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Spectroscopy and
Dynamics of Single
Molecules
Developments in Physical &
Theoretical Chemistry
Series Editor
James E. House
With the new series Developments in Physical & Theoretical Chemistry, Elsevier
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tions from those who are rising in prominence will also be included.
2018 Physical Chemistry of Gas–Liquid Interfaces

Jennifer A. Faust and James E. House, Editors
2019 Mathematical Physics in Theoretical Chemistry
S.M. Blinder and J.E. House, Editors
2019 Spectroscopy and Dynamics of Single Molecules
Carey K. Johnson, Editor
Developments in Physical &
Theoretical Chemistry
James E. House, Series Editor
Spectroscopy and
Dynamics of Single
Molecules
Methods and Applications
Edited by
Carey K. Johnson
Department of Chemistry, University of Kansas,
Lawrence, KS, United States
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Contributors
Charuni A. Amarasekara
Department of Chemistry and the Bioengineering Program, University of Kansas,
Peter M. Goodwin
Center for Integrated Nanotechnologies, Materials Physics and Applications,
Los Alamos National Laboratory, Los Alamos, NM, United States
George Hamilton
Department of Physics, Clemson University, Clemson, SC, United States
Colin D. Heyes
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville,
AR, United States
Carey K. Johnson
Department of Chemistry, University of Kansas, Lawrence, KS, United States
Daniel M. Kalb
Lydia Kisley
Department of Physics, Case Western Reserve University, Cleveland, OH,
United States
Don C. Lamb
Department of Chemistry, Center for NanoScience, Nanosystems Initiative
Munich (NIM), and Center for Integrated Protein Science Munich (CIPSM),
Ludwig Maximilian University of Munich, Munich, Germany
Marcia Levitus
School of Molecular Sciences; The Biodesign Institute, Arizona State University,
Tempe, AZ, United States
Demosthenes P. Morales
Evelyn Ploetz
Duncan P. Ryan
xi
xii Contributors
Clemens-B€
assem Salem
Hugo Sanabria
Department of Physics, Clemson University, Clemson, SC, United States
Steven A. Soper
Swarnagowri Vaidyanathan
Kumuditha M. Weerakoon-Ratnayake
James H. Werner
CHAPTER
Introduction
Carey K. Johnson
Department of Chemistry, University of Kansas, Lawrence, KS, United States
It has been thirty years since the detection of single molecules was first reported.1 In
the intervening decades the field has grown explosively. Applications vary widely,
from materials to cellular biology, and the field has spawned new and fruitful
research directions and methods, highlighted by super-resolution microscopy2,3—a
growing array of methods for peering into cells with unprecedented precision.
The goal of this volume is to provide a concise introduction to new techniques
and applications of single-molecule spectroscopy. My hope is that experimentalists
will value the volume as an introduction to new techniques they may wish to imple-
ment, while theoretical and computational chemists will find a description of tech-
niques and applications that may be potential candidates for theoretical treatment. A
further aim is to provide graduate students with a concise introduction to single-
molecule experimental techniques and methods of data analysis.
The added information gained by probing single molecules is well rehearsed
in the literature: removing the ensemble average to access the full distribution;
circumventing the need for synchronization in studies of dynamics; the possibility
to observe rare events; and accessibility to new photophysical and photochemical
phenomena. In addition, single-molecule approaches have allowed researchers to
ask and begin to answer new questions: How does the behavior of individual mol-
ecules lead to the overall properties observed in complex systems? How do molec-
ular machines work, step-by-step? What molecular interactions and events are
important for chemical catalysis or separations? How do molecular conformations
interchange?
Historical overview
Single-molecule spectroscopy emerged from three independent streams of research
in the early 1990s, each leading to detection at the single-molecule level. The first
was low-temperature spectroscopy in solids. In an effort to resolve the homogeneous
linewidth of molecules within an inhomogeneous broadened band, researchers inves-
tigated the spectroscopy of guest molecules in host matrices at low temperature by
Spectroscopy and Dynamics of Single Molecules. https://doi.org/10.1016/B978-0-12-816463-1.10000-8
3
# 2019 Elsevier Inc. All rights reserved.
4 CHAPTER Introduction
methods such as hole-burning spectroscopy.4,5 With lower and lower dopant concen-
tration, statistical fine structure could be detected in the dopant absorption band by
frequency-modulation spectroscopy.6,7 Spurred by the detection of structure trace-
able to statistical fluctuations in the numbers of absorbing molecules, Moerner
and Kador1,8 reported single-molecule absorption of pentacene molecules in a host
p-terphenyl crystal.
Although the detection of light absorption by single-molecules was a tour-de-
force of sensitive detection of a small signal riding on a large background, it soon
became clear that laser-induced fluorescence would be much more sensitive and ver-
satile for detection of single molecules. To detect fluorescence from single mole-
cules, it is necessary that the molecule have a high absorption cross section and
that it emit photons at a high rate compared to the rates of other photophysical pro-
cesses such as intersystem crossing or photochemical processes such as photobleach-
ing. In addition, a high-efficiency optical setup is required to collect fluorescence
photons over a large solid angle, and these photons must be sensitively registered
by a detector with low dark noise. In 1990, Orrit and Bernard reported single-
molecule detection of pentacene in p-terphenyl by fluorescence at low temperature,9
and in 1991 Ambrose and Moerner reported spectral jumps in the excitation band of
pentacene detected by fluorescence at low temperature.10
A second research stream leading to single-molecule detection had its source in
ultra-sensitive fluorescence detection of molecules in room-temperature liquids for
applications such as flow cytometry, DNA sequencing, immunofluorescence
assays,11 and fluorescence correlation spectroscopy (FCS). As early as 1976, Hirsch-
feld reported detection of polyethyleneimine conjugated with 80 to 100 fluorescein
dye molecules.12 The Keller group in 1987 and the Mathies group in 1989 reported
the detection of single molecules of phycoerythrin (containing 34 bilin chromo-
phores) in flowing liquid.13,14 Detection of single molecules of rhodamine 6G dye
molecules in solution was achieved initially with pulsed laser excitation and time-
gated detection to reduce background from Rayleigh, Raman, and specular scatter-
ing,15 and soon after with CW excitation.16 It soon became possible to observe fluo-
rescence bursts from single molecule in real time17,18 and to measure single-
molecule fluorescence lifetimes.19,20 FCS was first introduced in the 1970s,21–23
but applications surged in the 1990s with the availability of sensitive photon-
counting detectors such as avalanche photodiodes and the application of confocal
microscopy to reduce probe volumes in the labs of Rudolf Rigler and Elliot
Elson.24–27 Although not strictly a single-molecule technique—the correlation in
FCS is taken over contributions from many molecules—the raw data stream in
FCS when applied at sufficiently low concentrations contains photon bursts from
molecules diffusing through the probe volume.27,28
The third research stream contributing to single-molecule detection was scanning-
probe microscopy. Although single-molecules had been detected at low temperature,
it was not immediately obvious that single-molecules could be imaged at room
temperature. After all, the oscillator strength of molecules at room temperature is
spread over a broad absorption band, so that the absorption cross section and therefore
Beyond intensity: expanding techniques 5
the brightness of a single molecule at a given excitation wavelength are lower than in a
low-temperature solid. The fleeting passage of single molecules in solutions was
detected by 1990, as described above, but extended observation of single molecules
at room temperature was not reported until 1994 by scanning probe microscopy. Scan-
ning tunneling microscopy29 and atomic force microscopy30 had been developed in
the 1980s. With the goal of imaging single fluorophores with sub-diffraction spatial
resolution at interfaces at room temperature, Betzig and Chichester coupled scanning–
probe microscopy with near-field optics, a technique known as near-field scanning
optical microscopy (NSOM).31 Fluorophores were excited by the evanescent field
emerging from an aperture or tip in near-field, i.e. at distances much less than the wave-
length of the excitation light. Scanning of the tip allows resolution down to a few
nanometers. The method was applied to detection of the emission spectrum of a car-
bocyanine dye with near-field excitation,32 fluctuations in the fluorescence intensity
of single sulforhodamine dye molecules,33 and measurement of single-molecule
fluorescence lifetimes.34 Subsequent applications demonstrated imaging of photosyn-
thetic light harvesting complexes35 and allophycocyanin trimers.36
Beyond intensity: Expanding techniques

Far-field single-molecule imaging at room temperature was reported in 199637 set-
ting the stage for new applications of single-molecule fluorescence spectroscopy.
Far-field excitation quickly became the norm for single-molecule detection. Most
current implementations of single-molecule spectroscopy use far-field imaging at
room temperature.
Researchers attracted by the advantages of probing single molecules—resolving
distributions, tracking dynamics, detecting rare events or interactions—soon sought
additional dimensions of information from single-molecule experiments. Methods
well known from ensemble fluorescence were applied or adapted to single-molecule
detection. Fluorescence decays of single molecules were measured early.19,33 The
added information was used to identify molecules in solution,38 probe electron trans-
fer kinetics,39 and to detect conformational changes of RNA40 and DNA.41 Polari-
zation methods were also brought into play. The polarization of the excitation beam
can be modulated to probe the orientation31,33 or orientational mobility42 of single
molecules. Selection of fluorescence polarization permits detection of fluorescence
anisotropy43,44 and anisotropy decay.45
One of the most powerful fluorescence techniques at the single-molecule level is
F€orster resonance energy transfer (FRET). FRET with a single donor-acceptor pair
was introduced by Ha et al.46 coupled with NSOM. Since that time, single-pair FRET
coupled with wide-field imaging or confocal microscopy47,48 has become one of the
most widely used single-molecule methods.49–51
Methods were also developed to track single molecules in space. Inherent in
single-molecule tracking is localization of single molecules to a precision better
than the resolution limit.52–55 Localization techniques developed for single-
molecule applications are intrinsic to methods of super-resolution fluorescence

microscopy,56–59 the development of which led to the 2014 Nobel Prize in
Chemistry.2,3
New applications
Whereas initial publications in single-molecule spectroscopy demonstrated feasibil-
ity and introduced basic techniques, subsequent work expanded to an increasingly
wide range of applications. Single-molecule spectroscopy has by now been applied
in many fields of research (see Fig. 1). Its biggest impact has perhaps been felt in
molecular and cellular biology. (Among reviews too numerous to cite comprehen-
sively, see Refs. 60–64.) To cite a few additional examples, it has also been applied
to polymers,65,66 catalysis,67 and optoelectronics.68
Single-molecule spectroscopy is now a mature field, one where the technology
and methodology are largely established. Single-molecule methods have become a
resource in studies of physical, chemical, and biological systems, one very power-
ful tool among many. Advances involve applications to more and more complex
systems—living cells or chromatographic systems are two examples—and, by
and large, refinements of existing methods. One is now as likely to find single-
molecule spectroscopy mentioned in a conference session on enzyme function
or solar energy conversion as in a specialized session on single molecule spectros-
copy. In this way, the method will impact a broad and multidisciplinary array of
systems and fields.
FIG. 1
Distribution of papers in single-molecule fluorescence since 1989. Applications in chemistry
and molecular biology/biochemistry dominate, with significant activity in biophysics and cell
biology. Single-molecule fluorescence has also had important impacts in physics, materials,
and nanoscience. A topic search for “single-molecule fluorescence” was carried out in Web of
Science.
Topics covered in this book 7
Single-molecule spectroscopy has been the topic of numerous reviews. Only a

handful can be mentioned here, with apologies for the many excellent review articles
that are not mentioned. A retrospective review of the origins of single-molecule
detection can be found in Ref. 69. Other reviews include Refs. 50, 62, 63, 70–72.
Topics covered in this book

No book could comprehensively cover the field of single-molecule spectroscopy at
the depth the field deserves. This volume focuses on basic techniques and their appli-
cations in chemistry and molecular and cellular biology. Important topics not cov-
ered in this volume include single-molecule techniques other than fluorescence
(single-molecule absorption or Raman spectroscopy) and applications in fields such
as condensed matter physics or nanophotonics.
In Chapter 1, Marcia Levitus examines the photophysics of single-molecule
probes and methods to understand the role of probe photophysics in applications.
One of the earliest single-molecule observations was “blinking.”73 Professor Levitus
describes how blinking and other photophysical phenomena can be characterized and
taken into account in interpreting single-molecule results.
In Chapter 2, Clemens-B€assem Salem, Evelyn Ploetz, and Don C. Lamb describe
the application of FRET to probe dynamics in immobilized single molecules. FRET
has become one of the most powerful probes in the single-molecule toolkit. Salem
et al. describe methods to carry out single-molecule FRET experiments by wide-field
imaging and approaches to analyze results.
In Chapter 3, Lydia Kisley reviews single-molecule methods to find new infor-
mation about interactions and catalysis at interfaces. Professor Kisley shows that
single-molecule spectroscopy is particularly well suited to probing interfaces and
describes techniques and examples.
In Chapter 4, Colin D. Heyes introduces quantum dots as fluorophores for
single-molecule spectroscopy. While dyes and autofluorescent proteins are the most
common single-molecule fluorophores, Professor Heyes describes the fluorescent
properties and the advantages (and disadvantages) of quantum dots for single-
molecule spectroscopy.
In Chapter 5, Daniel M. Kalb, Duncan P. Ryan, Demosthenes P. Morales,
Peter M. Goodwin, and James H. Werner describes experimental approaches and
methods of analysis for three-dimensional single-molecule tracking in complex envi-
ronments such as living cells. Single-molecule fluorescence has made a particularly
strong impact in characterizing the translational mobility of molecules, first in two
dimensions and now in three dimensions.
In Chapter 6, George Hamilton and Hugo Sanabria describes the current state-of-
the-art in multi-parameter single-molecule fluorescence burst measurements. The
techniques developed for the detection of fluorescence bursts from single molecules
in solution have led to a rich and fruitful set of methods to characterize molecules in
solution by detection of single molecules bursts.
Chapter 7 by Kumuditha M. Weerakoon-Ratnayake, Swarnagowri Vaidyanathan,
Charuni A. Amarasekara, Carey K. Johnson, and Steven A. Soper describes the
application of single-molecule spectroscopy to detection and analysis of DNA in nano-
fluidic devices. Professor Soper and coworkers show how single-molecule detection
of DNA confined in nanochannels opens new dimensions for optical mapping and
diagnostic applications.
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CHAPTER
Photophysics of single-
molecule probes
1
Marcia Levitus*,†
*School of Molecular Sciences, Arizona State University, Tempe, AZ, United States
†
The Biodesign Institute, Arizona State University, Tempe, AZ, United States
1.1 Introduction
Researchers entering the field of single-molecule fluorescence face important deci-
sions that can potentially affect the outcome of the study, from selecting dyes, linker
types and labeling positions, to making decisions on whether and how to immobilize
molecules to surfaces, which buffers to use, etc. There are many variables that need
to be taken into account when designing and analyzing a single-molecule FRET
(smFRET) experiment, and new investigators are often disappointed to learn that
there is not always a consensus regarding optimal fluorescent probes and protocols.
A successful single-molecule fluorescence experiment requires a careful experi-
mental design to minimize artifacts and to ensure that the measured fluorescence
signals can be interpreted in terms of the biophysical or biochemical phenomena
of interest. Most commonly, smFRET experiments are performed to identify sub-
populations of molecules adopting different conformations within a sample,1–5 or
to investigate a dynamic process that changes the distance between two segments
of a biomolecule or between two interacting partners.6–12 A large number of
smFRET studies to date have focused on obtaining kinetic parameters for the inter-
conversion between different conformations that can be distinguished by their dif-
ferent FRET efficiencies (Fig.1.1). Kinetic information is typically obtained from
the analysis of time-dependent changes in fluorescence intensity (fluorescence tra-
jectories) obtained by acquiring the photons emitted by many individual molecules
over long times (usually seconds to minutes).14,15 A schematic example is given in
Fig. 1.1, which shows simulated donor and acceptor fluorescence trajectories (panel
B) for a hypothetical two-state system (panel A). Panel C shows the ratio of the
acceptor intensity to the sum of both intensities; a quantity usually referred to as
the proximity factor or the raw FRET efficiency (Eraw). In this scenario, the FRET
efficiencies are only used to distinguish between a small number of states with sig-
nificantly different donor-acceptor distances.16–20 Precise knowledge of the donor-
acceptor distances is not needed because the experiment is designed with sufficient
Spectroscopy and Dynamics of Single Molecules. https://doi.org/10.1016/B978-0-12-816463-1.00001-8
15
# 2019 Elsevier Inc. All rights reserved.
16 CHAPTER 1 Photophysics of single-molecule probes
FIG. 1.1
A- Hypothetical protein labeled with a donor (D) and acceptor (A) pair that can exist in two
conformations with donor-acceptor separations of R1 and R2. The two distances are assumed
to lead to clearly distinguishable FRET efficiencies. B- Hypothetical donor-acceptor single-
molecule traces (trajectories) for this protein. The traces were simulated assuming that the
laser excites preferentially the donor, so acceptor fluorescence arises exclusively from energy
transfer. The last transition is due to photobleaching of the acceptor, which leads to a recovery
of donor fluorescence. C- Raw energy transfer efficiency calculated as the ratio of the
acceptor intensity to the sum of the acceptor and donor intensities. For this assumed two-
state system, the value denoted by * is due to the conformation in which the inter-dye
distance is R1, and ** denotes the conformation in which the inter-dye distance is R2. The
reader should note that although the figure was constructed around known structures of
phosphoglycerate kinase, the labeling positions and simulated traces are hypothetical and not
necessarily consistent with the behavior of this protein.
prior structural knowledge of the system to allow assignment of the observed FRET
states to expected conformations of the proteins and nucleic acids (e.g. low FRET
for an open conformation and high FRET for a closed conformation, see Fig. 1.1). In
fact, such an experiment often does not require precise determinations of the FRET
efficiencies as long as the measurement can yield approximated values that can be
used to distinguish the different populations. This type of smFRET approach is
extremely powerful, but limited and even potentially misleading if the researcher
does not have sufficient knowledge of the possible conformations being probed
in the experiment.
With sufficient corrections and ancillary experiments, smFRET experiments can
yield accurate FRET efficiencies (i.e. the true quantum efficiency of energy transfer,
see Section 1.2.4), which can in turn be used to estimate donor-acceptor distances
with remarkable precision.22,23 These FRET-derived distances can be used as con-
straints in the determination of structural models in conjunction with computational
methods and other experimental biophysical techniques.23–27 As will be discussed in
detail in this chapter, the determination of a precise FRET efficiency and donor-
acceptor distance requires a thorough characterization of the photophysical proper-
ties of the fluorophores used to probe the system.
1.1 Introduction 17
There are many variables that need to be taken into account when designing a
single-molecule experiment. The choice of fluorescent dye, immobilization proto-
col, and even the buffer composition can affect the outcome of the experiment. Once
data is acquired, the correct interpretation of the fluorescence intensities in terms of
FRET efficiencies and ultimately in terms of distances, requires a profound under-
standing of the variables that affect the measured signals. The goal of this chapter is
to bring attention to the considerations and control experiments that are important to
avoid artifacts or misinterpretations of the data, while maximizing the accuracy of
the kinetic and structural information obtained from the experiment. This chapter
will focus on the critical role of fluorophore photophysics on the design and inter-
pretation of single-molecule experiments. Emphasis will be placed on understanding
how to prevent or account for contributions to the fluorescence signals that originate
from confounding photoinduced transitions between different electronic states of the
molecule (photophysical processes). Other variables such as surface effects or arti-
facts introduced by attachment of the extrinsic probe will be discussed only briefly
(Section 1.1.1). To motivate the need for this discussion, we will start by describing
two published examples of single-molecule experiments whose correct interpreta-
tion relies on considering the photophysical behavior of the probes
(Section 1.1.2). This will be followed by an introductory summary of photophysical
concepts and terms (Section 1.2), which will facilitate a rigorous quantitative discus-
sion of how the events that follow photoexcitation ultimately contribute to the mea-
sured fluorescence signals (Sections 1.3 and 1.4). Section 1.5 will then focus on
different aspects of how photophysical processes impact the interpretation of
single-molecule FRET experiments, and to conclude, the chapter will end with an
overview of the properties of the most common families of fluorophores used in
single-molecule research (Section 1.6).
1.1.1 Potential artifacts from surfaces and the use of extrinsic

probes
Proteins and nucleic acids are often immobilized on microscope slides to acquire
single-molecule trajectories over long timescales (seconds to minutes). It is critical,
however, to ensure that immobilization itself does not alter the conformation of the
biopolymer or its interactions with ligands and cofactors. Suggested protocols for
passivating surfaces and immobilizing proteins and nucleic acids exist in the litera-
ture.14,28,29 Yet, researchers should always perform their own control experiments to
ensure that results are not affected by surface interactions. In many cases, the
same experiment can be carried out in a traditional cuvette-type fluorescence exper-
iment to determine an average property (e.g. an average FRET efficiency, or an
average enzymatic reaction rate). This average should be consistent with the
average measured for many single-molecules on a surface provided that the surface
treatment does not affect the biochemical and biophysical properties of the system
under study.
In addition, and as with any other fluorescence-based experiment, control exper-
iments need to be performed to ensure that the outcome is not affected by the
fluorophore used to probe a structure, an interaction, or a conformational change.

There is limited information on this important subject because negative results
are unfortunately rarely shared in publications. Yet, a few studies have been
published that highlight the potentially distorting effects of fluorescent labels on
protein conformation,30,31 protein-protein interactions,32,33 peptide-membrane
interactions,34 DNA hairpin dynamics,35,36 and DNA duplex stability.37,38
Researchers should characterize the labeled materials as thoroughly as possible to
ensure that labeled proteins and nucleic acids have the same properties of their unla-
beled counterparts. This may involve measuring enzymatic activities, binding con-
stants between interacting partners, CD spectra, melting temperatures, and NMR or
crystallographic structures whenever possible. Ideally, single-molecule experiments
with more than one fluorescent probe should be performed to demonstrate that results
do not depend on the particular choice of fluorophore. Careful experiments with dif-
ferent FRET pairs, for example, allowed, DeVore et al. to recognize that one particular
FRET acceptor (Texas Red) altered the conformational landscape and dynamics of the
protein calmodulin (CaM).31 In this work, the authors performed identical smFRET
experiments with CaM labeled with one donor and three different acceptors (Alexa
Fluor 594, ATTO594, and Texas Red). The FRET distributions computed from the
measured data were consistent with the presence of multiple conformations of the pro-
tein, but surprisingly, the authors found that the dye Texas Red stabilized a high FRET
state consistent with a compact conformation of CaM. The three dyes used as acceptors
are all rhodamine dyes (see Fig. 1.9 for representative examples of rhodamine dyes),
but they bear different substitutions that result in different net charges. Alexa Fluor 594
and ATTO594 carry a net negative electrical charge, while Texas Red has a net neutral
charge. This difference could explain why Texas Red interacts with the protein more
strongly, biasing its conformational landscape to stabilize a compact conformation
that is not as populated when the system is probed with the Alexa dyes.
Artifacts due to probe interference or surface effects are somewhat unsurprising
in the sense that it is hardly surprising that adding a relatively bulky probe or attach-
ing a biomolecule to a surface can potentially modify its biochemical and biophysical
properties. Yet, there is nothing trivial about avoiding or even identifying these arti-
facts. In particular, the issue of probe interference does not often receive the attention
it deserves, as judged from often missing control experiments that should be carried
out to ease concerns about the potential impact of the fluorescent probe in the bio-
physical properties of the system of interest.
1.1.2 Potential artifacts due to the generation of dark states

What other types of considerations should a researcher take into account when
choosing labels, linkers, and sites of attachment? What sorts of control experiments
can be designed to exclude other potential artifacts, or to obtain complementary
information to properly analyze the measured signals? Addressing these questions
will be the focus of the remainder of the chapter. To motivate the discussion, let
us consider an early report of sm-FRET measurements on DNA duplexes by Meller
et al.21 Fig. 1.2 shows single-molecule fluorescence trajectories for a donor (TMR,
1.1 Introduction 19
FIG. 1.2
Fluorescence trajectories for a single dsDNA molecule labeled with TMR (donor, green) and
Cy5 (acceptor, red) as shown in the top figures. The fluorescence traces were obtained
by alternating a green and a red laser at a rate much faster than the transitions observed in
the figures. A- Donor (green) and acceptor (red) single molecule traces during green
excitation. B- Donor (green) and acceptor (red) single molecule traces during red excitation.
The acceptor intensity fluctuates between a high and a low intensity level corresponding
to the fluorescent state and a dark state, respectively. Comparison with the trace measured
during green excitation shows the exact same fluctuations for the acceptor, demonstrating
that loss of FRET under green excitation is due to acceptor blinking, and not to a change in
donor-acceptor distance.
Sm-FRET traces reprinted with permission from Sabanayagam, C. R.; Eid, J. S.; Meller, A. Long Time Scale
Blinking Kinetics of Cyanine Fluorophores Conjugated to DNA and Its Effect on Forster Resonance Energy
Transfer. J. Chem. Phys. 2005, 123, 224708, with the permission of AIP Publishing.
green) and acceptor (Cy5, red) pair bound covalently to a DNA duplex. The dyes are
covalently bound to the nucleic acid at positions separated by 14 nucleotides
(48 Å), and given the rigidity of duplex DNA at these short length-scales, changes
in distance are not expected beyond what results from the rapid diffusion of the
linkers used to attach the dyes to the DNA. Therefore, the donor and acceptor tra-
jectories for this system were expected to be constant (except for noise) with average
intensities consistent with the FRET efficiency expected for the 48 Å separation
between donor and acceptor. Instead, the measured traces suggest the existence of
two states with FRET efficiencies E1 0.6 and E2 0.1 interconverting in the
second-timescale. These large changes in FRET would be consistent with a change
in donor-acceptor distance of about 30 Å, which would indicate a significant confor-
mational change. Have the authors discovered a new DNA conformation? Have they
defied everything we know about DNA flexibility? Given the simplicity of the sys-
tem (duplex DNA), it is reasonable to consider a different origin for the observed
changes in FRET. Indeed, a carefully executed experiment shed light on the origin
of these fluctuations; as seen in Fig. 1.2B, the same fluctuations are observed when
the system is illuminated with a laser that excites the acceptor directly, but not the
donor. To obtain these data, the laser used to excite the donor (a 514 nm laser) was
alternated with a laser used to excite the acceptor directly (a 640 nm laser) at a rate
much faster than the timescale of the fluctuations observed in the figure. In this way,
the same state of the system could be probed with green and red excitation to produce
the two traces shown in the figure (that is, fluorescence from the donor and acceptor
under either green or red excitation). The results of Fig. 1.2A are indeed consistent
with changes in FRET due to changes in the donor-acceptor distance. However, if
this were the case, the fluorescence intensity of the acceptor when excited directly
with the red laser should have remained constant during the experiment. In contrast,
the results of Fig. 1.2B show that fluctuations are intrinsic to the acceptor, indicating
that the Cy5 molecule makes excursions to a nonemissive (dark) state that persists for
several seconds. This is an example of discrete reversible fluctuations in fluores-
cence intensity that originate from transitions between dark and bright states of
the fluorophore, a phenomenon commonly called ‘blinking’.
The example discussed above shows how fluorescence fluctuations of photophy-
sical origin could be easily misinterpreted as changes in donor-acceptor distance
if the researcher is not aware of this common artifact and thus does not design
appropriate control experiments. In the previous example, the researchers had
enough knowledge of the system (short dsDNA) to suspect that the observed signals
were not due to conformational changes. However, as the next example illustrates,
much greater care should be taken when the system is expected to display conforma-
tional dynamics. The trajectories shown in Fig. 1.3A were obtained with fluores-
cently labeled nucleosomes immobilized on a coverslip by Koopmans et al.13 In
this experiment, nucleosomal DNA was labeled with a donor (Cy3) and an acceptor
(Cy5) FRET pair to monitor the spontaneous unwrapping of the DNA from the
protein core, a phenomenon that had been previously described using fluorescence
correlation spectroscopy (FCS).39 In the closed conformation, the donor and
1.1 Introduction 21
FIG. 1.3
A and B- Observed fluorescence trajectories of single nucleosome particles labeled with
Cy3 (donor, green trace) and Cy5 (acceptor, red trace) under green laser excitation and
calculated FRET efficiency. The transition around 16 s is due to photobleaching of the
acceptor. C- A mechanism consistent with the measured trajectories shown in the top panel.
The close proximity between the two dyes in the closed conformation of the nucleosome
results in low donor emission and high acceptor emission. Loss of FRET and a concomitant
increase in donor emission occurs if a portion of the DNA unwraps from the protein core.
D- Hypothetical fluorescence trajectories under red excitation consistent with this proposed
mechanism. E- Another mechanism consistent with the measured trajectories shown in the
top panel. Loss of FRET and a concomitant increase in donor emission occurs when the
acceptor undergoes a transition to a nonemissive state. F- Fluorescence trajectories
measured under red excitation. The acceptor intensity fluctuates between two values
indicating transitions between a fluorescent and a nonemissive state. Comparison with the
trace measured during green excitation shows the same fluctuations for the acceptor,
demonstrating that loss of FRET under green excitation is due to acceptor blinking, and not to
a change in donor-acceptor distance. The data for panels A, B, and F were generously
provided by Prof. John van Noort (Leiden University) and were published in Ref. 13. The
traces in panel D are simulated (not measured).
acceptor are expected to be in close proximity, which would lead to efficient FRET.
If significant unwrapping of the DNA occurred as suggested by the published FCS
results, the increase in distance would result in a state with low FRET efficiency
(Fig. 1.3C). Experimentally, this would be consistent with a simultaneous decrease
and increase in the acceptor and donor intensities under green excitation, respec-
tively, which is in fact what the authors observed (Fig. 1.3A). Acceptor photobleach-
ing at approximately 15 s results in the recovery of the donor signal due to permanent
loss of FRET. The calculation of the FRET efficiency from these data gives the
trajectory shown in the figure, from which the researchers could have obtained, in
principle, the kinetic rates of the DNA opening and closing reactions. Of course, this
analysis results in the intended information only if the observed fluctuations were
indeed due to DNA unwrapping; if this were the case, however, the intensity of
the acceptor under direct (red) excitation should have remained constant as sketched
in Fig. 1.3D. In contrast, direct excitation of the acceptor was shown to result in
the same fluctuations observed under green excitation (Fig. 1.3F). This indicates
that the apparent FRET changes are not due to conformational fluctuations
(mechanism 1) but rather to excursions of the acceptor (Cy5) to a dark state (acceptor
blinking, mechanism 2). Without the trajectory measured under red excitation, it
would have been easy to misinterpret the trajectories obtained under green excitation
in terms of the conformational fluctuations sketched in the figure (proposed
mechanism 1).
Two other pieces of evidence further support the interpretation of these data in
terms of acceptor blinking (proposed mechanism 2). First, the authors point out that
very similar dynamics was reported by another laboratory in experiments that were
virtually identical except for the location of the probes.40 In this other study, the
authors labeled the DNA at positions where unwrapping events are expected to be
very rare when compared to the positions used by Koopmans et al. Yet, both labeling
schemes yielded the same FRET dynamics, suggesting that the signals were not due
to changes in donor-acceptor distance. In addition, Koopmans et al. reported that the
observed dynamics was nearly abolished when β-mercaptoethanol, which was added
to the buffer for its properties as a triplet quencher, was replaced by Trolox (another
triplet quencher). This result is consistent with the formation of a thiol-induced dark
state of Cy5, which resulted in the observed fluorescence fluctuations in the presence
of β-mercaptoethanol, but not Trolox. The nature of this dark state remained a mys-
tery until Dempsey et al. provided evidence of the formation of an adduct between
the thiol and the cyanine (see Section 1.6.3).41 Regardless of the nature of the dark
state, Koopmans’ results clearly indicate that most of the fluctuations observed in the
FRET trajectories are the result of thiol-induced Cy5 blinking and not DNA breath-
ing. Yet, it should be stressed that DNA breathing (Fig. 1.3C) was indeed detected in
some traces thanks to the careful control experiments and additional measurements
that allowed the researchers to remove the artifactual fluctuations (acceptor blinking)
from the true changes in donor-acceptor distance.
The two examples discussed in this section illustrate examples of control exper-
iments that a researcher should plan to avoid misinterpreting the data, and the types
of considerations that should guide the selection of dyes and buffers. As it will
become evident from the many examples described in this chapter, a basic under-
standing of the variables that affect the photophysical properties of the fluorescent
probes used in the experiment is essential to anticipate potential problems.
1.2 Photophysical processes and the generation of dark states 23
Section 1.6 of this chapter summarizes some known properties of the most common
chemical families of dyes used in single-molecule research, but it is important to
stress that our current understanding of how the environment of a fluorescent probe
within a biological polymer affects its photophysical properties is still incomplete.
The next sections focus on defining the concepts needed to discuss the photophysics
of single-molecule dyes and on how these properties influence the measured fluores-
cence intensities, from which FRET efficiencies are measured.
Before moving on, however, it is important to stress that although fluorophore
blinking was presented as a potential source of artifacts in the preceding discussion,
the on-off fluctuations caused by transitions to dark states are the basis of many
ground-breaking super-resolution microscopy approaches. The ability to transiently
switch fluorescent molecules ‘off’ is the basis of a family of super-resolution
methods based on stochastic readout. Examples include the techniques known as
STORM (stochastic optical reconstruction microscopy),42 PALM (photoactivated
localization microscopy),43 blink microscopy (BM),44 and related methods.45–47
A common feature of these approaches is that only one emitter is allowed to be in
the ‘on’ state within a diffraction limited area at any given time, so that its position
can be determined with high precision. Fluorophores are then switched back to the
dark state or photobleached permanently so another subset of fluorophores can be
activated and imaged. The temporal and spatial resolution that can be achieved in
these methods depends critically on the lifetimes of the ‘on’ and ‘off’ states. There-
fore, designing fluorophores and strategies for superresolution microscopy requires a
thorough understanding of the nature of the dark states that are generated upon
photoexciation, and how the kinetic rate constants that connect these states can be
manipulated to achieve ‘on-off’ rates suitable to achieve the needed resolution. This
chapter focuses on understanding the variables that affect the nature and dynamics of
the dark states that are commonly populated upon photoexcitation of organic dyes
commonly used in single molecule work. Although the chapter focuses on single-
molecule FRET and related approaches, the discussion and information presented
here will also interest readers with interest in superresolution technologies.
1.2 Photophysical processes and the generation of dark

states
State diagrams such as the one shown in Fig. 1.4 (frequently called Perrin-Jablonski
diagrams) are commonly used to depict molecular states and photophysical pro-
cesses. Thick horizontal lines represent molecular electronic states while thin lines
represent vibrational states. Note that only a few vibrational states are shown for clar-
ity within each electronic state, but importantly, higher vibrational levels of S0 over-
lap in energy with vibrational levels of S1 and T1. The vertical axis represents relative
energy, while the horizontal axis has no physical connotation and is merely used to
group states of the same multiplicity (i.e. singlet states, triplet states) in different col-
umns. Solid arrows are used to indicate radiative processes (absorption and
FIG. 1.4
Perrin-Jablonski diagram depicting molecular states and photophysical processes for a donor
(D) and acceptor (A) FRET pair. Only the singlet excited state of the acceptor (S1) is shown for
clarity. Thick horizontal lines represent molecular electronic states and thin lines represent
vibrational states (only a few vibrational states are shown for each electronic state). Radiative
process (absorption and emission) are represented with solid vertical lines. Wavy lines
indicate nonradiative transitions (see text for details).
emission), and wavy arrows indicate nonradiative transitions. In Fig. 1.4, D and
A represent a donor and acceptor pair in the context of FRET.
Absorption of light, depicted as ①, originates from the lowest vibrational state of
the lowest-energy singlet state (S0). A notable exception is molecular oxygen, O2,
which is a triplet in the ground state. In a typical single-molecule experiment, a laser
with output in the visible region of the spectrum is used to excite the molecule to the
first electronic excited state (S1). The transitions depicted in Fig. 1.4 are called vibro-
nic transitions because they involve a change in both the electronic and vibrational
quantum numbers. Transitions to singlet electronic states of higher energy (Sn,
n > 1) are also possible (not shown), but these states are rarely fluorescent because
molecules usually relax rapidly (in picoseconds) to the lowest singlet excited state
(S1). Direct excitation to the triplet state involves a change in spin and is therefore
considered forbidden (i.e. it occurs with a negligible probability).
In solution, excess vibrational energy is transferred to the solvent molecules
through collisions within a few picoseconds, a process commonly known as vibra-
tional relaxation (②). This process deactivates the excited state to its lowest vibra-
tional level (thick line of S1 in Fig. 1.4), which is further deactivated by several
possible mechanisms including fluorescence emission (③), internal conversion to
S0 (see below), intersystem crossing to the triplet state (⑤), energy transfer to an
acceptor molecule (⑧), and other nonradiative processes not depicted in Fig. 1.4 that
will be discussed later in this chapter. Internal conversion is an isoenergetic
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the road in all the dignified unconcern and ideal perfection of
mechanical conveyance. The Bath mail I had set my mind upon, and
I had missed it, as I missed every thing else, by my own absurdity, in
putting the will for the deed, and aiming at ends without employing
means. ‘Sir,’ said he of the Brentford, ‘the Bath mail will be up
presently, my brother-in-law drives it, and I will engage to stop him
if there is a place empty.’ I almost doubted my good genius; but, sure
enough, up it drove like lightning, and stopped directly at the call of
the Brentford Jehu. I would not have believed this possible, but the
brother-in-law of a mail-coach driver is himself no mean man. I was
transferred without loss of time from the top of one coach to that of
the other, desired the guard to pay my fare to the Brentford
coachman for me as I had no change, was accommodated with a
great coat, put up my umbrella to keep off a drizzling mist, and we
began to cut through the air like an arrow. The milestones
disappeared one after another, the rain kept off; Tom Turtle, the
trainer, sat before me on the coach-box, with whom I exchanged
civilities as a gentleman going to the fight; the passion that had
transported me an hour before was subdued to pensive regret and
conjectural musing on the next day’s battle; I was promised a place
inside at Reading, and upon the whole, I thought myself a lucky
fellow. Such is the force of imagination! On the outside of any other
coach on the 10th of December, with a Scotch mist drizzling through
the cloudy moonlight air, I should have been cold, comfortless,
impatient, and, no doubt, wet through; but seated on the Royal mail,
I felt warm and comfortable, the air did me good, the ride did me
good, I was pleased with the progress we had made, and confident
that all would go well through the journey. When I got inside at
Reading, I found Turtle and a stout valetudinarian, whose costume
bespoke him one of the Fancy, and who had risen from a three
months’ sick bed to get into the mail to see the fight. They were
intimate, and we fell into a lively discourse. My friend the trainer was
confined in his topics to fighting dogs and men, to bears and
badgers; beyond this he was ‘quite chap-fallen,’ had not a word to
throw at a dog, or indeed very wisely fell asleep, when any other
game was started. The whole art of training (I, however, learnt from
him,) consists in two things, exercise and abstinence, abstinence and
exercise, repeated alternately and without end. A yolk of an egg with
a spoonful of rum in it is the first thing in a morning, and then a walk
of six miles till breakfast. This meal consists of a plentiful supply of
tea and toast and beef-steaks. Then another six or seven miles till
dinner-time, and another supply of solid beef or mutton with a pint
of porter, and perhaps, at the utmost, a couple of glasses of sherry.
Martin trains on water, but this increases his infirmity on another
very dangerous side. The Gas-man takes now and then a chirping
glass (under the rose) to console him, during a six weeks’ probation,
for the absence of Mrs. Hickman—an agreeable woman, with (I
understand) a pretty fortune of two hundred pounds. How matter
presses on me! What stubborn things are facts! How inexhaustible is
nature and art! ‘It is well,’ as I once heard Mr. Richmond observe, ‘to
see a variety.’ He was speaking of cock-fighting as an edifying
spectacle. I cannot deny but that one learns more of what is (I do not
say of what ought to be) in this desultory mode of practical study,
than from reading the same book twice over, even though it should
be a moral treatise. Where was I? I was sitting at dinner with the
candidate for the honours of the ring, ‘where good digestion waits on
appetite, and health on both.’ Then follows an hour of social chat and
native glee; and afterwards, to another breathing over heathy hill or
dale. Back to supper, and then to bed, and up by six again—Our hero
‘Follows so the ever-running sun
With profitable ardour‘—
to the day that brings him victory or defeat in the green fairy circle. Is
not this life more sweet than mine? I was going to say; but I will not
libel any life by comparing it to mine, which is (at the date of these
presents) bitter as coloquintida and the dregs of aconitum!
The invalid in the Bath mail soared a pitch above the trainer, and
did not sleep so sound, because he had ‘more figures and more
fantasies.’ We talked the hours away merrily. He had faith in surgery,
for he had had three ribs set right, that had been broken in a turn-up
at Belcher’s, but thought physicians old women, for they had no
antidote in their catalogue for brandy. An indigestion is an excellent
common-place for two people that never met before. By way of
ingratiating myself, I told him the story of my doctor, who, on my
earnestly representing to him that I thought his regimen had done
me harm, assured me that the whole pharmacopœia contained
nothing comparable to the prescription he had given me; and, as a
proof of its undoubted efficacy, said, that, ‘he had had one gentleman
with my complaint under his hands for the last fifteen years.’ This
anecdote made my companion shake the rough sides of his three
great coats with boisterous laughter; and Turtle, starting out of his
sleep, swore he knew how the fight would go, for he had had a dream
about it. Sure enough the rascal told us how the three first rounds
went off, but ‘his dream,’ like others, ‘denoted a foregone conclusion.’
He knew his men. The moon now rose in silver state, and I ventured,
with some hesitation, to point out this object of placid beauty, with
the blue serene beyond, to the man of science, to which his ear he
‘seriously inclined,’ the more as it gave promise d’un beau jour for
the morrow, and showed the ring undrenched by envious showers,
arrayed in sunny smiles. Just then, all going on well, I thought on my
friend Toms, whom I had left behind, and said innocently, ‘There
was a blockhead of a fellow I left in town, who said there was no
possibility of getting down by the mail, and talked of going by a
caravan from Belcher’s at two in the morning, after he had written
some letters.’ ‘Why,’ said he of the lapels, ‘I should not wonder if that
was the very person we saw running about like mad from one coach-
door to another, and asking if any one had seen a friend of his, a
gentlemen going to the fight, whom he had missed stupidly enough
by staying to write a note.’ ‘Pray, Sir,’ said my fellow-traveller, ‘had
he a plaid-cloak on?’—‘Why, no,’ said I, ‘not at the time I left him, but
he very well might afterwards, for he offered to lend me one.’ The
plaid-cloak and the letter decided the thing. Joe, sure enough, was in
the Bristol mail, which preceded us by about fifty yards. This was
droll enough. We had now but a few miles to our place of destination,
and the first thing I did on alighting at Newbury, both coaches
stopping at the same time, was to call out, ‘Pray, is there a gentleman
in that mail of the name of Toms?’ ‘No,’ said Joe, borrowing
something of the vein of Gilpin, ‘for I have just got out.’ ‘Well!’ says
he, ‘this is lucky; but you don’t know how vexed I was to miss you;
for,’ added he, lowering his voice, ‘do you know when I left you I
went to Belcher’s to ask about the caravan, and Mrs. Belcher said
very obligingly, she couldn’t tell about that, but there were two
gentlemen who had taken places by the mail and were gone on in a
landau, and she could frank us. It’s a pity I didn’t meet with you; we
could then have got down for nothing. But mum’s the word.’ It’s the
devil for any one to tell me a secret, for it’s sure to come out in print.
I do not care so much to gratify a friend, but the public ear is too
great a temptation to me.
Our present business was to get beds and a supper at an inn; but
this was no easy task. The public-houses were full, and where you
saw a light at a private house, and people poking their heads out of
the casement to see what was going on, they instantly put them in
and shut the window, the moment you seemed advancing with a
suspicious overture for accommodation. Our guard and coachman
thundered away at the outer gate of the Crown for some time without
effect—such was the greater noise within;—and when the doors were
unbarred, and we got admittance, we found a party assembled in the
kitchen round a good hospitable fire, some sleeping, others drinking,
others talking on politics and on the fight. A tall English yeoman
(something like Matthews in the face, and quite as great a wag)—
‘A lusty man to ben an abbot able,’—
was making such a prodigious noise about rent and taxes, and the
price of corn now and formerly, that he had prevented us from being
heard at the gate. The first thing I heard him say was to a shuffling
fellow who wanted to be off a bet for a shilling glass of brandy and
water—‘Confound it, man, don’t be insipid!’ Thinks I, that is a good
phrase. It was a good omen. He kept it up so all night, nor flinched
with the approach of morning. He was a fine fellow, with sense, wit,
and spirit, a hearty body and a joyous mind, free-spoken, frank,
convivial—one of that true English breed that went with Harry the
Fifth to the siege of Harfleur—‘standing like greyhounds in the slips,’
&c. We ordered tea and eggs (beds were soon found to be out of the
question) and this fellow’s conversation was sauce piquante. It did
one’s heart good to see him brandish his oaken towel and to hear him
talk. He made mince-meat of a drunken, stupid, red-faced,
quarrelsome, frowsy farmer, whose nose ‘he moralized into a
thousand similes,’ making it out a firebrand like Bardolph’s. ‘I’ll tell
you what my friend,’ says he, ‘the landlady has only to keep you here
to save fire and candle. If one was to touch your nose, it would go off
like a piece of charcoal.’ At this the other only grinned like an idiot,
the sole variety in his purple face being his little peering grey eyes
and yellow teeth; called for another glass, swore he would not stand
it; and after many attempts to provoke his humourous antagonist to
single combat, which the other turned off (after working him up to a
ludicrous pitch of choler) with great adroitness, he fell quietly asleep
with a glass of liquor in his hand, which he could not lift to his head.
His laughing persecutor made a speech over him, and turning to the
opposite side of the room, where they were all sleeping in the midst
of this ‘loud and furious fun,’ said, ‘There’s a scene, by G—d, for
Hogarth to paint. I think he and Shakspeare were our two best men
at copying life.’ This confirmed me in my good opinion of him.
Hogarth, Shakspeare, and Nature, were just enough for him (indeed
for any man) to know. I said, ‘You read Cobbett, don’t you? At least,’
says I, ‘you talk just as well as he writes.’ He seemed to doubt this.
But I said, ‘We have an hour to spare: if you’ll get pen, ink, and
paper, and keep on talking, I’ll write down what you say; and if it
doesn’t make a capital ‘Political Register,’ I’ll forfeit my head. You
have kept me alive to-night, however. I don’t know what I should
have done without you.’ He did not dislike this view of the thing, nor
my asking if he was not about the size of Jem Belcher; and told me
soon afterwards, in the confidence of friendship, that ‘the
circumstance which had given him nearly the greatest concern in his
life, was Cribb’s beating Jem after he had lost his eye by racket-
playing.’—The morning dawns; that dim but yet clear light appears,
which weighs like solid bars of metal on the sleepless eyelids; the
guests drop down from their chambers one by one—but it was too
late to think of going to bed now (the clock was on the stroke of
seven), we had nothing for it but to find a barber’s (the pole that
glittered in the morning sun lighted us to his shop), and then a nine
miles’ march to Hungerford. The day was fine, the sky was blue, the
mists were retiring from the marshy ground, the path was tolerably
dry, the sitting-up all night had not done us much harm—at least the
cause was good; we talked of this and that with amicable difference,
roving and sipping of many subjects, but still invariably we returned
to the fight. At length, a mile to the left of Hungerford, on a gentle
eminence, we saw the ring surrounded by covered carts, gigs, and
carriages, of which hundreds had passed us on the road; Toms gave a
youthful shout, and we hastened down a narrow lane to the scene of
action.
Reader, have you ever seen a fight? If not, you have a pleasure to
come, at least if it is a fight like that between the Gas-man and Bill
Neate. The crowd was very great when we arrived on the spot; open
carriages were coming up, with streamers flying and music playing,
and the country people were pouring in over hedge and ditch in all
directions, to see their hero beat or be beaten. The odds were still on
Gas, but only about five to four. Gully had been down to try Neate,
and had backed him considerably, which was a damper to the
sanguine confidence of the adverse party. About two hundred
thousand pounds were pending. The Gas says, he has lost 3000l.
which were promised him by different gentlemen if he had won. He
had presumed too much on himself, which had made others presume
on him. This spirited and formidable young fellow seems to have
taken for his motto the old maxim, that ‘there are three things
necessary to success in life—Impudence! Impudence! Impudence!’ It
is so in matters of opinion, but not in the Fancy, which is the most
practical of all things, though even here confidence is half the battle,
but only half. Our friend had vapoured and swaggered too much, as if
he wanted to grin and bully his adversary out of the fight. ‘Alas! the
Bristol man was not so tamed!’—‘This is the grave-digger’ (would
Tom Hickman exclaim in the moments of intoxication from gin and
success, shewing his tremendous right hand), ‘this will send many of
them to their long homes; I haven’t done with them yet! ‘Why should
he—though he had licked four of the best men within the hour, yet
why should he threaten to inflict dishonourable chastisement on my
old master Richmond, a veteran going off the stage, and who has
borne his sable honours meekly? Magnanimity, my dear Tom, and
bravery, should be inseparable. Or why should he go up to his
antagonist, the first time he ever saw him at the Fives Court, and
measuring him from head to foot with a glance of contempt, as
Achilles surveyed Hector, say to him, ‘What, are you Bill Neate? I’ll
knock more blood out of that great carcase of thine, this day
fortnight, than you ever knock’d out of a bullock’s!’ It was not manly,
’twas not fighter-like. If he was sure of the victory (as he was not), the
less said about it the better. Modesty should accompany the Fancy as
its shadow. The best men were always the best behaved. Jem
Belcher, the Game Chicken (before whom the Gas-man could not
have lived) were civil, silent men. So is Cribb, so is Tom Belcher, the
most elegant of sparrers, and not a man for every one to take by the
nose. I enlarged on this topic in the mail (while Turtle was asleep),
and said very wisely (as I thought) that impertinence was a part of no
profession. A boxer was bound to beat his man, but not to thrust his
fist, either actually or by implication, in every one’s face. Even a
highwayman, in the way of trade, may blow out your brains, but if he
uses foul language at the same time, I should say he was no
gentleman. A boxer, I would infer, need not be a blackguard or a
coxcomb, more than another. Perhaps I press this point too much on
a fallen man—Mr. Thomas Hickman has by this time learnt that first
of all lessons, ‘That man was made to mourn.’ He has lost nothing by
the late fight but his presumption; and that every man may do as well
without! By an over-display of this quality, however, the public had
been prejudiced against him, and the knowing-ones were taken in.
Few but those who had bet on him wished Gas to win. With my own
prepossessions on the subject, the result of the 11th of December
appeared to me as fine a piece of poetical justice as I had ever
witnessed. The difference of weight between the two combatants (14
stone to 12) was nothing to the sporting men. Great, heavy, clumsy,
long-armed Bill Neate kicked the beam in the scale of the Gas-man’s
vanity. The amateurs were frightened at his big words, and thought
that they would make up for the difference of six feet and five feet
nine. Truly, the Fancy are not men of imagination. They judge of
what has been, and cannot conceive of anything that is to be. The
Gas-man had won hitherto; therefore he must beat a man half as big
again as himself—and that to a certainty. Besides, there are as many
feuds, factions, prejudices, pedantic notions in the Fancy as in the
state or in the schools. Mr. Gully is almost the only cool, sensible
man among them, who exercises an unbiassed discretion, and is not
a slave to his passions in these matters. But enough of reflections,
and to our tale. The day, as I have said, was fine for a December
morning. The grass was wet, and the ground miry, and ploughed up
with multitudinous feet, except that, within the ring itself, there was
a spot of virgin-green closed in and unprofaned by vulgar tread, that
shone with dazzling brightness in the mid-day sun. For it was now
noon, and we had an hour to wait. This is the trying time. It is then
the heart sickens, as you think what the two champions are about,
and how short a time will determine their fate. After the first blow is
struck, there is no opportunity for nervous apprehensions; you are
swallowed up in the immediate interest of the scene—but
‘Between the acting of a dreadful thing
And the first motion, all the interim is
Like a phantasma, or a hideous dream.’
I found it so as I felt the sun’s rays clinging to my back, and saw the
white wintry clouds sink below the verge of the horizon. ‘So, I
thought, my fairest hopes have faded from my sight!—so will the
Gas-man’s glory, or that of his adversary, vanish in an hour.’ The
swells were parading in their white box-coats, the outer ring was
cleared with some bruises on the heads and shins of the rustic
assembly (for the cockneys had been distanced by the sixty-six
miles); the time drew near, I had got a good stand; a bustle, a buzz,
ran through the crowd, and from the opposite side entered Neate,
between his second and bottle-holder. He rolled along, swathed in
his loose great coat, his knock-knees bending under his huge bulk;
and, with a modest cheerful air, threw his hat into the ring. He then
just looked round, and began quietly to undress; when from the
other side there was a similar rush and an opening made, and the
Gas-man came forward with a conscious air of anticipated triumph,
too much like the cock-of-the walk. He strutted about more than
became a hero, sucked oranges with a supercilious air, and threw
away the skin with a toss of his head, and went up and looked at
Neate, which was an act of supererogation. The only sensible thing
he did was, as he strode away from the modern Ajax, to fling out his
arms, as if he wanted to try whether they would do their work that
day. By this time they had stripped, and presented a strong contrast
in appearance. If Neate was like Ajax, ‘with Atlantean shoulders, fit
to bear’ the pugilistic reputation of all Bristol, Hickman might be
compared to Diomed, light, vigorous, elastic, and his back glistened
in the sun, as he moved about, like a panther’s hide. There was now a
dead pause—attention was awe-struck. Who at that moment, big
with a great event, did not draw his breath short—did not feel his
heart throb? All was ready. They tossed up for the sun, and the Gas-
man won. They were led up to the scratch—shook hands, and went at
it.
In the first round every one thought it was all over. After making
play a short time, the Gas-man flew at his adversary like a tiger,
struck five blows in as many seconds, three first, and then following
him as he staggered back, two more, right and left, and down he fell,
a mighty ruin. There was a shout, and I said, ‘There is no standing
this.’ Neate seemed like a lifeless lump of flesh and bone, round
which the Gas-man’s blows played with the rapidity of electricity or
lightning, and you imagined he would only be lifted up to be knocked
down again. It was as if Hickman held a sword or a fire in that right
hand of his, and directed it against an unarmed body. They met
again, and Neate seemed, not cowed, but particularly cautious. I saw
his teeth clenched together and his brows knit close against the sun.
He held out both his arms at full length straight before him, like two
sledge-hammers, and raised his left an inch or two higher. The Gas-
man could not get over this guard—they struck mutually and fell, but
without advantage on either side. It was the same in the next round;
but the balance of power was thus restored—the fate of the battle was
suspended. No one could tell how it would end. This was the only
moment in which opinion was divided; for, in the next, the Gas-man
aiming a mortal blow at his adversary’s neck, with his right hand,
and failing from the length he had to reach, the other returned it with
his left at full swing, planted a tremendous blow on his cheek-bone
and eyebrow, and made a red ruin of that side of his face. The Gas-
man went down, and there was another shout—a roar of triumph as
the waves of fortune rolled tumultuously from side to side. This was
a settler. Hickman got up, and ‘grinned horrible a ghastly smile,’ yet
he was evidently dashed in his opinion of himself; it was the first
time he had ever been so punished; all one side of his face was
perfect scarlet, and his right eye was closed in dingy blackness, as he
advanced to the fight, less confident, but still determined. After one
or two rounds, not receiving another such remembrancer, he rallied
and went at it with his former impetuosity. But in vain. His strength
had been weakened,—his blows could not tell at such a distance,—he
was obliged to fling himself at his adversary, and could not strike
from his feet; and almost as regularly as he flew at him with his right
hand, Neate warded the blow, or drew back out of its reach, and
felled him with the return of his left. There was little cautious
sparring—no half-hits—no tapping and trifling, none of the petit-
maitreship of the art—they were almost all knock-down blows:—the
fight was a good stand-up fight. The wonder was the half-minute
time. If there had been a minute or more allowed between each
round, it would have been intelligible how they should by degrees
recover strength and resolution; but to see two men smashed to the
ground, smeared with gore, stunned, senseless, the breath beaten out
of their bodies; and then, before you recover from the shock, to see
them rise up with new strength and courage, stand steady to inflict or
receive mortal offence, and rush upon each other ‘like two clouds
over the Caspian’—this is the most astonishing thing of all:—this is
the high and heroic state of man! From this time forward the event
became more certain every round; and about the twelfth it seemed as
if it must have been over. Hickman generally stood with his back to
me; but in the scuffle, he had changed positions, and Neate just then
made a tremendous lunge at him, and hit him full in the face. It was
doubtful whether he would fall backwards or forwards; he hung
suspended for a second or two, and then fell back, throwing his
hands in the air, and with his face lifted up to the sky. I never saw
any thing more terrific than his aspect just before he fell. All traces of
life, of natural expression, were gone from him. His face was like a
human skull, a death’s head, spouting blood. The eyes were filled
with blood, the nose streamed with blood, the mouth gaped blood.
He was not like an actual man, but like a preternatural, spectral
appearance, or like one of the figures in Dante’s Inferno. Yet he
fought on after this for several rounds, still striking the first
desperate blow, and Neate standing on the defensive, and using the
same cautious guard to the last, as if he had still all his work to do;
and it was not till the Gas-man was so stunned in the seventeenth or
eighteenth round, that his senses forsook him, and he could not
come to time, that the battle was declared over.[3] Ye who despise the
Fancy, do something to shew as much pluck, or as much self-
possession as this, before you assume a superiority which you have
never given a single proof of by any one action in the whole course of
your lives!—When the Gas-man came to himself, the first words he
uttered were, ‘Where am I? What is the matter?’ ‘Nothing is the
matter, Tom,—you have lost the battle, but you are the bravest man
alive.’ And Jackson whispered to him, ‘I am collecting a purse for
you, Tom.’—Vain sounds, and unheard at that moment! Neate
instantly went up and shook him cordially by the hand, and seeing
some old acquaintance, began to flourish with his fists, calling out,
‘Ah you always said I couldn’t fight—What do you think now?’ But all
in good humour, and without any appearance of arrogance; only it
was evident Bill Neate was pleased that he had won the fight. When
it was over, I asked Cribb if he did not think it was a good one? He
said, ‘Pretty well!’ The carrier-pigeons now mounted into the air,
and one of them flew with the news of her husband’s victory to the
bosom of Mrs. Neate. Alas, for Mrs. Hickman!
Mais au revoir, as Sir Fopling Flutter says. I went down with
Toms; I returned with Jack Pigott, whom I met on the ground. Toms
is a rattle brain; Pigott is a sentimentalist. Now, under favour, I am a
sentimentalist too—therefore I say nothing, but that the interest of
the excursion did not flag as I came back. Pigott and I marched along
the causeway leading from Hungerford to Newbury, now observing
the effect of a brilliant sun on the tawny meads or moss-coloured
cottages, now exulting in the fight, now digressing to some topic of
general and elegant literature. My friend was dressed in character for
the occasion, or like one of the Fancy; that is, with a double portion
of great coats, clogs, and overhauls: and just as we had agreed with a
couple of country lads to carry his superfluous wearing apparel to the
next town, we were overtaken by a return post-chaise, into which I
got, Pigott preferring a seat on the bar. There were two strangers
already in the chaise, and on their observing they supposed I had
been to the fight, I said I had, and concluded they had done the
same. They appeared, however, a little shy and sore on the subject;
and it was not till after several hints dropped, and questions put, that
it turned out that they had missed it. One of these friends had
undertaken to drive the other there in his gig: they had set out, to
make sure work, the day before at three in the afternoon. The owner
of the one-horse vehicle scorned to ask his way, and drove right on to
Bagshot, instead of turning off at Hounslow: there they stopped all
night, and set off the next day across the country to Reading, from
whence they took coach, and got down within a mile or two of
Hungerford, just half an hour after the fight was over. This might be
safely set down as one of the miseries of human life. We parted with
these two gentlemen who had been to see the fight, but had returned
as they went, at Wolhampton, where we were promised beds (an
irresistible temptation, for Pigott had passed the preceding night at
Hungerford as we had done at Newbury), and we turned into an old
bow-windowed parlour with a carpet and a snug fire; and after
devouring a quantity of tea, toast, and eggs, sat down to consider,
during an hour of philosophic leisure, what we should have for
supper. In the midst of an Epicurean deliberation between a roasted
fowl and mutton chops with mashed potatoes, we were interrupted
by an inroad of Goths and Vandals—O procul este profani—not real
flash-men, but interlopers, noisy pretenders, butchers from Tothill-
fields, brokers from Whitechapel, who called immediately for pipes
and tobacco, hoping it would not be disagreeable to the gentlemen,
and began to insist that it was a cross. Pigott withdrew from the
smoke and noise into another room, and left me to dispute the point
with them for a couple of hours sans intermission by the dial. The
next morning we rose refreshed; and on observing that Jack had a
pocket volume in his hand, in which he read in the intervals of our
discourse, I inquired what it was, and learned to my particular
satisfaction that it was a volume of the New Eloise. Ladies, after this,
will you contend that a love for the Fancy is incompatible with the
cultivation of sentiment?—We jogged on as before, my friend setting
me up in a genteel drab great coat and green silk handkerchief
(which I must say became me exceedingly), and after stretching our
legs for a few miles, and seeing Jack Randall, Ned Turner, and
Scroggins, pass on the top of one of the Bath coaches, we engaged
with the driver of the second to take us to London for the usual fee. I
got inside, and found three other passengers. One of them was an old
gentleman with an aquiline nose, powdered hair, and a pigtail, and
who looked as if he had played many a rubber at the Bath rooms. I
said to myself, he is very like Mr. Windham; I wish he would enter
into conversation, that I might hear what fine observations would
come from those finely-turned features. However, nothing passed,
till, stopping to dine at Reading, some inquiry was made by the
company about the fight, and I gave (as the reader may believe) an
eloquent and animated description of it. When we got into the coach
again, the old gentleman, after a graceful exordium, said, he had,
when a boy, been to a fight between the famous Broughton and
George Stevenson, who was called the Fighting Coachman, in the
year 1770, with the late Mr. Windham. This beginning flattered the
spirit of prophecy within me and rivetted my attention. He went on
—‘George Stevenson was coachman to a friend of my father’s. He was
an old man when I saw him some years afterwards. He took hold of
his own arm and said, “there was muscle here once, but now it is no
more than this young gentleman’s.” He added, “well, no matter; I
have been here long, I am willing to go hence, and I hope I have done
no more harm than another man.” Once,’ said my unknown
companion, ‘I asked him if he had ever beat Broughton? He said Yes;
that he had fought with him three times, and the last time he fairly
beat him, though the world did not allow it. “I’ll tell you how it was,
master. When the seconds lifted us up in the last round, we were so
exhausted that neither of us could stand, and we fell upon one
another, and as Master Broughton fell uppermost, the mob gave it in
his favour, and he was said to have won the battle. But,” says he, “the
fact was, that as his second (John Cuthbert) lifted him up, he said to
him, ‘I’ll fight no more, I’ve had enough;’ which,” says Stevenson,
“you know gave me the victory. And to prove to you that this was the
case, when John Cuthbert was on his death-bed, and they asked him
if there was any thing on his mind which he wished to confess, he
answered, ‘Yes, that there was one thing he wished to set right, for
that certainly Master Stevenson won that last fight with Master
Broughton; for he whispered him as he lifted him up in the last
round of all, that he had had enough.’”’ ‘This,’ said the Bath
gentleman, ‘was a bit of human nature;’ and I have written this
account of the fight on purpose that it might not be lost to the world.
He also stated as a proof of the candour of mind in this class of men,
that Stevenson acknowledged that Broughton could have beat him in
his best day; but that he (Broughton) was getting old in their last
rencounter. When we stopped in Piccadilly, I wanted to ask the
gentleman some questions about the late Mr. Windham, but had not
courage. I got out, resigned my coat and green silk handkerchief to
Pigott (loth to part with these ornaments of life), and walked home in
high spirits.
P.S. Toms called upon me the next day, to ask me if I did not think
the fight was a complete thing? I said I thought it was. I hope he will
relish my account of it.
MERRY ENGLAND
The New Monthly Magazine.]

[December, 1825.
‘St. George for merry England!’
This old-fashioned epithet might be supposed to have been

bestowed ironically, or on the old principle—Ut lucus a non lucendo.
Yet there is something in the sound that hits the fancy, and a sort of
truth beyond appearances. To be sure, it is from a dull, homely
ground that the gleams of mirth and jollity break out; but the streaks
of light that tinge the evening sky are not the less striking on that
account. The beams of the morning sun shining on the lonely glades,
or through the idle branches of the tangled forest, the leisure, the
freedom, ‘the pleasure of going and coming without knowing where,’
the troops of wild deer, the sports of the chase, and other rustic
gambols, were sufficient to justify the well-known appellation of
‘Merry Sherwood,’ and in like manner, we may apply the phrase to
Merry England. The smile is not the less sincere because it does not
always play upon the cheek; and the jest is not the less welcome, nor
the laugh less hearty, because they happen to be a relief from care or
leaden-eyed melancholy. The instances are the more precious as they
are rare; and we look forward to them with the greater good will, or
back upon them with the greater gratitude, as we drain the last drop
in the cup with particular relish. If not always gay or in good spirits,
we are glad when any occasion draws us out of our natural gloom,
and disposed to make the most of it. We may say with Silence in the
play, ‘I have been merry once ere now,’—and this once was to serve
him all his life; for he was a person of wonderful silence and gravity,
though ‘he chirped over his cups,’ and announced with characteristic
glee that ‘there were pippins and cheese to come.’ Silence was in this
sense a merry man, that is, he would be merry if he could, and a very
great economy of wit, like very slender fare, was a banquet to him,
from the simplicity of his taste and habits. ‘Continents,’ says Hobbes,
‘have most of what they contain’—and in this view it may be
contended that the English are the merriest people in the world,
since they only show it on high-days and holidays. They are then like
a schoolboy let loose from school, or like a dog that has slipped his
collar. They are not gay like the French, who are one eternal smile of
self-complacency, tortured into affectation, or spun into languid
indifference, nor are they voluptuous and immersed in sensual
indolence, like the Italians; but they have that sort of intermittent,
fitful, irregular gaiety, which is neither worn out by habit, nor
deadened by passion, but is sought with avidity as it takes the mind
by surprise, is startled by a sense of oddity and incongruity, indulges
its wayward humours or lively impulses, with perfect freedom and
lightness of heart, and seizes occasion by the forelock, that it may
return to serious business with more cheerfulness, and have
something to beguile the hours of thought or sadness. I do not see
how there can be high spirits without low ones; and every thing has
its price according to circumstances. Perhaps we have to pay a
heavier tax on pleasure, than some others: what skills it, so long as
our good spirits and good hearts enable us to bear it?
‘They’ (the English), says Froissart, ‘amused themselves sadly after
the fashion of their country’—ils se réjouissent tristement selon la
coutume de leur pays. They have indeed a way of their own. Their
mirth is a relaxation from gravity, a challenge to dull care to be gone;
and one is not always clear at first, whether the appeal is successful.
The cloud may still hang on the brow; the ice may not thaw at once.
To help them out in their new character is an act of charity. Any
thing short of hanging or drowning is something to begin with. They
do not enter into their amusements the less doggedly because they
may plague others. They like a thing the better for hitting them a rap
on the knuckles, for making their blood tingle. They do not dance or
sing, but they make good cheer—‘eat, drink, and are merry.’ No
people are fonder of field-sports, Christmas gambols, or practical
jests. Blindman’s-buff, hunt-the-slipper, hot-cockles, and snap-
dragon, are all approved English games, full of laughable surprises
and ‘hairbreadth ‘scapes,’ and serve to amuse the winter fire-side
after the roast-beef and plum-pudding, the spiced ale and roasted
crab, thrown (hissing-hot) into the foaming tankard. Punch (not the
liquor, but the puppet) is not, I fear, of English origin; but there is no
place, I take it, where he finds himself more at home or meets a more
joyous welcome, where he collects greater crowds at the corners of
streets, where he opens the eyes or distends the cheeks wider, or
where the bangs and blows, the uncouth gestures, ridiculous anger
and screaming voice of the chief performer excite more boundless
merriment or louder bursts of laughter among all ranks and sorts of
people. An English theatre is the very throne of pantomime; nor do I
believe that the gallery and boxes of Drury Lane or Covent Garden
filled on the proper occasions with holiday folks (big or little) yield
the palm for undisguised, tumultuous, inextinguishable laughter to
any spot in Europe. I do not speak of the refinement of the mirth
(this is no fastidious speculation) but of its cordiality, on the return
of these long looked-for and licensed periods; and I may add here, by
way of illustration, that the English common people are a sort of
grown children, spoiled and sulky perhaps, but full of glee and
merriment, when their attention is drawn off by some sudden and
striking object. The May-pole is almost gone out of fashion among
us: but May-day, besides its flowering hawthorns and its pearly
dews, has still its boasted exhibition of painted chimney-sweepers
and their Jack-o’-the-Green, whose tawdry finery, bedizened faces,
unwonted gestures, and short-lived pleasures call forth good-
humoured smiles and looks of sympathy in the spectators. There is
no place where trap-ball, fives, prison-base, foot-ball, quoits, bowls
are better understood or more successfully practised; and the very
names of a cricket bat and ball make English fingers tingle. What
happy days must ‘Long Robinson’ have passed in getting ready his
wickets and mending his bats, who when two of the fingers of his
right hand were struck off by the violence of a ball, had a screw
fastened to it to hold the bat, and with the other hand still sent the
ball thundering against the boards that bounded Old Lord’s cricket-
ground! What delightful hours must have been his in looking
forward to the matches that were to come, in recounting the feats he
had performed in those that were past! I have myself whiled away
whole mornings in seeing him strike the ball (like a countryman
mowing with a scythe) to the farthest extremity of the smooth, level,
sun-burnt ground, and with long, awkward strides count the notches
that made victory sure! Then again, cudgel-playing, quarter-staff,
bull and badger-baiting, cock-fighting are almost the peculiar
diversions of this island, and often objected to us as barbarous and
cruel; horse-racing is the delight and the ruin of numbers; and the
noble science of boxing is all our own. Foreigners can scarcely
understand how we can squeeze pleasure out of this pastime; the
luxury of hard blows given or received; the joy of the ring; nor the
perseverance of the combatants.[4] The English also excel, or are not
excelled in wiring a hare, in stalking a deer, in shooting, fishing, and
hunting. England to this day boasts her Robin Hood and his merry
men, that stout archer and outlaw, and patron saint of the sporting-
calendar. What a cheerful sound is that of the hunters, issuing from
the autumnal wood and sweeping over hill and dale!
——‘A cry more tuneable
Was never halloo’d to by hound or horn.’
What sparkling richness in the scarlet coats of the riders, what a

glittering confusion in the pack, what spirit in the horses, what
eagerness in the followers on foot, as they disperse over the plain, or
force their way over hedge and ditch! Surely, the coloured prints and
pictures of these, hung up in gentlemen’s halls and village alehouses,
however humble as works of art, have more life and health and spirit
in them, and mark the pith and nerve of the national character more
creditably than the mawkish, sentimental, affected designs of
Theseus and Pirithous, and Æneas and Dido, pasted on foreign
salons à manger, and the interior of country-houses. If our tastes are
not epic, nor our pretensions lofty, they are simple and our own; and
we may possibly enjoy our native rural sports, and the rude
remembrances of them, with the truer relish on this account, that
they are suited to us and we to them. The English nation, too, are
naturally ‘brothers of the angle.’ This pursuit implies just that
mixture of patience and pastime, of vacancy and thoughtfulness, of
idleness and business, of pleasure and of pain, which is suited to the
genius of an Englishman, and as I suspect, of no one else in the same
degree. He is eminently gifted to stand in the situation assigned by
Dr. Johnson to the angler, ‘at one end of a rod with a worm at the
other.’ I should suppose no language can show such a book as an
often-mentioned one, ‘Walton’s Complete Angler,’—so full of
naïveté, of unaffected sprightliness, of busy trifling, of dainty songs,
of refreshing brooks, of shady arbours, of happy thoughts and of the
herb called Heart’s Ease! Some persons can see neither the wit nor
wisdom of this genuine volume, as if a book as well as a man might
not have a personal character belonging to it, amiable, venerable
from the spirit of joy and thorough goodness it manifests,
independently of acute remarks or scientific discoveries: others
object to the cruelty of Walton’s theory and practice of trout-fishing
—for my part, I should as soon charge an infant with cruelty for
killing a fly, and I feel the same sort of pleasure in reading his book
as I should have done in the company of this happy, child-like old
man, watching his ruddy cheek, his laughing eye, the kindness of his
heart, and the dexterity of his hand in seizing his finny prey! It must
be confessed, there is often an odd sort of materiality in English
sports and recreations. I have known several persons, whose
existence consisted wholly in manual exercises, and all whose
enjoyments lay at their finger-ends. Their greatest happiness was in
cutting a stick, in mending a cabbage-net, in digging a hole in the
ground, in hitting a mark, turning a lathe, or in something else of the
same kind, at which they had a certain knack. Well is it when we can
amuse ourselves with such trifles and without injury to others! This
class of character, which the Spectator has immortalised in the
person of Will Wimble, is still common among younger brothers and
gentlemen of retired incomes in town or country. The Cockney
character is of our English growth, as this intimates a feverish fidgety
delight in rural sights and sounds, and a longing wish, after the
turmoil and confinement of a city-life, to transport one’s-self to the
freedom and breathing sweetness of a country retreat. London is half
suburbs. The suburbs of Paris are a desert; and you see nothing but
crazy wind-mills, stone-walls, and a few straggling visitants in spots
where in England you would find a thousand villas, a thousand
terraces crowned with their own delights, or be stunned with the
noise of bowling-greens and tea-gardens, or stifled with the fumes of
tobacco mingling with fragrant shrubs, or the clouds of dust raised
by half the population of the metropolis panting and toiling in search
of a mouthful of fresh air. The Parisian is, perhaps, as well (or better)
contented with himself wherever he is, stewed in his shop or his
garret; the Londoner is miserable in these circumstances, and glad to
escape from them.[5] Let no one object to the gloomy appearance of a
London Sunday, compared with a Parisian one. It is a part of our
politics and our religion: we would not have James the First’s ‘Book
of Sports’ thrust down our throats: and besides, it is a part of our
character to do one thing at a time, and not to be dancing a jig and
on our knees in the same breath. It is true the Englishman spends his
Sunday evening at the alehouse—
——‘And e`en on Sunday
Drank with Kirton Jean till Monday’—
but he only unbends and waxes mellow by degrees, and sits soaking
till he can neither sit, stand, nor go: it is his vice, and a beastly one it
is, but not a proof of any inherent distaste to mirth or good-
fellowship. Neither can foreigners throw the carnival in our teeth
with any effect: those who have seen it (at Florence, for example),
will say that it is duller than any thing in England. Our Bartholomew-
Fair is Queen Mab herself to it! What can be duller than a parcel of
masks moving about the streets and looking as grave and
monotonous as possible from day to day, and with the same lifeless
formality in their limbs and gestures as in their features? One might
as well expect variety and spirit in a procession of wax-work. We
must be hard run indeed, when we have recourse to a pasteboard
proxy to set off our mirth: a mask may be a very good cover for
licentiousness (though of that I saw no signs), but it is a very bad
exponent of wit and humour. I should suppose there is more drollery
and unction in the caricatures in Gilray’s shop-window, than in all
the masks in Italy, without exception.[6]
The humour of English writing and description has often been
wondered at; and it flows from the same source as the merry traits of
our character. A degree of barbarism and rusticity seems necessary
to the perfection of humour. The droll and laughable depend on
peculiarity and incongruity of character. But with the progress of
refinement, the peculiarities of individuals and of classes wear out or
lose their sharp, abrupt edges; nay, a certain slowness and dulness of
understanding is required to be struck with odd and unaccountable
appearances, for which a greater facility of apprehension can sooner
assign an explanation that breaks the force of the seeming absurdity,
and to which a wider scope of imagination is more easily reconciled.
Clowns and country people are more amused, are more disposed to
laugh and make sport of the dress of strangers, because from their
ignorance the surprise is greater, and they cannot conceive any thing
to be natural or proper to which they are unused. Without a given
portion of hardness and repulsiveness of feeling the ludicrous cannot
well exist. Wonder, and curiosity, the attributes of inexperience,
enter greatly into its composition. Now it appears to me that the
English are (or were) just at that mean point between intelligence
and obtuseness, which must produce the most abundant and
happiest crop of humour. Absurdity and singularity glide over the
French mind without jarring or jostling with it; or they evaporate in
levity:—with the Italians they are lost in indolence or pleasure. The
ludicrous takes hold of the English imagination, and clings to it with
all its ramifications. We resent any difference or peculiarity of
appearance at first, and yet, having not much malice at our hearts,
we are glad to turn it into a jest—we are liable to be offended, and as
willing to be pleased—struck with oddity from not knowing what to
make of it, we wonder and burst out a laughing at the eccentricity of
others, while we follow our own bent from wilfulness or simplicity,
and thus afford them, in our turn, matter for the indulgence of the
comic vein. It is possible that a greater refinement of manners may
give birth to finer distinctions of satire and a nicer tact for the
ridiculous: but our insular situation and character are, I should say,
most likely to foster, as they have in fact fostered, the greatest
quantity of natural and striking humour, in spite of our plodding
tenaciousness, and want both of gaiety and quickness of perception.
A set of raw recruits with their awkward movements and unbending
joints are laughable enough: but they cease to be so, when they have
once been drilled into discipline and uniformity. So it is with nations
that lose their angular points and grotesque qualities with education
and intercourse: but it is in a mixed state of manners that comic
humour chiefly flourishes, for, in order that the drollery may not be
lost, we must have spectators of the passing scene who are able to
appreciate and embody its most remarkable features,—wits as well as
butts for ridicule. I shall mention two names in this department,
which may serve to redeem the national character from absolute
dulness and solemn pretence,—Fielding and Hogarth. These were
thorough specimens of true English humour; yet both were grave
men. In reality, too high a pitch of animal spirits runs away with the
imagination, instead of helping it to reach the goal; is inclined to take
the jest for granted when it ought to work it out with patient and

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Spectroscopy and Dynamics of Single

Molecules: Methods and Applications

2018 Physical Chemistry of Gas–Liquid Interfaces

Library of Congress Cataloging-in-Publication Data

For information on all Elsevier publications visit our

Publisher: Susan Dennis

Typeset by SPi Global, India

Beyond intensity: Expanding techniques

molecule applications are intrinsic to methods of super-resolution fluorescence

Single-molecule spectroscopy has been the topic of numerous reviews. Only a

Topics covered in this book

70. von Diezmann, A.; Shechtman, Y.; Moerner, W. E. Three-Dimensional Localization of

1.1.1 Potential artifacts from surfaces and the use of extrinsic

fluorophore used to probe a structure, an interaction, or a conformational change.

1.1.2 Potential artifacts due to the generation of dark states

1.2 Photophysical processes and the generation of dark

If I had stopped to inquire at the White Horse Cellar, which would

The New Monthly Magazine.]

This old-fashioned epithet might be supposed to have been

What sparkling richness in the scarlet coats of the riders, what a

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