. With our values of wd2u = u, = 50 - 90 MHz, only rigid molecules such as pentacene with homogeneous widths less than -100 MHz will be detected. One particular background signal from FMS, however, must be avoided. Because the method directly senses the conversion of FM into AM, any residual AM (also called RAM) from imperfections in the modulator can give rise to a spurious background signal. To overcome this limitation, we used a secondary modulation of the spectral feature itself. Figure 4 illustrates the case wherein an electric field oscillating at a low frequency in the kHz range produces the second modulation. The electric field shifk the absorption profde twice during each cycle via the quadratic Stark effect. Then the output of the mixer, processed by a final lock-in amplifier, LIA, at Zf, yields a detected signal that is free from RAM background limitations. In addition to this FM/Stark technique, we used ultrason-
ic stress (US) modulation separately. This technique operates in the same way as FM/Stark hy shifting the homogeneous line periodically. It is clear that other local perturbations of the impurity molecule might also be used for double-modulationdetection. The size of the expected absorption signal from a single molecule is straightforward to estimate (5). The change in absorbance, (Aa)L, is given by the probability of absorption of a photon in the incident beam by the molecule, u/A, where u is the peak absorption cross section and A is the area of the laser beam. Clearly, tightly focused laser spots and molecules with strong absorptions are preferable. In our experiment, the focal spot is -3 pm in diameter, and the peak (lowtemperature) absorption cross section for pentacene is 9.3 X 10-l2em2,yielding an absorbance change of -W4. This is not an extremely small signal, except that detection must be performed with a light intensity that does not produce extreme power broadening. To meet this constraint in a tightly focused spot, we performed measurementa with only 0.1 pW of light at the detector. Even at this level we chose to accept some amount of power hroadening. Such a low light level requires an
C DL
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ll
LO
R
u LIA
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Schematic showing the principle of frequency modulation spectroscopy with Stark secondary modulation. The upper part of lhe WaWe s b w s lhe dye laser (DL) specbum behm the elecmwlic mOduiamr EO).after lhe EO, and aner me sample in me crycstatC. Legend: rf-rf o~clllalo~ at ,w ,, AFQav-arnyumd 4.
I m h e photcdiodedetector. Mdcubl%balancedmixer. HV-high-voltage source. LIA-lock-in amplllier. D S d i g b l stwage and averaging nsciIioscope. LO--local oscillalw. R--rt p a t I-intermediate tre quency pm.
ANALYTICAL CHEMISTRY, VOL. 61. NO. 21, NOVEMBER 1, I989
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This realization-in conjunction with the shape of the observed features, the pasition relative to the pentacene in p-terphenyl origins, the lack of such signals in undoped samples, and the appearance of single-molecule spectra with both the FM/Stark and FMWS techniques-leads us to conclude that the recorded spectra result from single molecules of pentacene. Absolute calibration of the observed signals is difficult to obtain with zerobackground techniques. We attempted to do this carefully by using an FM signal of known amplitude, but the amount by which the secondary modulation shifted absorption lines could only be estimated. In spite of these limitations, the size of the single-molecule signals appears to be somewhat larger than expected, given our level of power broadening. This intriguing observation (which needs to be confirmed in future experiments) suggests that pentacene molecules in the highly strained, improbable sites far out in the wings of the inhomogeneous line may have reduced intersystem crossing rates. The attainment of single-molecule detection and spectroscopy in solids opens up a new frontier of single-absorber experiments in which the measured properties of the absorbing center are not averaged over many “equivalent” absorbers. The absorbing entity is exquisitely sensitive to the symmetry and perturbations introduced by the local environment, such as the lwal vibrational modes and the true local fields. Although the method presented in this article is not applicable as an analytical technique to all molecular impurities, it can be applied to a large number of absorbing ions and molecules in solids with zero-phonon transitions. The detectability of the resulting single-center signal, which ultimately depends on the absorption strength and on quantum noise limits, must he evaluated in each case. For situations in which the molecular linewidth is large, recent important advances in two-tone FM spectroscopy (19. 20) make FMS practical with very large sideband spacings. Thcaro.konSFSwasperformedwithTom Carter. who is currently with the Chemistry Department. Florida Stste University. T a l l a h a m . This work was auppnrled in part hy the I1.S. Office of Naval Research.
Aeterences (1) Itano. W. M.;
Bergquist, J. C.; Wine-
land. D. J. Science 1987,237,612. (2) Diedrich, F.; Peik. E.; Chen. J. M.; Quint. W.; Walther, H. Pkyr. Reu. Lett.
1987.235.1517. (4) Npuyen. D. C.; Keller. R. A,; Jett. J. H.; Martin, J. C. Ann1 Ckem. 1987.59,2158. (5) Moerner. W. E.;Kador. L. Phys. Reu. Lett. 1989.62,2535. (6) Kador. L.; Horne,D. E.;Moemer,W. E.,
submitted for publication in J. Phys. Chom -. ......
(7) Olson, R. W.; Fayer. M. D. J. Phys.
...~
C ~19Rn. R 4 ~2 M 1 .h e m ~. ~
(81
Raudour. J. I..; Delupeard. Y.; Cailleau.
H. Arlo C r w . 1976,R32. 1 . W . (9) Stoncham. A. M Rev. Mod. Phya. 1965. 41,82.
(10) Moerner, W. E.; Carter, T. P. Pkys.
Reu. Lett. l987,59,2705. Carter. T. P.; Manavi, M.; Moemer. W. E. J. Ckem. Phys. 1988.89,1768. (12) Carter. T. P.;Home, D. E.; Moerner, W. E. Chem. Pkys. Left. 1988.151.102. (13) Bjorklund, G. C. Opt. Left. 1980,5.15. (11)
(14) Patterson.F.G.;Lee.H.W.H.;Wilson, W. I..; Fayer, M. D. Ckem. Phys. 1984.84, 51. (15) Lange, R.;Grill, W.; Martienssen, W. Europhys. Lett. 1988.6.499. (16) Yen, W. M. In Loser Spectroscopy of Solids 11; Yen. W . M., Ed.; Springer Topin in Applied Physics; Springer: Berlin, 1989: Vol. 65,p. 23. (17) Bjorklund, G. C.; Levenson. M. D.; Lenth. W.; Ortiz, C. Appl. Pkys. R 1983.
1988.1% 719
W. E. Moerner (left) is a researcher in IRMS Polymer Science and Technology Department. He receiued his Ph.D. in physics from Cornel1 Uniuersity in 1981. His research interests include persistent spectral hole-burning; low-temperature photochemistry in solids; physics of optoelectronic nonlinear materials; and fundamental properties of spectral lines in solids. L. Kador (right),a World Trade Visiting Scientist a t IBM while this work was performed, received his Ph.D. in physics from the University of Bayreutk (West Germany) in 1988. He is now a researcher at the University of Bayreuth. supported by the Deutsche Forschungagemeinschaft. His interests include single-molecule detection and persistent spectral hole-burning as a probe of Stark and external field effects in amorphous solids.
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