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 AN INTRODUCTION
TO SINGLE MOLECULE
BIOPHYSICS
Foundations of Biochemistry and Biophysics
Currently available
Quantitative Understanding of Biosystems: An Introduction to Biophysics
Thomas M. Nordlund

Biomolecular Thermodynamics: From Theory to Application

Douglas E. Barrick

Biomolecular Kinetics: A Step-by-Step Guide

Clive R. Bagshaw

An Introduction to Single Molecule Biophysics

Yuri L. Lyubchenko (Ed.)

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David E. Draper

RNA Biophysics
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 AN INTRODUCTION
TO SINGLE MOLECULE
BIOPHYSICS

Edited by

Yuri L. Lyubchenko
College of Pharmacy
University of Nebraska Medical Center
Cover: Image by Salvatore Chiantia, courtesy of Petra Schwille, Max Planck Institute of Biochemistry.
CRC Press
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Library of Congress Cataloging‑in‑Publication Data

Names: Lyubchenko, Yuri L., editor.

Title: Introduction to single molecule biophysics / [edited by] Yuri L.
Lyubchenko.
Other titles: Foundations of biochemistry and biophysics.
Description: Boca Raton : Taylor & Francis, 2017. | Series: Foundations of
biochemistry and biophysics
Identifiers: LCCN 2017033078 | ISBN 9781439806944 (hardback : alk. paper)
Subjects: | MESH: Single Molecule Imaging--methods | Biophysical Phenomena
Classification: LCC QH345 | NLM WN 190 | DDC 572--dc23
LC record available at https://lccn.loc.gov/2017033078

Visit the Taylor & Francis Web site at

http://www.taylorandfrancis.com
and the CRC Press Web site at
http://www.crcpress.com
Contents

Series Preface vii

Preface ix
Editor xiii
Contributors xv

1. Single Molecule Fluorescence 1

Ashok Deniz

2. Atomic Force Microscope for Topographic Studies 21

Yuri L. Lyubchenko

3. Atomic Force Microscope Force Spectroscopy 79

Eric A. Josephs, Piotr E. Marszalek, and Zackary N. Scholl

4. Magnetic Tweezers 115

Piero R. Bianco, Yuri L. Lyubchenko, and Zhiqiang Sun

5. Optical Tweezers 141

Marco Capitanio

6. Nanofluidic Transport and Sensing in Biological

and Artificial Nanopores 197
Aleksandr Noy and Meni Wanunu

Index 229

v
Series Preface

B iophysics encompasses the application of the principles, tools, and techniques

of the physical sciences to problems in biology, including determination and
analysis of structures, energetics, dynamics, and interactions of biological mole-
cules. Biochemistry addresses the mechanisms underlying the complex reactions
driving life, from enzyme catalysis and regulation to the structure and function
of molecules. Research in these two areas is having a huge impact in pharmaceu-
tical sciences and medicine.
These two highly interconnected fields are the focus of this book series. It cov-
ers both the use of traditional tools from physical chemistry, such as nuclear mag-
netic resonance (NMR), X-ray crystallography, and neutron diffraction, as well as
novel techniques, including scanning probe microscopy, laser tweezers, ultrafast
laser spectroscopy, and computational approaches. A major goal of this series is
to facilitate interdisciplinary research by training biologists and biochemists in
quantitative aspects of modern biomedical research, and teaching core biological
principles to students in physical sciences and engineering.
Proposals for new volumes in the series may be directed to Lu Han, Executive
editor, at CRC Press/Taylor & Francis Group (lu.han@taylorandfrancis.com).

vii
Preface

S ingle molecule biophysics (SMB) is a relatively new branch of molecular bio-

physics, which utilizes a set of methods that enable imaging or probing of
individual molecules. The attractiveness of SMB is defined by two major features.
The first is biological systems, including cells, which consist of a limited number
of biological molecules or complexes. A study of this kind requires techniques
capable of probing single molecules. Second are the intricate functions of biologi-
cal molecules and systems as they assemble in transient states. Characterization
of these states is critical in understanding the molecular mechanism of the bio-
logical process under study. As they become available, SMB techniques have
been applied to numerous biological problems associated with understanding
the molecular mechanisms of DNA replication, transcription, and translation, as
well as the functions of molecular machines. Many of these examples and others
are discussed in the book in association with a specific method.
The discoveries of major SMB techniques have historically been ­associated
with Nobel Prizes. For example, the atomic force microscope (AFM) belongs
to the family of scanning probe microscopes, the discovery of which was
awarded the Nobel Prize in Physics in 1986 to G. Binnig and H. Rohrer at IBM,
Zurich, who published their discovery only 4 years prior to the award. Current
single molecule fluorescence methods originate from the 1989 publication
by W. Moerner and L. Kador, with a Nobel Prize awarded to W. Moerner in
2014 for the discovery. Optical traps or tweezers utilize the phenomenon of
manipulating atoms and small particles by focused light beam. By applying
this method in physics to the cooling and trapping of atoms with laser light, the
assembly of a Bose–Einstein condensate was achieved, leading to Nobel Prizes
in Physics in 1997 and 2001.
This introductory book broadly covers the motivations, history, and meth-
odologies of the major single molecule imaging and probing techniques, which
can be applied to numerous important biological systems and questions.

ix
 x Preface

Furthermore, this book highlights the most useful and exciting strengths of
these evolving methods through examples of several recent biological applica-
tions. The primary target audience for this book is undergraduate and graduate
students, academics, and professionals who are looking to learn more about this
rapidly growing field and may benefit from some of its many research capabili-
ties. An important focus of the book is to introduce the key concepts and ideas of
the major approaches in the single molecule field in a relatively simple manner.
A defining characteristic of the book’s style is that large sets of specific examples
have been assembled for each biological application described. The goal of this
layout is to better familiarize readers with techniques they may be interested
in pursuing for their own research. Additionally, up-to-date information about
several different applications is discussed, and a substantial reference list is pro-
vided, which even seasoned practitioners in the field should find useful both as
a personal reference and as an introductory guide to the field for incoming stu-
dents and colleagues.
The book is assembled around six different SMB techniques, with each chapter
dedicated to a specific method. The first chapter covering single molecule fluo-
rescence techniques is written by Deniz. This chapter provides basic knowledge
in fluorescence and the Förster resonance energy transfer (FRET) phenomenon,
and describes principles of instrumentation enabling implementation of the
techniques at the single molecule level. Brief discussions of applications of the
methods round out the chapter and highlight the power of the methods to answer
fundamental questions in biophysics and biology. In Chapter 2, Lyubchenko pro-
vides an overview of AFM as a tool for single molecule imaging. In addition to
a description of the instrument, its main properties, and the spatial resolution
limit, the chapter describes the use of AFM for direct time-lapse visualization of
dynamics of molecular complexes at the nanoscale. A great deal of attention was
given to high-speed AFM capable of data acquisition at a video rate. This emerg-
ing AFM technique has already produced new knowledge in a large number of
applications, with a few of them described. Josephs, Marszalek, and Scholl in
Chapter 3 review the applications of AFM as a nanotool for measuring the inter-
actions between molecules and the mechanical properties of isolated molecules
and their complexes. The authors describe the principles of AFM single molecule
force spectroscopy (SMFS) experiments, interpretation of data, and general prin-
ciples of force spectroscopy data analysis. Although AFM force spectroscopy is
a mode of operation of essentially all commercially available AFM instruments,
the authors describe principles for designing a force spectroscopy instrument.
As in other chapters, a few applications of this instrumentation are described.
In Chapter 4, Bianco, Lyubchenko, and Sun describe the principles and opera-
tion of magnetic tweezers. In this technique, the system under study to which
a small magnetic bead is attached is interrogated with an external magnet. This
method, essentially established and developed by Bensimon and Croquette in
France, caught the broad attention of the SMB community due to the relative
simplicity of the instrument construction. Compared with other probing tech-
niques, AFM and optical tweezers, the major attractive feature of magnetic twee-
zers is the ability to control the rotational dynamics of the system. Advances in
 Preface xi

this application of magnetic tweezers are described, and a few applications are
presented. Capitanio outlines the principles of optical tweezers methodology in
Chapter 5. This is one of the most widely used single molecule techniques that
attracts investigators through its ability to probe numerous biological systems,
discover their dynamic behavior, and investigate force dependence. Principles of
the instrument assembly, the fundamentals of force and position measurements,
and the operation of optical tweezers in different configurations are described.
Capitanio guides the researcher to the most appropriate optical tweezers con-
figuration to be used with a given biological system, how to optimize the mea-
surements, and what resolution limits should be expected. Most examples are
presented on the application of optical tweezers to single biological molecules
in vitro, with a final illustration of recent applications of the technique to quan-
titative studies to probing a single cell. Protein nanopores occupy a central role
in biology as the primary conduit for a cell’s communication with the outside
world, and synthetic nanopores have long aimed to replicate this functionality
in engineered assemblies. In Chapter 6, Noy and Wanunu go over the basic con-
cepts of nanopore transport, and the most common types of biological and arti-
ficial nanopores, including the recently developed carbon nanotube porins. They
also review some of the unique transport phenomena that are observed in these
structures, and describe the physical and technological basis of nanopore-based
sensing. Particular emphasis is given to the nanopore-based DNA sequenc-
ing technology that will be underpinning the next generation of genomics and
health care applications.
The set of techniques assembled in this book includes the most widely used
experimental SMB experiments. These methods have already led to a large num-
ber of discoveries, with some selected examples described in this book. A major
trend in the SMB field is to utilize a combination of the described techniques
in one integrated instrument, and a few such integrations are described in this
book. However, even these methods are in flux and undergo constant improve-
ments and modifications. Novel methods appear as well, and these advances set
the basis for future editions of such an introductory book.

Yuri L. Lyubchenko
University of Nebraska Medical Center
Editor

Yuri L. Lyubchenko is professor of Pharmaceutical Sciences at the University

of Nebraska Medical Center, Omaha, Nebraska. His research focuses on under-
standing fundamental mechanisms underlying health and disease, which are key
to developing new and more effective diagnostics and medications. This primar-
ily basic research allows him not only to identify new drug targets for small mol-
ecule drugs, but also to develop the nanotools and methods needed to discover
novel approaches for diagnosis, treatment, and disease prevention, and to more
rapidly determine their efficacy at the molecular level.

xiii
Contributors

Piero R. Bianco Piotr E. Marszalek

Department of Microbiology and
Immunology
Center for Single Molecule Biophysics
University at Buffalo
Buffalo, New York
Marco Capitanio
LENS (European Laboratory for
Non-Linear Spectroscopy)
University of Florence
Florence, Italy
Ashok Deniz
The Scripps Research Institute
La Jolla, California
Eric A. Josephs
Department of Mechanical
Engineering and Materials Science
Duke University
Durham, North Carolina
Yuri L. Lyubchenko
Department of Pharmaceutical
Sciences
College of Pharmacy
University of Nebraska Medical Center
Omaha, Nebraska
Department of Mechanical
Engineering and Materials Science
Duke University
Durham, North Carolina
Aleksandr Noy
Lawrence Livermore National Laboratory
Livermore, California
and
University of California, Merced
Merced, California
Zackary N. Scholl
Department of Computational
Biology and Bioinformatics
Duke University
Durham, North Carolina
Zhiqiang Sun
Department of Pharmaceutical Sciences
College of Pharmacy
University of Nebraska Medical Center
Omaha, Nebraska
Meni Wanunu
Northeastern University
Boston, Massachusetts

xv
 1 Single Molecule
Fluorescence
Ashok Deniz
The Scripps Research Institute
La Jolla, California

Contents
1.1 Single Molecule Fluorescence Observables and Detection Modes . . . . . . . 2
1.1.1 Observables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.1.1.1 Fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.1.1.2 Single-Dye Measurements (Intensity and
Other Properties) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.1.1.3 Single Molecule FRET (Two or More Dyes) . . . . . . . . . . . . 5
1.1.1.4 Single Molecule Localization and Tracking . . . . . . . . . . . . 8
1.1.2 Instrumentation and Measurement Modalities . . . . . . . . . . . . . . . . 9
1.1.2.1 Total Internal Reflection Fluorescence . . . . . . . . . . . . . . . 10
1.1.2.2 Confocal Imaging/Spectroscopy . . . . . . . . . . . . . . . . . . . . 11
1.1.2.3 Attachment of Fluorescent Dyes for
Single Molecule Fluorescence Experiments . . . . . . . . . . . 12
1.2 Surface Attachment of Biomolecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
1.3 Some Key Examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
1.3.1 Enzyme Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
1.3.2 Structural Biophysics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
1.3.3 Molecular Structure, Binding, and Function in Cells . . . . . . . . . . 16
1.4 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

T he past couple of decades have seen a veritable explosion of single molecule

studies of biological phenomena, along with concurrent improvements in
the technologies needed for such studies. Such studies provide several ­substantial
benefits compared with standard ensemble studies, and on a practical level they
provide complementary information for answering important biophysical and

1
 2 Single Molecule Fluorescence

biological questions. For example, these methods have provided important

new insights into the functioning of enzymes, dynamic structural complexity
in proteins and nucleic acids, protein folding and misfolding, the function of
motor and gene regulation proteins, and even the entry and dynamics of viruses
into cells.
Single molecule methods offer several important strengths. While many
ensemble methods average properties over millions or billions of molecules
resulting in information loss, Single molecule methods measure the properties
of molecules one at a time, allowing more detailed information to be recorded.
For example, conformational distributions of proteins and nucleic acids can
be recorded. Moreover, these molecules are often quite dynamic, and single
­molecule methods can better capture heterogeneity in these dynamics. In this
regard, most importantly, complex, multistate, stochastic dynamics can be cap-
tured, which would be virtually impossible in ensemble experiments because
different molecules would be proceeding through different steps of reaction tra-
jectories, even if all reactions were initiated at the same time. In this chapter, we
discuss some of the important single molecule fluorescence methods and follow
up with short discussions of some interesting biological problems that have been
tackled using these methods.
Figure 1.1 illustrates a few important parameters that are of interest to
students and practitioners of biophysics in its broadest context (including
biologists, physicists, and chemists). Single molecule methods can provide
information about many such biological properties over a large dynamic
range, mainly in vitro, but increasingly in living cells and in vivo (Deniz et al.
2008). For example, distance measurements can be performed with resolu-
tions from mesoscopic to atomic dimensions by using different methods. The
single molecule practitioner will ideally choose a particular method to be
used based on the specific problem or biological system under study. Here,
we first briefly list several molecular properties of interest, along with a brief
discussion of a few common single molecule methods that can be used to
make measurements of these properties.
In the next section, we introduce individual types of common single molecule
measurements briefly, highlighting their importance in biophysical and biologi-
cal studies.

1.1 Single Molecule Fluorescence

Observables and Detection Modes
1.1.1 Observables
1.1.1.1 Fluorescence
Fluorescence is a relaxation process available to single electronic excited
states of molecules, and this process involves the emission of a photon with an
energy corresponding to the energy difference between the excited state and
the ground state (Figure 1.2). Typically, emission occurs from a lower vibra-
tional level of the excited state; hence, the emission is red shifted in wavelength
 1.1 Single Molecule Fluorescence Observables and Detection Modes 3

Fluorescence Force

Binding and In vitro

organization

Structure Rotation
and
fluctuations ing Movement
ld
Fo

Mi
sfo
ldi
ng

Enzyme function In vivo

Figure 1.1 Single molecule fluorescence (and force/manipulation) methods

­ rovide a wide variety of information about biological systems. In this chapter,
p
we focus on fluorescence methods.

compared to the excitation light. Because of this feature, the excitation light can
be blocked while letting the emission light be detected, which is a huge advan-
tage, contributing to the very high sensitivity of the method for single molecule
measurements.
In addition, the timescale of fluorescence is such that it typically competes
with other excited state relaxation processes, which in turn allows it to report
molecular properties of interest. For example, relaxation can also occur by non-
radiative means; hence, the “brightness” of the fluorescence is often modulated
by intramolecular or intermolecular quenchers, thus reporting on the proxim-
ity of such quenchers. In the special case of Förster resonance energy transfer
(FRET), the proximity of a second chromophore allows longer range distances to
be measured. Tumbling of large molecules can also occur on a timescale similar
to fluorescence, which allows for the measurement of such tumbling and molec-
ular size via polarization measurements (Figure 1.3). Finally, photon emission
can be used to localize the location of molecules down to nanometer (10 –9 m)
 4 Single Molecule Fluorescence

S1
T1

Intersystem
Non-radiative Crossing
decay

S0 Fluorescence
essscc

Figure 1.2 Jablonski diagram depicting fundamental electronic states and pro-
cesses relevant to fluorescence.

Molecular tumbling
randomizes polarization

Detect fluorescence
light of one or more
polarizations

Excite fluorophore Information about

with polarized light molecular
tumbling (size and shape)
Polarization
direction
Polarizer

Figure 1.3 The principle of fluorescence polarization experiments and measure-

ment of molecular tumbling.

precision, which can be used to follow the details of movement of molecules

in vitro and in live cells.

1.1.1.2 Single-Dye Measurements (Intensity and Other Properties)

A number of single molecule measurements have used fluorescence emission
from a single dye to report on molecular properties. For example, in pioneering
single molecule experiments on enzyme molecules, Xie and coworkers used the
fact that a redox enzyme cofactor’s fluorescence was turned on and off depend-
ing on whether it was oxidized or reduced (Lu et al. 1998). Since the enzymatic
cycle involved oxidation and reduction of the cofactor, recording the “blinking”
in emission intensity of the cofactor fluorescence allowed them to record the
 1.1 Single Molecule Fluorescence Observables and Detection Modes 5

catalytic activity of these enzymes in real time. Even before these measurements,
others had used a substrate that turned fluorescent upon reaction to the prod-
uct to measure the activity of individual enzymes (Xue and Yeung 1995; Craig
et al. 1996), although here individual turnovers were not recorded. These kinds of
experiments have resulted in the generation of entirely new kinds of information
about the complexity of enzyme function.
Emission from one kind of dye (but from multiple molecules) has also
been used in pioneering experiments, for example, to observe the rotation
of molecular motors. Finally, photons have a direction associated with them
(polarization). This feature can be used to measure the orientation or rota-
tion of molecules, which has also been used in a number of single molecule
experiments. For example, polarization information can report on the size
of a molecule, because tumbling rate scales with the size, and tumbling of
the molecule following excitation and before emission results in randomiza-
tion of the polarization of the emitted light. Molecular size can give infor-
mation about conformation and binding—indeed, ensemble polarization is
commonly used as a sensitive tool for screening binding, for example, in the
search for drug candidates.
Yet another useful single-dye analysis that has been used extensively is
fluorescence correlation spectroscopy (Magde et al. 1972; Rigler et al. 1993;
Eigen and Rigler 1994; Thompson et al. 2002; Kim et al. 2007). In some ways,
this was a precursor of modern fluorescence single molecule experiments.
The experiment essentially consists of using confocal detection to monitor
emission from a small number of molecules as they diffuse into and out of a
focal volume. A correlation analysis then is used to extract information about
the fluctuation timescales and amplitudes from the apparently noisy data
(Figure 1.4). This method has been widely used to study diffusion times (and
hence binding or sometimes conformational changes). It can also be used to
study chain fluctuations using an appropriate quencher, as for the disordered
state of the protein Sup35-NM (Figure 1.4). More generally, this represents a
class of correlation or fluctuation analysis methods.

1.1.1.3 Single Molecule FRET (Two or More Dyes)

Single molecule FRET is the single molecule analog of the well-known ensemble
technique commonly referred to as FRET (Förster Resonance Energy Transfer)
that is useful for measuring distances (and, hence, structure) in molecules.
Named after Theodor Förster, it is also commonly known as fluorescence reso-
nance energy transfer, which may be a bit misleading because transfer is non-
radiative and not via the emission and reabsorption of a photon. Pioneering
work several decades ago by Stryer and Haughland resulted in demonstration
of the strong distance dependence of the transfer (Stryer and Haugland 1967).
In the mid-1990s to late 1990s, the first demonstration of single molecule FRET
(Ha et al. 1996) and its distance dependence (Deniz et al. 1999) were followed by
quite extensive use of the method in the single molecule community.
As shown in Figure 1.5, the phenomenon refers to the nonradiative transfer of
excitation energy from a donor molecule to an acceptor molecule by the coupling
 6 Single Molecule Fluorescence

30
25
20

Counts/ms
15
10
5
0
0 100 200 300 400
Time (ms)

Correlation
analysis

0.40
Timescale and other Conformational Diffusion

Autocorrelation (G(t)-1)
0.35 fluctuation
quantitative information 0.30

about 0.25

molecular 0.20
0.15
fluctuations
0.10
0.05

0.00
0.05
1E-5 1E-4 1E-3 0.01 0.1 1 10 100 1000
Delay time (ms)

Figure 1.4 The basic principles behind fluorescence correlation spectroscopy

measurements. (Adapted from Mukhopadhyay, S., et al., Proc. Natl. Acad. Sci.
U S A, 104, 2649, 2007.)

S1 1.0
1
S1 0.8 E=
1+(R/R0)6
FRET efficiency

0.6
FRET coupling
0.4

0.2
Excitation Fluorescence S0 Fluorescence
following FRET
S0 Acceptor
Donor 0 20 40 60 80 100 120
Distance R (Å)
(a) (b)

Figure 1.5 Jablonski diagram of the FRET process (a) and distance dependence
of FRET (b).

between their transition dipoles. The transfer rate has a strong distance dependence
(r6). This translates into a strong distance dependence for the transfer efficiency
(EFRET), which is often measured in experiments. EFRET represents the fraction of
donor excited states that result in energy transfer to the acceptor (vs. fluorescence
of the donor or nonradiative decay). As can be seen, the transfer efficiency, which is
a function of a competition in rate constants for the various decay processes of the
donor, can report on the distance between donor and acceptor dyes (30–70 Å range).
 1.1 Single Molecule Fluorescence Observables and Detection Modes 7

By labeling a protein, RNA, or other molecule with such donor and acceptor dyes, it
is then possible to measure distance and hence conformational properties for these
molecules at single molecule resolution. Labeling methods and instrumentation
are briefly discussed in Sections 2.2.3 and 2.2.2, respectively.
As an example, we briefly discuss the simple case of the first direct observation
of two-state protein folding by FRET by Deniz et al. (2000). The protein studied
was chymotrypsin inhibitor 2 (CI-2), which was a protein believed to fold via a two-
state folding mechanism. In other words, the protein resided either in a denatured
state or a folded state, but not significantly in intermediate conformations. To carry
out this study, the protein was first produced in a dual-labeled form with donor
and acceptor sites at positions expected to report on denatured and folded states
with different FRET signatures. Site-specific labeling was achieved using synthetic
and ligation methods (Deniz et al. 2000). Freely diffusing proteins were examined;
hence, potential perturbation due to interactions with surfaces was avoided. Each
single molecule experiment consisted of measuring FRET efficiencies for a large
number (>1000) of single molecules and collecting these as a histogram depicting
the number of events versus EFRET. As the examples in Figure 1.6 show, these histo-
grams provide information about populations of proteins in solution.
Cy5
S
S
GdmCI Cysteine 40
Denatured state Folded state
N-terminus

TMR

1.0
[Denaturant]

200
3M 0.8
100
Fraction folded

0.6
Number of events

0
200 4M
0.4
100
Ensemble Trp.
Filens Trp.
0 0.2 Ensemble FRET
200 6M Filens FRET
Single-mol. FRET
Fil sm. FRET
100 0.0

0 1 2 3 4 5 6
0.4 0.6 0.8 1.0 Guanidinium chloride
FRET efficiency

Figure 1.6 Two-state protein folding directly validated by smFRET experiments.

(Adapted from Deniz, A. A., et al., Proc. Natl. Acad. Sci. U S A, 97, 5179, 2000.)
Shown are the two-state model; protein with attached dyes; smFRET histograms
at low, intermediate, and high denaturant concentration; and extracted denatur-
ation curves with ensemble curves for comparison.
 8 Single Molecule Fluorescence

Under native conditions (absence of denaturant), proteins adopted a high

EFRET or compact conformation as expected. Under unfolding conditions, a peak
was observed ~0.65 EFRET , which corresponded to a more expanded denatured
state. Interestingly, under intermediate levels of denaturant, the experiment
provided snapshots of either folded or denatured proteins, with no significant
intermediates observed, thereby providing a direct observation of a two-state
folding mechanism. An interesting feature was that a denaturation curve could
be directly extracted from the data by using areas of the peaks (as a control, curve
corresponded well with a denaturation curve from ensemble data on an unlabeled
protein calculated using a two-state model). In addition, there may have been a
small shift in the denatured peak position, which would indicate that the dena-
tured state was expanding as a function of increasing denaturant, although the
small shift did not allow this conclusion to be made unequivocally. More recent
experiments on other protein systems have revealed more dramatic shifts, for
example, in the case of an intrinsically disordered yeast prion protein monomer
(Mukhopadhyay et al. 2007). Furthermore, it should be noted that the denatured
and native states can be more generally described as ensembles in structures rap-
idly interconverting on the timescale of these experiments. Experiments directly
probing the fluctuations within these ensembles have been carried out using cor-
relation techniques.
This single molecule FRET (smFRET) method has found wide use in the
single molecule community, with applications ranging from probing folding of
proteins and complexes, movement of molecular motors on their DNA or other
tracks, and formation or dissociation of complexes in vitro and in cells. More
recently, this method has even been extended to additional dimensions, for exam-
ple, three-color smFRET (Hohng et al. 2004; Clamme and Deniz 2005; Gambin
and Deniz 2010). These methods will allow more global analysis of molecules and
complexes at single molecule resolution.

1.1.1.4 Single Molecule Localization and Tracking

Another important feature of single molecule experiments is the possibility of
tracking the position of a molecule as it moves (Brandenburg and Zhuang 2007;
Greenleaf et al. 2007; Chang et al. 2008). For example, the movement of protein
motors on molecular tracks, of protein receptors on cell membranes, or cellu-
lar internalization of viruses can be a key part of their functions or infection;
hence, a huge effort has gone into understanding the complicated characteristics
of these motions. Tracking in its simplest form consists of monitoring photons
from a single molecule using a high-sensitivity charge-coupled device (CCD)
camera. Since molecules are typically much smaller than the wavelength of light
(~450–650 nm for visible light used in many single molecule experiments), the
detected emission profile (point spread function) is determined by the optical
properties of the excitation and collection. This can often be approximated rea-
sonably by a two-dimensional (2D) Gaussian (Figure 1.7), which is used to fit
and pinpoint the location of the molecule. By performing this procedure as a
function of time, the path of motion of the molecule can be determined with
nanometer precision.
 1.1 Single Molecule Fluorescence Observables and Detection Modes 9

Molecule localization by
analysis of single molecule
movie images

High-resolution trajectory
of molecular movement

Figure 1.7 Single-particle or single molecule tracking. At the top are 2D

Gaussian fits of image data; the center of the 2D Gaussian provides a highly accu-
rate measure of the particle position, as depicted in the trajectory in red.

A related set of methods that has emerged over the last few years are the so-
called super-resolution imaging methods. This is a neat way to get around the
classical diffraction limit of imaging, which was the dogma in the imaging field
until about a decade back. The idea is that diffraction limits the resolution of two
objects below about half the wavelength of the emitted light. However, as noted
above, if one images the objects (molecules) one at a time, their positions can
be localized with much higher precision (arbitrarily high, depends on the num-
ber of collected photons). Hence, groups (initially of Xioawei Zhuang [Rust et al.
2006] and Eric Betzig [Betzig et al. 2006]) have developed methods to stochasti-
cally turn on a small fraction of single molecules in a sample, localize them by
imaging, turn them off, then turn on another small subset, and so on. Hence, by
imaging an ensemble of molecules, one at a time, high-resolution imaging can be
performed, which is providing detailed views of cellular architecture that were
previously unavailable from fluorescence imaging (Bates et al. 2008; Patterson
et al. 2010). Indeed, the impact of this advance is large enough that the 2014
Nobel Prize in Chemistry in part went to Betzig for his above contribution.

1.1.2 Instrumentation and Measurement Modalities

Several types of instruments are used for single molecule fluorescence measure-
ments. These were often lab assembled until recently, although some are now
available commercially. Key features required for single molecule measurements
are high-quality illumination and detection to allow rejection of background
photons, and high-efficiency detection of the small number of photons emitted
by single molecules. The light source for excitation is usually a laser, which pro-
vides illumination with advantageous optical properties, including beam pro-
file and narrow spectral bandwidth. The former permits focusing the beam to a
very small volume, and the latter permits better rejection of the intense excita-
tion light prior to measurement of the very low-intensity single molecule fluo-
rescence light. The excitation geometry usually seeks to limit the detection area
or volume to nanometer-micron dimensions. Correspondingly, measurement
 10 Single Molecule Fluorescence

modes include total internal reflection fluorescence (TIRF), confocal and near-
field detection, and epi imaging. Detection is via high-efficiency detectors such
as high-end CCD cameras (TIRF and epi) and avalanche photodiodes (APDs,
confocal). Confocal and TIRF microscopies are the most commonly used modes,
and these are discussed in more detail later.

1.1.2.1 Total Internal Reflection Fluorescence

Total internal reflection (TIRF) is a phenomenon where light passing from a higher
to a lower refractive index medium is completely reflected if the incident angle
is greater than a “critical angle” defined by the difference in refractive indices.
Although 100% reflection is achieved in the far field, a very narrow evanescent elec-
tromagnetic field is generated at the far side of the interface. This field decays expo-
nentially with a length scale of ~100 nm. The basic features of a TIRF instrument
are depicted in Figure 1.8. Laser light passes through a coupling prism, coupling
medium, and fused silica slide, then encounters an interface with a lower refrac-
tive index medium (aqueous sample). If the incident angle is adjusted appropriately,
TIR occurs, generating a thin layer of illumination in the sample solution. The mol-
ecules of interest are usually immobilized on the slide and are illuminated by the
evanescent field. Fluorescent impurities and so on in the solution that are outside
of the evanescent field are not illuminated and hence do not contribute to fluores-
cent background. Fluorescence emitted by the single molecules is then collected by
a high numerical-aperture objective, which permits high collection efficiency. The
light then passes through filters to reject excitation light; additional optics (e.g., to
separate different wavelength regions for FRET detection) is then focused onto a
CCD camera to produce an image. Intensified CCD cameras were often used in the
past; these have typically been replaced by electron-multiplying CCDs (with better

Laser

Low conc.
~ nM sample

High N.A. objective

2-color/FRET usually water immersion

Additional filters/optics

CCD CCD
High efficiency
camera camera
CCD camera

Figure 1.8 The basic instrumental setup for total internal reflection fluorescence
(TIRF) single molecule experiments.
 1.1 Single Molecule Fluorescence Observables and Detection Modes 11

detection characteristics), and newer cameras and detectors are under development.
The camera provides an image of the detection area, with bright spots correspond-
ing to signals from single molecules. A movie of such images then provides temporal
information, typically with a time resolution of 10–100 ms. Analyses of the movies
provide time trajectories about the fluorescence characteristics and hence molecular
properties of these single molecules. For example, a FRET experiment can provide
information about distance as a function of time, and provide information about
conformational fluctuations of the molecules of interest. An important character-
istic of these types of measurements is the ability to follow hundreds of single mol-
ecules simultaneously and in real time for extended periods (usually up to seconds
or minutes). Detailed analyses can be performed on the detected time trajectories.
Common analyses for smFRET experiments include determining donor and accep-
tor intensity trajectories, converting them to a FRET time trajectory, assigning
states to different FRET levels, and then extracting rate constants using dwell-time
analysis. More complex analyses have been developed, for example, using Hidden
Markov Modeling (McKinney et al. 2006), to uncover information about the FRET
and interconversion characteristics of states in complex systems.

1.1.2.2 Confocal Imaging/Spectroscopy

Confocal microscopy is also quite widely used in single molecule fluorescence
studies. The instrumental setup (Figure 1.9) is quite similar to that used for TIRF.

Low concentration
<nM sample

High N.A. objective

Usually water immersion

Dichroic mirror:
Reflect excitation
transmit emission
Laser

Pinhole Filters/optics

30
25
Count g/mg

20
15
10
5
High efficiency 0
0 100 200 300 400 500
APD detector Time (ms)

Computer

Figure 1.9 Basic instrumental setup for confocal single molecule experiments.
 12 Single Molecule Fluorescence

Here, laser illumination is via a high numerical aperture (N.A.) objective, which
focuses the beam to a diffraction-limited spot. Fluorescence is collected by the
same objective, separated from excitation light by a dichroic mirror, and then
passes through a pinhole that further narrows the detection volume, primarily
in the Z-dimension. The light is then treated with additional optics, for example,
to split it into donor and acceptor components for FRET detection, and is then
imaged onto APD detectors, which are used in a single-photon counting mode.
For many applications, the number of detected photons is recorded per unit time
(e.g., 1 ms or fewer), and this “intensity” is recorded as a function of time. If
this is done for donor and acceptor channels, a FRET efficiency can be calcu-
lated as E = Ia/(Ia + Id), with various corrections being applied for background,
cross-talk between channels, and differences in detection and emission quan-
tum yields. A more powerful approach for recording photon events is to record
detection times for each photon. From this information, intensity time-traces
with arbitrarily high time resolution (down to the ~10 ns recording resolution of
commonly used hardware) can be computed. In addition, correlation informa-
tion can be computed from the same data. Seidel and coworkers (Sisamakis et al.
2010) have done extensive work on multiparameter fluorescence detection, where
for each photon, multiple fluorescence properties are recorded, including mac-
roscopic detection time (as above), microscopic detection time (with respect to a
picosecond laser excitation pulse), polarization, and color can be simultaneously
recorded using a multidetector setup. These data can then be used to recreate an
intensity time-trace and measure lifetimes, properties, and FRET efficiencies for
single molecules, thus proving to be a powerful advanced data collection method.
Confocal data collection can be carried out on immobilized molecules as for
TIRF, but is often also used to detect diffusing or flowing molecules. This lat-
ter type of measurement largely eliminates potential perturbation from surface
immobilization and allows collection of molecular properties for large numbers
of single molecules in a relatively short time (Mukhopadhyay and Deniz 2007;
Deniz et al. 2008). On the contrary, long time-traces cannot be obtained by this
method. Data for immobilized molecules in this format are usually acquired by
scanning the sample using a piezoelectric or other scanner. Scanning is used to
find single molecules and position the excitation/detection volume over the sin-
gle molecule, collect photons until photobleaching, then repeat the search/posi-
tioning. Time trajectories of fluorescence signals are collected in this manner and
can be analyzed by methods as discussed in the TIRF section in Section 1.1.2.

1.1.2.3 Attachment of Fluorescent Dyes for Single Molecule

Fluorescence Experiments
Single molecule fluorescence experiments usually require the use of bright exter-
nally introduced dyes. These dyes need to be attached at a specific location in
the molecule, thus providing a specific probe for the properties of the molecule.
Several good chemistries exist for this purpose (Ferreon et al. 2010).
For short DNA and RNA oligonucleotides that can be made synthetically (com-
mercially available), many dyes can be attached during synthesis, hugely simpli-
fying this process. Alternatively, primary amine or thiol functionalities may be
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 BROILING.

Broiling is the best possible mode of

cooking and of preserving the flavour of
several kinds of fish, amongst which we
may specify mackerel and whitings; it is
also incomparably superior to frying for
steaks and cutlets, especially of beef and
mutton; and it is far better adapted also, to
the preparation of food for invalids; but it
should be carefully done, for if the heat be A Conjuror.
too fierce, the outside of the meat will be
scorched and hardened so as to render it
uneatable; and if, on the contrary, it be too gentle, the gravy will be
drawn out, and yet the flesh will remain so entirely without firmness,
as to be unpleasant eating. A brisk fire, perfectly free from smoke, a
very clean gridiron, tender meat, a dish and plates as hot as they
can be, and great despatch in sending it to table when done, all are
essential to the serving of a good broil. The gridiron should be
heated, and rubbed with mutton suet before the meat is laid on, and
it should be placed slopingly over the fire, that the fat may run off to
the back of the grate, instead of falling on the live coals and smoking
the meat; if this precaution should not prevent its making an
occasional blaze, lift the gridiron quickly beyond the reach of the
smoke, and hold it away until the fire is clear again. Steaks and
chops should be turned often, that the juices may be kept in, and
that they may be equally done in every part. If, for this purpose, it
should be necessary for want of steak-tongs to use a fork, it should
be passed through the outer skin or fat of the steak, but never stuck
into the lean, as by that means much of the gravy will escape. Most
eaters prefer broiled beef or mutton, rather underdressed; but lamb
or pork cutlets should always be thoroughly cooked. When a fowl or
any other bird is cut asunder before it is broiled, the inside should
first be laid to the fire: this should be done with kidneys also. Fish is
less dry and of better flavour, as well as less liable to be smoked, if it
be wrapped in a thickly buttered sheet of writing paper before it is
placed on the gridiron. For the more delicate-skinned kinds, the bars
should be rubbed with chalk instead of suet when the paper is
omitted. Cutlets, or meats of any other form, when egged and
crumbed for broiling, should afterwards be dipped into clarified butter
or sprinkled with it plentifully, as the egg-yolk and bread will
otherwise form too dry a crust upon it. French cooks season their
cutlets both with salt and pepper, and brush a little oil or butter over
them to keep them moist; but unless this be done, no seasoning of
salt should be given them until they are just ready to be dished: the
French method is a very good one. Steaks or cutlets may be quickly
cooked with a sheet or two of lighted paper only, in the apparatus
shown in the preceding page, and called a conjuror. Lift off the cover
and lay in the meat properly seasoned, with a small slice of butter
under it, and insert the lighted paper in the aperture shown in the
plate; in from eight to ten minutes the meat will be done, and found
to be remarkably tender, and very palatable: it must be turned and
moved occasionally during the process. This is an especially
convenient mode of cooking for persons whose hours of dining are
rendered uncertain by the nature of their avocations. For medical
men engaged in extensive country practice it has often proved so;
and we would especially recommend it to the notice of emigrants, to
whom it would often prove invaluable. The part in which the meat is
placed is of block tin, and fits closely into the stand, which is of sheet
iron. The conjuror from which our design was drawn, was purchased
in a country town in Essex, and was exceedingly well made, and
very cheap. We find on inquiry that the maker has quitted the place,
or we would insert his address.
 FRYING.

This is an operation, which, though

apparently very simple, requires to be more
carefully and skilfully conducted than it
commonly is. Its success depends
principally on allowing the fat to attain the
Sauté Pan. exact degree of heat which shall give
firmness, without too quick browning or
scorching, before anything is laid into the
pan; for, if this be neglected, the article fried will be saturated with
fat, and remain pale and flaccid. When the requisite degree of colour
is acquired before the cooking is complete, the pan should be placed
high above the fire, that it may be continued slowly to the proper
point. Steaks and cutlets should be seasoned with salt and pepper
and dredged on both sides lightly with flour before they are laid into
the pan, in which they should be often moved and turned that they
may be equally done, and that they may not stick nor burn to it. From
ten to fifteen minutes will fry them. They should be evenly sliced,
about the same thickness as for broiling, and neatly trimmed and
divided in the first instance. Lift them into a hot dish when done; pour
the fat from the pan, and throw in a small slice of butter; stir to this a
large teaspoonful of flour, brown it gently, and pour in by degrees a
quarter of a pint of hot broth or water; shake the pan well round, add
pepper, salt, and a little good catsup, or any other store sauce which
may be preferred to it, and pour the gravy over the steaks: this is the
most common mode of saucing and serving them.
Minute directions for fish, vegetables, omlets, and different
preparations of batter, are given in their proper places; but we must
again observe, that a very small frying pan (scarcely larger than a
dinner-plate) is necessary for many of these; and, indeed, the large
and thick one suited to meat and fish, and used commonly for them
is altogether unfit for nicer purposes.
The sauté-pan, shown in the preceding page, is much used by
French cooks instead of a frying-pan; it is more particularly
convenient for tossing quickly over the fire small collops, or aught
else which requires but little cooking.
All fried dishes, which are not sauced, should be served extremely
dry upon a neatly-folded damask cloth: they are best drained upon a
sieve reversed placed before the fire.
A wire basket of this form is convenient
for frying parsley and other herbs. It must
be placed in a pan well filled with fat, and
lifted out quickly when the herbs are done:
they may likewise be crisped in it over a
Wire Basket for
clear fire, without any fat.
Frying.
The frying-pans
fitted with wire
linings that lift in
and out of them,
Wire Lining of Frying- which have lately
pan. come much into Modern Sauté Pan.
use in good
kitchens, are so
excellently adapted to save trouble, and so convenient for preparing
delicately light patties, croquettes, rissoles, and other dishes of a
similar nature, that no cook who is expected to serve them in the
best manner should be without one. They should all be arranged
upon this wire lining, and plunged together into the boiling fat; and
well drained on it when they are lifted out.
 BAKING, OR OVEN COOKERY.

The improved construction of the ovens

connected with all modern cooking stoves,
gives great facility at the present day for
home baking, even in very small
establishments; and without this
convenience it is impossible for justice to be
done to the person who conducts the
cookery; as many and great disadvantages
attend the sending to a public oven; and it is
Nottingham Jar. very discouraging to a servant who has
prepared her dishes with nicety and skill, to
have them injured by the negligence of
other persons. One of the best modes of cooking with which we are
acquainted is by means of a jar, resembling in form that shown
above, well pasted down, and covered with a fold of thick paper, and
then placed in a gentle oven. Rice is most excellent when thus
slowly baked with a certain proportion of liquid, either by itself, or
mingled with meat, fish, or fruit; but we must reserve for another
volume particulars of this little system of slow oven-cookery, in which
for some years past we have had numberless experiments made
with almost uniform success: it is especially suited to invalids, from
preserving the entire amount of nourishment contained in the articles
of food dressed by it; and it is to their use that we hope to
appropriate it.
73. We have scarcely done justice in the former editions
of this work to these very useful little ovens, which
we have found, after long trial, better adapted to
some purposes than brick or iron ones, because
preparations which require it, (those of Indian corn,
for example) can be heated in them more gradually;
and when once the management of them is
understood, they will answer admirably for delicate
sweet puddings, and for cakes, with the advantage
American Oven.[73] of requiring but a very moderate fire.
 The oven may be used with advantage for many purposes for
which it is not commonly put into requisition. Calves’ feet, covered
with a proper proportion of water, may be reduced to a strong jelly if
left in it for some hours; the half-head, boned and rolled, will be
found excellent eating, if laid, with the bones, into a deep pan and
baked quite tender in sufficient broth or water, to keep it covered in
every part until done; good soup also may be made in the same way,
the usual ingredients being at once added to the meat, with the
exception of the vegetables, which will not become tender if put into
cold liquid, and should therefore be thrown in after it begins to
simmer. Baking is likewise one of the best modes of dressing various
kinds of fish: pike and red mullet amongst others. Salmon cut into
thick slices, freed from the skin, well seasoned with spice, mixed with
salt (and with minced herbs, at pleasure), then arranged evenly in a
dish, and covered thickly with crumbs of bread, moistened with
clarified butter, as directed in Chapter II., for baked soles, and placed
in the oven for about half an hour, will be found very rich and highly
flavoured. Part of the middle of the salmon left entire, well cleaned,
and thoroughly dried, then seasoned, and securely wrapped in two
or three folds of thickly buttered paper, will also prove excellent
eating, if gently baked. (This may likewise be roasted in a Dutch
oven, either folded in the paper, or left without it, and basted with
butter.) Hams, when freshly cured, and not over salted, if neatly
trimmed, and covered with a coarse paste, are both more juicy, and
of finer flavour baked than boiled. Savoury or pickled beef too, put
into a deep pan with a little gravy, and plenty of butter or chopped
suet on the top, to prevent the outside from becoming dry; then
covered with paste, or with several folds of thick paper, and set into a
moderate oven for four or five hours, or even longer, if it be of large
weight, is an excellent dish. A goose, a leg of pork, and a sucking
pig, if properly attended to while in the oven, are said to be nearly, or
quite as good as if roasted; but baking is both an unpalatable and an
unprofitable mode of cooking joints of meat in general, though its
great convenience to many persons who have but few other facilities
for obtaining the luxury of a hot dinner renders it a very common
one. It is usual to raise meat from the dish in which it is sent to the
oven by placing it, properly skewered, on a stand, so as to allow
potatoes or a batter pudding to be baked under it. A few button
onions, freed from the outer skin, or three or four large ones, cut in
halves, are sometimes put beneath a shoulder of mutton. Two
sheets of paper spread separately with a thick layer of butter,
clarified marrow, or any other fat, and fastened securely over the
outside of a joint, will prevent its being too much dried by the fierce
heat of the oven. A few spoonsful of water or gravy should be poured
into the dish with potatoes, and a little salt sprinkled over them. A
celebrated French cook recommends braising in the oven; that is to
say, after the meat has been arranged in the usual manner, and just
brought to boil over the fire, that the braising pan, closely stopped,
should be put into a moderate oven, for the same length of time as
would be required to stew the meat perfectly tender.
 BRAISING.

Braising is but a more expensive mode of

stewing meat. The following French recipe
will explain the process. We would observe,
however, that the layers of beef or veal, in
which the joint to be braised is imbedded,
can afterwards be converted into excellent
soup, gravy, or glaze; and that there need,
English Braising-pan. in consequence, be no waste nor any
unreasonable degree of expense attending
it; but it is a troublesome process, and quite
as good a result may be obtained by simmering the meat in very
strong gravy. Should the flavour of the bacon be considered an
advantage, slices of it can be laid over the article braised, and
secured to it with a fillet of tape.
“To braise the inside (or small fillet, as it is called in France) of a
sirloin of beef: Raise the fillet clean from the joint; and with a sharp
knife strip off all the skin, leaving the surface of the meat as smooth
as possible; have ready some strips of unsmoked bacon, half as
thick as your little finger, roll them in a mixture of thyme finely
minced, spices in powder, and a little pepper and salt. Lard the fillet
quite through with these, and tie it round with tape in any shape you
choose. Line the bottom of a stewpan (or braising-pan) with slices of
bacon; next put in a layer of beef or veal, four onions, two bay-
leaves, two carrots, and a bunch of sweet herbs, and place the fillet
on them. Cover it with slices of bacon, put some trimmings of meat
all round it, and pour on to it half a pint of good beef broth or gravy.
Let it stew as gently as possible for two hours and a half; take it up,
and keep it very hot; strain, and reduce the gravy by quick boiling
until it is thick enough to glaze with; brush the meat over with it; put
the rest in the dish with the fillet, after the tape has been removed
from it, and send it directly to table.”
Equal parts of Madeira and gravy are sometimes used to moisten
the meat.
 No attempt should be made to braise a joint in any vessel that is
not very nearly of its own size.
74. The line which passes round this stewpan just
above the handle, is a mistake of the designer,
and conveys an erroneous idea of the form of
the cover, and it ought to have been omitted.

A round of buttered paper is generally put

over the more delicate kinds of braised Copper Stewpan.[74]
meat, to prevent their being browned by the
fire, which in France is sometimes put
round the lid of the braising-pan, in a groove made on purpose to
contain it. The embers of a wood fire mixed with the hot ashes, are
best adapted to sustain the regular but gentle degree of heat
required for this mode of cooking.
Braising pans are of various forms. They are often shaped like a
ham-kettle, and sometimes like the design at the commencement of
this section; but a stewpan of modern form, or any other vessel
which will admit of embers being placed upon the lid, will answer for
the purpose as well. Common cooks sometimes stew meat in a
mixture of butter and water, and call it braising.
 LARDING.

Larding Pins.

Cut into slices, of the same length and thickness, some bacon of
the finest quality; trim away the outsides, place the slices evenly
upon each other, and with a sharp knife divide them obliquely into
small strips of equal size. For pheasants, partridges, hares, fowls,
and fricandeaux, the bacon should be about the eighth of an inch
square, and two inches in length; but for meat which is to be larded
quite through, instead of on the outside merely, the bits of bacon
(properly called lardoons) must be at least the third of an inch
square.
In general, the breasts only of birds are larded, the backs and
thighs of hares, and the whole of the upper surface of a fricandeau:
these should be thickly covered with small lardoons, placed at
regular intervals, and in lines which intersect each other, so as to
form rather minute diamonds.
The following directions for larding a pheasant will serve equally
for poultry, or for other kinds of game:—
Secure one end of the bacon in a slight larding-needle, and on the
point of this take up sufficient of the flesh of the bird to hold the
lardoon firmly; draw the needle through it, and part of the bacon, of
which the two ends should be left of equal length. Proceed thus, until
the breast of the pheasant is entirely garnished with lardoons, when
it ought to resemble in appearance a cake thickly stuck with strips of
almonds.
 The larger strips of bacon, after being rolled in a high seasoning of
minced herbs and spices, are used to lard the inside of meat, and
they should be proportioned to its thickness, as they must be passed
quite through it. For example: a four-inch slice from a rump of beef
will require lardoons of very nearly that length, which must be drawn
through with a large larding-pin, and left in it, with the ends just out of
sight on either side.
In France, truffles, anchovies, slices of tongue, and of fat, all
trimmed into proper shape, are occasionally used for larding. The
bacon employed there for the purpose is cured without any saltpetre
(as this would redden the white meats), and it is never smoked: the
receipt for it will be found in Chapter XIII.
A turkey is sometimes larded with alternate lardoons of fat bacon
and of bullock’s tongue, which has been pickled but not dried: we
apprehend that the lean of a half-boiled ham, of good colour, would
answer the purpose quite as well, or better.
Larding the surface of meat, poultry, or game, gives it a good
appearance, but it is a more positive improvement to meat of a dry
nature to interlard the inside with large lardoons of well-seasoned,
delicate, striped English bacon.
 BONING.

Very minute directions being given in other parts of our volume for
this, we confine ourselves here to the following rules:—in
disengaging the flesh from it, work the knife always close to the
bone, and take every care not to pierce the outer skin.
 TO BLANCH MEAT OR VEGETABLES.

This is merely to throw either into a pan of boiling water for a few
minutes, which gives firmness to the first, and is necessary for some
modes of preparing vegetables.
The breast only of a bird is sometimes held in the water while it
boils, to render it firm for larding. To preserve the whiteness of meat,
and the bright green of vegetables, they are lifted from the water
after they have boiled a few minutes, and are thrown immediately
into spring water, and left till cold.
5 to 10 minutes.
 GLAZING

This process we have explained at the

article Glaze, Chapter IV. The surface of the
meat should be covered evenly, with two or
three separate layers of the glaze, which, if
properly made, soon becomes firm. A ham
should be well dried in the oven before it is
laid on. Cutlets of all kinds may be glazed
before they are sent to table, with very good
effect. The figure above represents a glaze-
pot and brush, used for heating and
applying the preparation: a jar placed in a pan of boiling water may
be substituted for the first, when it is not at hand.
 TOASTING.

A very cheap apparatus, by which chops can be dressed before a

clear fire, is shown by the first of these figures; and the second is
peculiarly convenient when bread or muffins are required to be
toasted expeditiously and in large quantities, without much time and
attention being bestowed upon them.
 TO BROWN THE SURFACE OF A DISH WITHOUT BAKING OR
PLACING IT AT THE FIRE.

This is done with a salamander, as it is called, formed like the

engraving below; it is heated in the fire, and held over the dish
sufficiently near to give it colour. It is very much used in a superior
order of cookery. A kitchen shovel is sometimes substituted for it on
an emergency.
CHAPTER X.

Beef.
No.
1. Sirloin.
2. Rump.
3. Edge-bone.
4. Buttock, or Round.
5. Mouse Buttock.
6. Veiny Piece.
7. Thick Flank.
8. Thin Flank.
9. Leg.
10. Fore Rib. (Five Ribs.)
11. Middle Rib. (Four Ribs.)
12. Chuck Rib. (Three Ribs.)
13. Shoulder, or Leg of Mutton Piece.
14. Brisket.
15. Clod.
16. Neck.
17. Shin.
18. Cheek.
 TO CHOOSE BEEF.

Beef is in reality in season through the entire year, but it is best during the
winter months, when it will hang a sufficient time to become tender before it is
dressed. Meat of a more delicate nature is better adapted for the table in summer.
The Christmas beef of England is too much celebrated to require any mention
here.
If young and freshly killed, the lean of ox-beef will be smoothly
grained, and of a fine, healthy, carnation-red, the fat rather white
than yellow, and the suet white and firm. Heifer-beef is more closely
grained, and rather less bright of colour, the bones are considerably
smaller, and the fat of a purer white.
Of bull-beef we only speak to warn our readers that it is of all meat
the coarsest and the most rank in flavour. It may be known by its
dark hue, its close tough fibre, and the scanty proportion, bad
appearance, and strong odour of its fat.
In choice and well-fed beef, the lean will be found intergrained with
fat: very lean meat is generally of an inferior quality.
The ribs, the sirloin, and the rump, are the proper joints for
roasting. The round, or buttock, the edgebone, the second round, or
mouse-buttock, the shin, the brisket, the shoulder or leg of mutton
piece, and the clod, may be boiled or stewed. The neck is generally
used for soup or gravy; and the thin flank for collaring. The best
steaks are cut from the middle of the rump; the next best from the
veiny piece, or from the chuck-rib. The inside of the sirloin,
commonly used for the purpose in France, makes by far the most
delicate steaks; but though exceedingly tender, they are considered
by some English epicures to be wanting in flavour.
The finest part of the sirloin is the chump-end, which contains the
larger portion of the fillet; of the ribs, the middle ones are those
generally preferred by experienced housekeepers.