Anne Myers Kelley, Xavier Michalet, Shimon
Single-Molecule Spectroscopy Comes of Age
In the summer of 1999, researchers working in the field
of single-molecule biophysics gathered for a 1-week workshop in the French
town of Tours (1). Sensing the birth of a new discipline,
the participants termed the workshop "the Woodstock of single molecules,"
but their enthusiasm was mainly about future promise: It was clear that
a considerable amount of work would be required to extend and validate
methodologies originally developed for solids at cryogenic temperatures
(2) to materials and biological systems (3).
In April of this year, a symposium at the American Chemical Society
Meeting in San Diego (4) demonstrated that single-molecule
spectroscopy is beginning to deliver on its promise. Chemists, physicists,
and biologists gathered for nearly 60 talks on topics as diverse as low-temperature
dynamics of single-dye molecules embedded in crystals (M. Orrit, Bordeaux
I University), optical tracking of the entry of individual viruses into
live cells (C. Bräuchle, Ludwig-Maximilian-University, Munich), single-photon
light sources from single molecules (W. E. Moerner, Stanford University)
(5), polymer conformations and dynamics (P. Barbara,
University of Texas, Austin; R. Dickson, Georgia Tech) (6-8)
and the mechanisms of single enzymatic motors (K. Thorn, University of
California, San Francisco; K. Kinosita Jr., Keio University; T. Yanagida,
Osaka University) (9, 10). The
broad range of topics illustrates the power of optical approaches for studying
single molecules or molecular assemblies. Very few talks emphasized methodologies
but rather concentrated on the new scientific knowledge being gained, demonstrating
that single-molecule spectroscopy is reaching maturity (11).
One reason for the burgeoning interest in single-molecule optical
techniques is that photons may be the least perturbing probe of the state
of a molecule (although in different power regimes, lasers can be used
to exert a substantial force on atoms, molecules, and beads). Different
spectroscopic signatures can be used, of which fluorescence is the most
popular because of its inherent sensitivity. Fluorescence studies can be
performed on biomolecules that have intrinsic fluorophores, such as photosynthetic
light-harvesting complexes (J. Schmidt, Leiden University) or enzymes containing
flavin cofactors (S. Xie, Harvard University). More often, however, a fluorescent
moiety is attached to the species under study by genetic engineering such
as tagging with green fluorescent protein (Thorn; T. Schmidt, Leiden University),
by targeted chemical functionalization as in site-specific protein labeling
(P. Lu, Pacific Northwest National Laboratory), or by chemical modification
of DNA or RNA bases (S. Chu, Stanford; M. Ishikawa, Joint Research Center
for Atom Technology, Tsukuba).
Another powerful spectroscopic tool at the single-molecule level
is Raman (T. Basché, University of Mainz) or surface-enhanced Raman
scattering (M. Käll, Chalmers University of Technology; S. Nie, Indiana
University; L. Brus, Columbia University). Raman spectra contain more detailed
information about molecular structure than fluorescence spectra and can,
for the most active surface-enhanced sites, provide photon count rates
competitive with fluorescence (K. Kneipp, Massachusetts Institute of Technology).
The most fundamental reason for the excitement about single-molecule
spectroscopy is that ensemble studies give only an average picture of the
properties exhibited by individual molecules that are not identical. In
some cases, the heterogeneity arises from definable chemical differences
such as the extent of glycosylation, as seen in the intermolecular variations
in catalytic rates in mammalian alkaline phosphatase (N. Dovichi, University
of Washington). But intermolecular variations often persist even when there
are no chemical differences. This seems to fly in the face of the ergodic
theorem, which states that a measurement of some property of an ensemble
at a given time should be equivalent to the long-time average of the same
property on any one member.
The key is the time scale over which the averaging is performed.
Single-molecule measurements are interesting only for systems in which
different molecules retain distinguishably different behavior on time scales
longer than that necessary (from signal-to-noise ratio considerations)
to make a measurement. It turns out that a great majority of biochemical
and material systems fall in this category. Single-molecule studies often
reveal temporal fluctuations of the recorded quantity ("dynamic disorder"),
which provide insight into fluctuation time scales hidden in bulk measurements.
There may also be large variations from molecule to molecule ("static disorder").
The distinction between dynamic and static disorder depends on the time
resolution of the experiment, and intermediate situations may arise.
Studies of single embedded molecules are providing a wealth of
information on the local properties of complex materials such as biological
and biomimetic membranes (G. Harms, Pacific Northwest National Laboratories;
R. Dunn, University of Kansas) (12), inorganic semiconductors
(S. Buratto, University of California, Santa Barbara), and light-emitting
organic polymers (Barbara) (7). The emission spectra
and diffusion constants of single embedded dyes are valuable probes of
the highly heterogeneous local environments in polymer or glass matrices
(Deschenes; Dickson; D. Higgins, Kansas State University) (6,
(see the first figure).
Single-molecule dynamics in polymers. Orientational trajectories
of three dye molecules embedded in poly(methyl methacrylate) are probed
with wide-field single-molecule optical microscopy (6).
The different orientational dynamics of the different molecules illustrate
the heterogeneity observed within polymer hosts even 80°C below the
glass transition temperature. The cartoon shows how differences in local
free volume might give rise to these differences in rotational mobility.
CREDIT: TRAJECTORY PLOT/ROBERT DICKSON
Time-resolved measurements on single molecules also provide powerful
insights into the rates and mechanisms of kinetic processes that are fundamentally
stochastic: Even if all members of the ensemble could be synchronized to
begin at the same time, they would rapidly run out of phase with one another,
obscuring the kinetic steps. Beautiful illustrations of single-molecule
dynamical studies of this type were provided by several speakers (Yanagida,
Chu, Xie, and Kinosita). Kinosita, for instance, illustrated the power
of a simple dark-field laser illumination to monitor the stepwise submillisecond
dynamics of the F1-adenosine triphosphate (ATP) synthase rotary
motor (10) (see the second figure).
Single-molecule dynamics in biomolecules. When isolated, the
F1 part of ATP synthase hydrolyzes ATP, leading to rotation
of its shaftlike g subunit within an ab-trimer.
The system has a threefold rotational symmetry, and the g
subunit is therefore expected to rotate by discrete 120° steps. Because
of the stochastic nature of the motion, single-molecule techniques are
required to resolve these individual steps. Yasuda et al. (10)
attached a fluorescent molecule or gold bead to individual protein molecules
and imaged them with a laser dark-field microscope and an ultrafast camera,
allowing the 120° jumps of individual molecules to be observed on the
submillisecond time scale. At low ATP concentrations, each 120° step
can be resolved into substeps of 90° and 30°, which correspond
to ATP binding and the release of the hydrolysis products, respectively
CREDIT: DATA PLOT, ADAPTED FROM (10)
Several papers indicated that multiparameter studies (measuring, for
example, fluorescence intensity, lifetime, polarization anisotropy, and
spectral peak position at the same time) are needed to develop a complete
picture of the molecular state and environment of single molecules [Harms;
C. Seidel, Max Planck Institute, Göttingen; M. Sauer, University of
Heidelberg; C. Hübner, Eidgenössische Technische Hochschule (ETH)]
(14-16). The finer characterization
gained by multiparameter observation allows variations resulting from multiple
states of a single molecule to be distinguished more easily from variations
arising from multiple molecular species. This allows distinct species to
be identified with a reduced number of detected photons, increasing the
effective time resolution of single-molecule identification schemes (Sauer;
Several speakers described optical configurations that enable
simultaneous time-and wavelength-resolved measurements, which will probably
become standard in future studies. The additional wealth of data will,
in turn, call for new data reduction schemes. Hidden-Markov models (D.
Talaga, Rutgers University), multidimensional correlation functions (J.
Cao, Massachusetts Institute of Technology), and other techniques will
have to be developed to go beyond mere comparison of bulk average values
to single-molecule histograms (17). These and other
methods will have to be imported from research fields accustomed to dealing
with large, complex data sets. In addition, more elaborate physical models
will be needed to interpret the increasingly detailed data that will emerge
from future experiments (E. Geva, University of Michigan; T. Plakhotnik,
ETH; P. Wolynes, University of California, San Diego).
Single-molecule optical studies have clearly come of age. These
techniques are being applied to an increasing number of problems in chemistry,
physics, and biology. In combination with single-molecule manipulation,
microfluidics, and microelectromechanical systems, they will open up ever
more possibilities for new discoveries.
References and Notes
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"Optical Studies of Single Molecules and Molecular Assemblies in Chemical
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For recent reviews, see the special issues of Science [283,
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Physics [247, 1-184 (1999)], and Cytometry [36,
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A. M. Kelley is in the Department of Chemistry, Kansas State
University, Manhattan, KS 66506, USA. E-mail: email@example.com.
X. Michalet and S. Weiss are in the
Materials Sciences and Biophysical Sciences Divisions, Lawrence Berkeley
National Laboratory, Berkeley, CA 94720, USA.