Table Of ContentSpringer Series in
CHEMICAL PHYSICS
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Springer Series in
CHEMICAL PHYSICS
Series Editors: F. P. Schäfer J. P. Toennies W. Zinth
The purpose of this series is to provide comprehensive up-to-date monographs
in both weH established disciplines and emerging research areas within the broad
fields of chemical physics and physical chemistry. The books deal with both fun
damental science and applications, and may have either a theoretical or an experi
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in chemical physics and related fields.
63 Ultrafast Phenomena XI
Editors: T. Elsaesser, J.G. Fujimoto, D.A. Wiersma, and W. Zinth
64 Asymptotic Methods in Quantum Mechanics
Application to Atoms, Molecules and Nuclei
By S.H. Patil and K.T. Tang
65 Fluorescence Correlation Spectroscopy
Theory and Applications
Editors: R. Rigler and E.S. Eison
66 Ultrafast Phenomena XII
Editors: T. Elsaesser, S. Mukamel, M.M. Murnane, and N.F. Scherer
67 Single Moleeule Spectroscopy
Nobel Conference Lectures
Editors: R. Rigler, M. Orrit, T. Basche
Series homepage - http://www.springer.de/phys/books/chemical-physics/
Volumes 1-62 are listed at the end ofthe book
R. Rigler M. Orrit T. Basche
Single
Moleeule
Spectroscopy
Nobel Conference Lectures
With 179 Figures
i
Springer
Professor Dr. Rudolf Rigler
Karolinska Institutet, Department of Medical Biophysics,
S-17177 Stockholm, Sweden
e-mail: [email protected]
Dr. Michel Orrit
CNRS et Universite Bordeaux I, C.P.M.O.H.
351 Cours de la Liberation, 33405 Talence, France
e-mail: [email protected]
Professor Dr. Thomas Basche
Johannes Gutenberg-Universität, Institut für Physikalische Chemie
Jakob-Welder-Weg 11, 55099 Mainz, Germany
e-mail: [email protected]
Series Editors:
Professor EP. Schäfer Professor W. Zinth
Max-Phinck-Institut für Biophysikalische Chemie Universität München,
D-37077 Göttingen-Nikolausberg, Germany Institut für Medizinische Optik
Öttingerstr. 67
Professor T.P. Toennies D-80538 München, Germany
Max-Planck-Institut für Strömungsforschung
Bunsenstrasse 10
D-37073 Göttingen, Germany
ISSN 0172-6218
ISBN 978-3-642-62702-6
Library of Congress Cataloging-in-Publication Data
Single molecule spectroscopy : nobel conference lectures / [edited by 1R . Rigler, M. Orrit, T. Basche.
p. cm. --(Springer series in chemical physics, ISSN 0172-6218 ; 67)
ISBN 978-3-642-62702-6 ISBN 978-3-642-56544-1 (eBook)
DOI 10.1007/978-3-642-56544-1
1. Molecular spectroscopy. I. Rigler, Rudolf. 11. Orrit, M. (Michel), 1956-III. Besche, T. IV. Springer series in
chemical physics ; v. 67.
QC454.M6 S57 2002
535.8'4--dC21 2001049365
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Preface
One often hears that nanoscience or, in other words, the knowledge and
control of matter at length scales of a few nanometers, will be the scientific
frontier of the 21st century. Although it has become almost commonplace, this
prediction deserves some justification. The technological and scientific stakes
of nanoscience indeed encompass many fields of science: they include the
ultimate miniaturization of electronic devices to acquire, store, and process
information, and also such basic endeavors as understanding the microscopic
processes and patterns responsible for the physical properties of materials, or
the many unsolved questions raised by the astoundingly intricate workings
of living matter. Although the dream of observing and controlling matter
at molecular scales is nearly as old as the very concept of molecules, earlier
attempts at practical realizations were hampered by a scarcity of suitable
access to the nanoworld.
During the last two decades of the 20th century, owing to the several
new tools which have been developed to address objects at nanometer scales,
the nanoworld appears closer than ever, within our reach! A major class
of methods in nanoscience are local probe microscopies such as scanning
tunnelling or atomic force microscopies. They require scanning a sharp tip
with molecular dimensions across the surface of the sample under study and,
by direct action of the tip on the sample, they make nano-manipulations
possible.
The present book is devoted to another class of methods, the selection
and study of single, optically active nano-objects by purely optical means.
The selected objects often are organic molecules, but the same techniques
apply to nano-crystals, self-assembled quantum dots, metal nano-particles,
etc. The central idea of these methods is to isolate the optical signal from
a small area of a sample, where at most one active molecule can interact
with the exciting laser light. The optical observable is most often fluores
cence or luminescence, but other responses can also be used, for instance
optical absorption, or surface-enhanced or resonance Raman scattering. The
optical addressing of single objects offers specific capabilities that make these
methods complementary, and sometimes even superior to scanning local mi
croscopies, although their direct spatial resolution is inherently limited by
the wavelength of light.
Preface
VI
Optical illumination of a sample is usually non-intrusive, because it re
quires only very weak perturbations, which is essential for delicate biologi
cal structures. In contrast to scanning tips, photons can penetrate beyond
surfaces and interrogate single molecules in their natural environments. In
addition, a wide variety of spectroscopic optical techniques have been de
veloped since the advent of lasers, for frequency-resolved and time-resolved
spectroscopy. By applying laser radiation to single molecules, one can in prin
ciple transfer this whole toolbox of spectroscopies down to the nanoworld.
Single molecule spectroscopy and microscopy have spread at a quick pace
in the past decade, and are poised for further expansion as major avenues in
nanoscience, either by themselves or in combination with scanning probe mi
croscopy and manipulation. Their specific advantages are those of optics com
bined with those of microscopy in general. Addressing single objects radically
removes all averaging involved in conventional measurements on populations.
Not only does this provide a much clearer picture of individual behaviors, and
facilitate comparison with theoretical models, it also delivers novel observ
abIes such as statistical distributions, correlations, dynamical fluctuations,
etc., which by definition vanish upon averaging over macroscopic popula
tions.
The observation of fluctuations is particularly new and important, since all
earlier kinetic studies of large populations in chemistry and biology invariably
required one necessary step: the synchronization of all individuals. Single
molecules for the first time provide direct insight into natural fluctuations,
and the potential of this insight for biomolecules is tremendous. Similarly,
following single molecules as a function of time can reveal rare but significant
events that would disappear upon averaging. Rutherford's discovery of the
atomic nucleus is a well-known illustration of the far-reaching insight provided
by individual events. The same argument could soon apply to the fleeting
intermediates involved in chemical reactions or in protein folding.
Much of the recent progress in optical science and applications is owed to
lasers. More than four decades after its discovery, laser radiation still finds
new uses, notably in studying and manipulating small objects in condensed
matter. By trapping small particles optically (with so-called optical traps
and optical tweezers), we can now measure the tiny forces causing bonding
or conformational changes in single molecules, even when they are weak in
termolecular interactions. A. Ashkin (Chap. 1) gives a historical perspective
on the development of laser trapping, which has led to new insights in bio
physics, as well as to the burgeoning physics of ultracold atoms. The general
trend of addressing smaller and smaller particles has ultimately led to the
manipulation and observation of single molecules, for which laser radiation is
also central. The first single-molecule experiments were done with absorption,
as W.E. Moerner recalls (Chap. 2), and later pursued by fluorescence excita
tion, which is the major method employed currently. The problem of how to
measure the absorption signal of a single molecule under ambient conditions
Preface vii
remains one of the major challenges in the field. The first single-molecule
fluorescence experiments were done initially in 1990, either in a flowing so
lution at room temperature or in a solid matrix at cryogenic temperatures.
In 1993, near-field optical microscopy led to the observation of single mole
cules immobilized on surfaces at room temperature. Soon thereafter, around
1994, confocal microscopy was successfully applied to the same end. Since
confocal microscopy is a standard method, several groups started working on
single molecules and observed the attending phenomena of blinking, photo
bleaching, bunching, etc. A major motivation in the work on single molecules
under ambient conditions is the drive towards the exploration of biological
processes. This has now become the most important field of application of
single molecule spectroscopy.
At liquid helium temperatures, a particular object can often be observed
for a long time, and be used as a test object for high-precision physics, as
illustrated by several contributions. By combining cryogenic temperatures
with single-frequency laser excitation, one achieves very high spectral reso
lutions, which can be used to investigate molecular physics very accurately.
Some examples are provided in the review by Moerner (Chap. 2), in partic
ular the magnetic resonance of single molecules with well resolved excitation
and emission lines. Lower spectral resolutions (of the order of 1 cm-1) can
be extremely useful to elucidate the electronic structure of complex systems,
as demonstrated in the case of single bacterial antenna complexes (Chap. 3).
In the field of biophysics, low temperatures can help stabilize a structure and
resolve complex electronic spectra. The work presented in Chap. 3 is a spec
tacular demonstration of the power of single-molecule spectroscopy: although
the B800 and B850 absorption bands of a large population look similar, the
spectra of single complexes and bandwidths differ profoundly, highlighting
the differences in intermolecular interactions in these two coupled electronic
systems.
A single molecule is also a sensitive probe for all dynamical processes
taking place in its neighborhood. Using single molecules, it has been possible
to investigate tunneling in crystals and in disordered solids, or to study the
bistable conformations of a molecule and of its first solvent shells, as nicely
illustrated with terrylene in p-terphenyl crystals. Several experiments and
simulations converge to give a picture of the molecular motion involved, a flip
of the central phenyl ring of a neighbor p-terphenyl molecule (Chap. 4). If the
molecule and its solvent shell are stable enough, they can serve as a model
optical two-level system, and be used to investigate light-matter interactions.
This is illustrated with the description of a triggered source of single photons
based on a single molecule (Chap. 5), where an adiabatic passage is used to
bring the molecule into its excited state with high probability.
In physical chemistry, single molecules or small particles can probe in
homogeneous systems. The photophysical properties of conjugated polymers
reflect the sample heterogeneities which are caused by molecular weight dis-
viii Preface
tributions and conformational defects on the polymer backbone. By studying
single fluorescent conjugated polymer molecules (Chap. 6) a more detailed
picture of intramolecular electronic energy relaxation is obtained, as well
as a correlation between photophysical properties and specific polymer con
formation. An artificial liposome is a minuscule test tube, in which single
molecules can be trapped and preserved from external reactants. Wilson et
al. (Chap. 7) show how such liposomes can be made and manipulated with
micropipettes. Metal nanostructures can lead to strong enhancement of local
optical fields, which enables surface-enhanced Raman scattering. As Kneipp
et al. show (Chap. 8), SERS can reach single molecule sensitivity. It thus pro
vides molecular fingerprints, with the added advantage that photob leaching is
eliminated. In many fluorescence experiments, it is important to distinguish
between different fluorophores. This is usually done by spectral filtering, or by
lifetime measurements. Hubner et al. (Chap. 9) demonstrate that by combin
ing spectral and time-resolved information, the number of photons required
to discriminate two fluorescent labels can be reduced dramatically.
The developments which lead to the observation of single molecules in
solution at room temperature have opened the field of single-molecule ana
lysis in biosciences (Chap. 10). Fluorescence correlation spectroscopy (FCS)
has proven to be a powerful technique which permits the observation of the
dynamics ·of biomolecules down to the single-molecule level. Wohland et al.
(Chap. 11) employ FCS to characterize receptor proteins. Extensions of the
technique to dual-color cross-correlation schemes enhance the throughput
rate for screening applications in biochemistry and evolutionary optimiza
tion processes (Chap. 12). The feasibility of analyzing the behavior of a sin
gle biomolecule in relation to an ensemble has given novel information on the
function of biomolecules such as enzymes. Several contributions show that
single enzyme molecules undergo a series of conformational transitions be
fore catalysis can occur. Single molecule experiments show the existence of
conformational fluctuations in the enzyme-cosubstrate complex (Chap. 13)
and product formation (Chap. 10), as well in turnover rates (Chap. 14).
These results are consistent with and lend strong support to the model of
conformational substates proposed by Frauenfelder (Chap. 15). Closely re
lated is the study of intramolecular dynamics in small enzyme populations
(Chap. 16). Spectacular results have been achieved by the analysis of molecu
lar motors at the single molecule level (Chap. 17), as well as from the analysis
of the folding of single protein molecules (Chap. 18). The rapid development
of single-molecule analysis now covers a broad variety of biological mole
cules, including nucleic acids (Chap. 19), and proteins of differing complexity,
such as enzymes, contractile elements, and light-emitting proteins like GFP
(Chap. 20). For biological systems, single-molecule analysis is of prime inter
est, since ensemble behavior can be deduced from the single molecule scenario
but not vice versa. Fundamental biological processes such as self-replication,
including evolution and selection, are basically single-molecule processes. In
Preface ix
summary, the contributions in this field can be seen to be the starting point
of very dynamic development in the near future.
This volume presents the current picture of activities in single-molecule
spectroscopy at the dawn of the 21st century. This is an active and interdisci
plinary field, which is likely to profoundly change in coming years, as single
molecule techniques propagate into different areas of application. Therefore,
a prediction of future developments is particularly risky. The optical probing
of complex systems with single molecules gives a detailed picture of molecular
movements and structure at nanometer scale. This new tool can be applied to
many problems in physical chemistry and material science, such as wetting,
adhesion, and friction. A particularly promising application will be the com
bination of optical single-molecule microscopy on the one hand, and raster
scanning techniques Or electron microscopy on the other, to arrive at a COr
relation between the spectroscopic and structural properties of one and the
same single nanoparticle.
Interestingly, while biochemical reactions have been studied at the single
molecule level, experiments at the very heart of chemistry, i.e. the study of
classical chemical reaction mechanisms Or product distributions, still appear
mainly as a challenging endeavor. The main area of activity will probably re
main molecular biology. The many unanswered mechanistic questions about
the structure, dynamics, and functions of biomolecules in their natural envi
ronments could start to be answered in the next few years. Already, single en
zymes have been investigated over long time scales, the docking of a substrate
onto an enzymatic site and the subsequent conformational changes of the en
zyme can be followed by fluorescence resonance energy transfer (FRET), in
combination with force measurements, and the working of molecular mo
tors has been considerably clarified, if not elucidated. The current challenges
of single-molecule methods under ambient conditions are the blinking and
photo-bleaching of the active particles, molecules, or nanocrystals. If these
problems can be solved, the future of optical single-molecule methods will be
quite bright indeed!
Mainz, Talence, Stockholm T. Basche
August 2001 M. Grrit
R. Rigler
Contents
Preface................................. .. ................ .... v
1 History of Optical Trapping and Manipulation of Small
Neutral Particles, Atoms, and Molecules
A. Ashkin...................................................... 1
2 Thirteen Years of Single-Molecule Spectroscopy
in Physical Chemistry and Biophysics
W. E. Moerner .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . .3 2. . . . . . . . . . . .
3 The Electronic Structure of Single Photosynthetic
Pigment-Protein Complexes
A. M. van Oijen, M. Ketelaars, J. Kohler, T. J. Aartsma, J. Schmidt.. 62
4 Single-Molecule Optical Switching: A Mechanistic Study
of Nonphotochemical Hole-Burning
F. Kulzer, T. Basche ........... .......... .. .... .. ............... 82
5 Triggered Emission of Single Photons by a Single Molecule
C. BruneI, P. Tamarat, B. Lounis, M. Orrit ........................ 99
6 Photophysics of Conjugated Polymers Unmasked by Single
Molecule Spectroscopy
J. Yu, D.-H. Hu, P. F. Barbara .. ... .. ..... ... ... .............. ... 114
7 Confining and Probing Single Molecules
in Synthetic Liposomes
C. F. Wilson, D. T. Chiu, R. N. Zare, A. Stromberg, A. Karlsson,
O. Orwar ..... .. ................... ... ......... ....... ..... .... 130
8 Single Molecule Detection Using Near Infrared
Surface-Enhanced Raman Scattering
K. Kneipp, H. Kneipp, 1. Itzkan, R. R. Dasari, M. S. Feld .. .......... 144
9 Single-Molecule Fluorescence - Each Photon Counts
C. G. Hiibner, V. Krylov, A. Renn, P. Nyffeler, U. P. Wild ........... 161
