FINDING A SINGLE MOLECULE IN A HAYSTACK Optical Detection and Spectroscopy of Single Absorbers in Solids W. E. Moerner IBM Research Division Almaden Research Center K95/801, 650 Harry Rd. San Jose, CA 95120-6099
L. Kador University of Bayreuth Lehrstuhl für Experimental Physik IV Bayreuth, 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 (1,2). 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 absorbing molecular (or ionic) impurity in a solid (called single-molecule detection, or SMD) would provide a novel probe 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 problem 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 (=:10 12 -10 18 ) 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 absorbing molecule could be 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 be 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
FOCUS be useful to have a detection technique that could operate at relatively large impurity concentrations (10~7 mol/ 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-absorbing impurities in a solid, using the mod-
ANALYTICAL CHEMISTRY, VOL. 661, NO. 21, NOVEMBER 1, 1989 · 1217 A
FOCUS tes
Polymeric Materials
Packa
XcSt
interconnet"
1
joi»aW»
M,,, nd
*
Se,, S. **>"=
j
A
Glossary Quantum jumps: 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-phonon transitions: 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.
ÉH
Polymeric Materials for Electronics Packaging
W
hile there has been a number of books addressing packaging, and to some extent polymers, virtually none has emphasized the synthetic and physical chemistry of these systems. This new volume fills that gap, addressing the many aspects relating to the development of novel polymeric materials and processes. The emphasis is on chemistry and materials science, rather than circuitry, its electrical capabilities, or its design characteristics. Covering a broad spectrum of subjects, this 38-chapter book focuses on four general areas: • physical chemistry of materials • properties and applications of encapsulants • properties and applications of gels • printed circuit board substrates and materials for circuit board substrates. Also included is a review of the marketing trends which drive packaging technology. This unique volume will be helpful to scientists engaged in materials development for electronic packaging, electronic packaging engineers, and to technologists who monitor developments in the electronics industry and related fields. John H. Lupinski, Editor, General Electric Company Robert S. Moore, Editor, Eastman Kodak Company Developed from a symposium sponsored by the Division of Polymeric Materials: Science and Engineering and of Polymer Chemistry of the American Chemical Society ACS Symposium Series No. 407 512 pages (1989) Clothbound ISBN 0-8412-1679-9 LC 89-37412 US & Canada $99.95 Export $119.95
American Chemical Society Distribution Office, Dept. 45 1155 Sixteenth St., N.W. Washington, DC 20036 or CALL TOLL FREE
800-227-5558 (in Washington, D.C. 872-4363) and use your credit card!
Homogeneous absorption line: The absorption profile of a single impurity center. In most cases, the homogeneous lines of all of the impurity centers in the solid are identical in width; hence, this width is called the homogeneous width. Inhomogeneous absorption line: The type of absorption line that occurs for zero-phonon transitions of impurity centers in solids at low temperatures. The inhomogeneous line is almost always much wider than the homogeneous width because of the spread of center frequencies for the various impurities. Number fluctuations: For an additive 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 transient techniques: Methods for measuring the homogeneous width using pulses shorter than all the excited-state lifetimes, such as photon echoes, stimulated photon echoes, and free-induction decays. Quantum limit: In optical spectroscopy, the signal-to-noise regime where the noise is dominated by Poisson shot noise fluctuations of the light beam itself. Quadratic Stark effect: 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.
el system composed of pentacene substitutional impurities in p-terphenyl crystals at 1.5 Κ (Figure 1). A penta cene molecule can substitute for any one of the four p-terphenyl molecules in the low-temperature unit cell (7), giving rise to four Si *- So 0-0 optical absorption origins near 593 nm: Οι, Ο2, O3, and O4. We will focus on the origins Oi and 02, because the homogeneous linewidths are smaller for these than for the other origins. (See Reference 8 for details of the low-temperature crys tal structure.) In our ^ΙΟΟ-μπι-thick samples at impurity concentrations of 10~7 mol/ mol, therefore, the background "hay stack" is composed of 1013 host mole cules plus 106 additional pentacene molecules. Our method of selecting a single resonant impurity for optical probing relies on the phenomenon of inhomogeneous broadening that occurs for all zero-phonon transitions at low temperatures. Zero-phonon transitions can become very narrow as the tem perature is lowered, because broaden ing processes in which a host phonon scatters off the electronic excited state are quenched. In essence, the absorp
1218 A · ANALYTICAL CHEMISTRY, VOL. 6 1 , NO. 2 1 , NOVEMBER 1, 1989
tion lines for the various guests become so narrow that the normally hidden distribution of center frequencies be comes apparent (Figure 2). The lowtemperature inhomogeneous profile (for a particular origin) is composed of many narrow homogeneous (usually Lorentzian) absorption lines with a dis tribution of center frequencies caused by dislocations, point defects, or ran dom internal electric and strain fields and field gradients in the host material. Clearly, one absorber may be selected for spectroscopy by proceeding out into the wings of the inhomogeneous line, 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 inhomogen eous line. We chose the approach de scribed herein because by tuning to the center of the inhomogeneous line where Ν is large, we can use a conve niently large alignment signal to opti mize the detection system. Then, by tuning out into the wings, single-mole cule absorptions can be observed. Because of the randomness associat ed with imperfections in the host mate rial, 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 inhomogeneous profile, the true shape of the inhomogeneous line cannot, in reality, be 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 t h a t scales as the square root of the mean number of centers in resonance. To be precise, if we define the average number of centers in
the probed volume within one homogeneous width of the laser wavelength as N H , there should be a statistical fine structure (SFS) present on the absorption profile scaling in absolute magnitude as VNH (in the limit of N H » 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 N H es 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
SAMPLE PREPARATION BOMBS FOR TRACE METAL ANALYSIS Available in a variety of sizes and styles, our # teflon-lined Acid Digestion Bombs are designed for microwave and convection heating. Use them to treat inorganic samples in HF, HO and other strong mineral acids.Or to digest organic samples in strong alkali or oxidizing acid at elevated temperatures and pressures.
Our Oxygen Combustion Bombs decompose any combustible solid or liquid — quickly and completely. The sealed bomb contains and converts materials to a soluble form while allowing complete recovery of all elements. Now available in a new longer-lasting material for high halogencontaining substances. For complete details ask for our free Brochure #4700.
PARR INSTRUMENT COMPANY
Figure 1. Pentacene molecules may substitute for any one of the four inequivalent p-terphenyl molecules in the low-temperature p-terphenyl unit cell. (See Reference 8 for details.)
211 Fifty-Third Street Moline, IL 61265 309-762-7716 Telex: 270226 Fax: 309-762-9453
CIRCLE 125 ON READER SERVICE CARD
ANALYTICAL CHEMISTRY, VOL. 61, NO. 21, NOVEMBER 1, 1989 · 1219 A
FOCUS
Inhomogeneous line (Gaussian)
.α
c is passed through an electroopticphase modulator EO to produce light that is frequency-modulated at an rf frequency m in the MHz range. This FM light beam has the spectrum shown in the upper center of the figure: a car rier at the original frequency, plus two sidebands displaced by ±a>m from the carrier. If this FM light beam were sent directly to a high-speed photodiode de tector (which measures the envelope of the power absorbed), no photocurrent at m; and (3) for wide spectral features, the FMS signal is proportional to the de rivative of the feature, and with in creasing width the amplitude ap proaches zero. The first property is re sponsible for the quantum-limited performance of FM spectroscopy that
has been achieved with simple (nonavalanche) photodiodes (18). Proper ties 2 and 3 are particularly important for SMD, because any undesired opti cal absorption from other impurities or from the p-terphenyl host that is broad compared with wm is not detected with appreciable amplitude. With our val ues of wm/2ir = vm = 50 - 90 MHz, only rigid molecules such as pentacene with homogeneous widths less than ^ 1 0 0 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 residu al AM (also called RAM) from imper fections in the modulator can give rise to a spurious background signal. To overcome this limitation, we used a sec ondary modulation of the spectral fea ture itself. Figure 4 illustrates the case wherein an electric field oscillating at a low frequency in the kHz range pro duces the second modulation. The elec tric field shifts the absorption profile twice during each cycle via the quad ratic Stark effect. Then the output of the mixer, processed by a final lock-in amplifier, LIA, at 2f, yields a detected signal that is free from RAM back ground limitations. In addition to this FM/Stark technique, we used ultrason
--
ω
— ι
m, APD—ava lanche photodiode detector, M—double-balanced mixer, HV—high-voltage source. LIA—lock-in amplifi er, DS—digital storage and averaging oscilloscope, LO—local oscillator, R—rf port, I—intermediate fre quency port.
ANALYTICAL CHEMISTRY, VOL. 6 1 , NO. 2 1 , NOVEMBER 1, 1989 · 1221 A
FOCUS Zeolite Synthesis
!
H»
Over 600 pages
Zeolite Synthesis
H
ere in one volume is a complete prog ress report on the various aspects of zeolite synthesis on a molecular level. No other source covers this subject so completely! This new title provides many examples that illustrate how zeolites can be crystallized and what the important parameters are that con trol crystallization. A variety of topics covered in this important book include: • • • • •
avalanche photodiode to avoid detec tor Johnson noise. However, avalanche photodiodes have an excess multiplica tive noise t h a t requires operation somewhat above the quantum limit. In spite of these limitations, the FM/ Stark and FM/US methods can be used to detect the optical absorption of a single molecule of pentacene in a solid crystal of p-terphenyl. Figure 5 shows examples of the spectra for the FM/ Stark case. The first three traces show simulations to explain the expected single-molecule lineshape for either Stark or ultrasonic double modulation. Trace 5(a) shows a Lorentzian absorp tion profile of width y, and trace 5(b) shows the expected simple FM signal (17): two copies of the absorption line with opposite sign, spaced by 2vm =150 MHz (in the limit y « vm). With sec ondary modulation that causes fre quency shifts less than the linewidth, the resulting double-modulation lineshape is the derivative of the simple FM lineshape (trace 5(c)). Thus the signature of a single molecule is a " W"shaped feature with a large negative slope and a large positive slope separat ed by 2vm.
crystallization techniques gel chemistry crystal signs and morphology the role of organic compounds novel synthesis procedures
With forty-two chapters, this valuable title of fers a complete review of zeolite synthesis as well as the latest findings in this important field. Valuable new methods are highlighted such as VPI-5—the first 18-membered ring molecular sieve. In addition, examples of mod ern spectroscopic characterization of reactants and reaction intermediates are provided. Also covered is the chemistry of silicate solutions and reactant effects on crystallization products. This essential volume contains benchmark contributions from many notable pioneers in the field including R.M. Barrer, H. Robson, and Robert Milton. Mario L. Occelli, Editor, Unocal Corporation Harry E. Robson, Editor, Louisiana State University Developedfroma symposium sponsored by the Divi sion of Colloid and Surface Chemistry of the American Chemical Society ACS Symposium Series No. 398 643 pages (1989) Clothbound ISBN 0-8412-1632-0 LC 89-6884 US & Canada $139.95 Export $167.95
O.R.D.E.R.F.R.O.M American Chemical Society Distribution Office, Dept 33 1155 Sixteenth St, N.W. Washington, DC 20036 or CALL TOLL FREE
800-227-5558 (in Washington, D.C. 872-4363) and use your credit card!
-o ο Ε .Si
-0.4
χι Ο Q
(g). *4
-0.8
0
200
400
600
Laser frequency (MHz)
Figure 5. Single-molecule spectra using FM/Stark technique (quadratic Stark ef fect). (a) Simulation of absorption line, (b) Simulation of FM spectrum for (a). c m = 75 MHz. (c) Simulation of FM/Stark double-modulation lineshape. (d) SMD spectra at 592.423 n m , 512 averages, 8 traces overlaid; bar shows value of 2c m = 150 MHz. (O, line center is at 592.326 nm.) (e) Average of traces in (d) with fit to the in-focus molecule (smooth curve), (f) Signal very far off line at 597.514 n m , same conditions, (g) Traces of SFS at the 0 2 line center, 592.186 n m , 128 av erages each.
1222 A · ANALYTICAL CHEMISTRY, VOL. 6 1 , NO. 2 1 , NOVEMBER 1, 1989
In a typical experiment, the laser fre quency was set near the center of the inhomogeneous line, and the resulting strong SFS signal was used to optimize the optical and electronic configura tion. Then as the laser wavelength was moved out into the wings of the line, the SFS amplitude dropped uniformly. Eventually, spectra that appear to be superpositions of 2-5 single-molecule spectra such as trace 5(c) were ob served. Finally, sufficiently far out into the wings of the line, true single-mole cule spectra were recorded. Trace 5(d) shows a set of eight FM/ Stark double modulation spectra of a strong in-focus molecule far out in the long-wavelength edge of Oi, along with several unavoidable weak repeatable features from out-of-focus molecules at the left and right edges of the laser scan range. These out-of-focus features were caused by molecules not located at the laser waist position (Figure 2) and may be suppressed in future ex periments with thinner samples. The fiducial bar marks a spectral range equal to 2vm. Trace 5(e) shows the average of the eight scans shown in trace 5(d), along with a fit to the central feature generat ed by a simple model for the doublemodulation process (6). The fit to the essential features of the SMD lineshape is reasonable, and the homoge neous width required by the fitting process is somewhat larger than the low-power homogeneous width, as ex pected. Trace 5(f) shows the detected signal from a laser wavelength so far away from the pentacene site origins that no molecules are expected to lie in the la ser scan range; this is the background shot and avalanche noise. In samples of undoped pure p-terphenyl, only a base line noise level similar to the off-line data in Figure 5(f) is observed, even near the center of the inhomogeneous line. Trace 5(g) shows spectra of the strong SFS observed near the center of the inhomogeneous line using a smaller number of averages. This spectrum is composed of a superposition of many "W" profiles similar to Figure 5(d) with many different center frequen cies, illustrating the qualitative differ ence between spectra of large numbers of molecules (Figure 5(g)) and spectra of one molecule (Figure 5(d)). Using the related FM/US technique, variations in the modulating frequency fm caused the single-molecule lineshapes to expand and contract so that proper spacing between the inner edges of the " W" could be maintained. Such clear variations in the recorded spec trum cannot be observed when many molecules are present in the laser scan.
This realization—in conjunction with the shape of the observed features, the position 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 FM/US 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 modu lation 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 experi ments) suggests that pentacene mole cules in the highly strained, improba ble 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-ab sorber experiments in which the mea sured properties of the absorbing cen ter are not averaged over many "equi valent" absorbers. The absorbing entity is exquisitely sensitive to the symmetry and perturbations intro duced by the local environment, such as the local vibrational modes and the true local fields. Although the method presented in this article is not applicable as an ana lytical technique to all molecular impu rities, it can be applied to a large num ber of absorbing ions and molecules in solids with zero-phonon transitions. The detectability of the resulting sin gle-center signal, which ultimately de pends on the absorption strength and on quantum noise limits, must be eval uated in each case. For situations in which the molecular Iinewidth is large, recent important advances in two-tone FM spectroscopy (19, 20) make FMS practical with very large sideband spacings. The work on SFS was performed with Tom Carter, who is currently with the Chemistry Department, Florida State University, Tallahassee. This work was supported in part by the U.S. Office of Naval Research.
References (1) Itano, W. M.; Bergquist, J. C; Wineland, D. J. Science 1987,237,612. (2) Diedrich, F.; Peik, E.; Chen, J. M.; Quint, W.; Walther, H. Phys. Rev. Lett. 1987,59,2931. (3) Ashkin, Α.; Dziedzic, J. M. Science
1987 235 1517 (4) Nguyen, D. C.; Keller, R. Α.; Jett, J. H.; Martin, J. C. Anal Chem. 1987,59,2158. (5) Moerner, W. E.; Kador, L. Phys. Rev. Lett.1989,62, 2535. (6) Kador, L.; Home, D. E.; Moerner, W. E., submitted for publication in J. Phys. Chem. (7) Olson, R. W.; Fayer, M. D. J. Phys. Chem. 1980,84,2001. (8) Baudour, J. L.; Delugeard, Y.; Cailleau, H. Acta Cryst. 1976, B32,150-54. (9) Stoneham, A. M. Rev. Mod. Phys. 1969, 47.82. (10) Moerner, W. E; Carter, T. P. Phys. Rev. Lett. 1987,59,2705. (11) Carter, T. P.; Manavi, M.; Moerner, W. E. J. Chem. Phys. 1988,89,1768. (12) Carter, T. P.; Home, D. E.; Moerner, W. E. Chem. Phys. Lett. 1988,151,102. (13) Bjorklund, G. C. Opt. Lett. 1980,5,15. (14) Patterson, F. G.; Lee, H.W.H.; Wilson, W. L.; Fayer, M. D. Chem. Phys. 1984,84, 51. (15) Lange, R.; Grill, W.; Martienssen, W. Europhys. Lett. 1988,6,499. (16) Yen, W. M. In Laser Spectroscopy of Solids II; Yen, W. M., Ed.; Springer Top ics in Applied Physics; Springer: Berlin, 1989; Vol. 65, p. 23. (17) Bjorklund, G. C; Levenson, M. D.; Lenth, W.; Ortiz, C. Appl. Phys. Β 1983, 32,145. (18) Levenson, M. D.; Moerner, W. E.; Home, D. E. Opt. Lett. 1983,8,108. (19) Janik, G. R.; Carlisle, C. B.; Gallagher, T. F. J. Opt. Soc. Am. Β 1986,3,1070. (20) Cooper, D. E.; Carlisle, C. B. Opt. Lett. 1988,13, 719.
W. E. Moerner (left) is a researcher in IBM's Polymer Science and Tech nology Department. He received his Ph.D. in physics from Cornell Univer sity in 1981. His research interests in clude persistent spectral hole-burn ing; low-temperature photochemistry in solids; physics of optoelectronic nonlinear materials; and fundamental properties of spectral lines in solids. L. Kador (right), a World Trade Vis iting Scientist at IBM while this work was performed, received his Ph.D. in physics from the University of Bayreuth (West Germany) in 1988. He is now a researcher at the University of Bayreuth, supported by the Deutsche Forschungagemeinschaft. His inter ests include single-molecule detection and persistent spectral hole-burning as a probe of Stark and external field effects in amorphous solids.
OUR STANDARD DELIVERY IS ANYTHING BUT STANDARD.
Conostan oil-based cali
day longer. Conostan
Conostan standards for the analysis of metals in oils.
bration standards are just a
orders are shipped UPS, air
telephone call away. Stan
express, or as you designate.
dard orders are filled the
For service that's anything
day they are received, and
but standard, call or write
are on the way to you within
for our free brochure
24 hours. Orders for custom
describing the full line of
blends usually take only a
single and multi-element
Standards to Burn ®
CONOSTAN Conostan Division Conoco Specialty Products Inc. P.O. Box 1267 PoncoCity, OK Phone:(405)767-3078 Fax, (405) 767-5843
CIRCLE 30 ON READER SERVICE CARD
ANALYTICAL CHEMISTRY, VOL. 61, NO. 21, NOVEMBER 1, 1989 · 1223 A
