Introduction to experimental biophysics, second edition biological methods for physical scientists

Introduction to Experimental Biophysics
Biological Methods for Physical Scientists, Second Edition


FOUNDATIONS OF BIOCHEMISTRY AND BIOPHYSICS SERIES
Introduction to Experimental Biophysics:
Biological Methods for Physical Scientists,
Second Edition
Jay L. Nadeau
Introduction to Single Molecule Biophysics
Yuri L. Lyubchenko
Biomolecular Thermodynamics: From Theory to Application
Douglas Barrick
Biomolecular Kinetics: A Step-by-Step Guide
Clive R. Bagshaw
An Introduction to Biophysics:
Quantitative Understanding of Biosystems,
Second Edition
Thomas M. Nordlund and Peter M. Hoffmann


Introduction to Experimental Biophysics
Biological Methods for Physical Scientists, Second Edition

Jay L. Nadeau


CRC Press
Taylor & Francis Group
6000 Broken Sound Parkway NW, Suite 300

Boca Raton, FL 33487-2742
© 2018 by Taylor & Francis Group, LLC
CRC Press is an imprint of Taylor & Francis Group, an Informa business
No claim to original U.S. Government works
Printed on acid-free paper
International Standard Book Number-13: 978-1-4987-9959-1 (Paperback); 978-1-138-08815-3 (Hardback)
This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish reliable
data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of
their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged
please write and let us know so we may rectify in any future reprint.
Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by
any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in
any information storage or retrieval system, without written permission from the publishers.
For permission to photocopy or use material electronically from this work, please access www.copyright.com ( />or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit
organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by
the CCC, a separate system of payment has been arranged.
Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and
explanation without intent to infringe.
Library of Congress Cataloging‑in‑Publication Data
Names: Nadeau, Jay L., author.
Title: Introduction to experimental biophysics : biological methods for physical scientists / Jay L. Nadeau.
Other titles: Experimental biophysics | Foundations of biochemistry and biophysics.
Description: Second edition. | Boca Raton, FL : CRC Press, Taylor & Francis Group, [2017] |
Series: Foundations of biochemistry and biophysics
Identifiers: LCCN 2017010261| ISBN 9781138088153 (hardback) | ISBN 1138088153 (hardback) |
ISBN 9781498799591 (pbk. ; alk. paper) | ISBN 1498799590 (pbk. ; alk. paper)
Subjects: LCSH: Biophysics--Experiments--Technique.
Classification: LCC QH505 .N247 2017 | DDC 572--dc23
LC record available at />Visit the Taylor & Francis Web site at

and the CRC Press Web site at



v

Contents

Series Preface

xvii

Prefacexix
Acknowledgmentsxxi
Authorxxiii
Contributorsxxv
Chapter 1

Introduction and Background

1

Chapter 2

Basic Molecular Cloning of DNA and RNA

43

Chapter 3

Expression of Genes in Bacteria, Yeast,
and Cultured Mammalian Cells

75

Chapter 4

Advanced Topics in Molecular Biology

129

Chapter 5

Protein Expression Methods

157

Joshua A. Maurer
Chapter 6

Protein Crystallization
Oliver M. Baettig and Albert M. Berghuis

187


vi

Contents

Chapter 7

Introduction to Biological Light Microscopy

225

Coauthored with Michael W. Davidson
Chapter 8

Advanced Light Microscopy Techniques

279

Coauthored with Lina Carlini
Chapter 9

Advanced Topics in Microscopy II:
Holographic Microscopy

305

Coauthored with Manuel Bedrossian
Chapter 10 Quantitative Cell Culture Techniques

325

Chapter 11 Semiconductor Nanoparticles (Quantum Dots)

361

Chapter 12 Gold Nanoparticles

395

Edward S. Allgeyer, Gary Craig,
Sanjeev Kumar Kandpal, Jeremy Grant,
and Michael D. Mason
Chapter 13 Advanced Topics in Gold Nanoparticles:
Biomedical Applications

429

Chapter 14 Surface Functionalization Techniques

453

Chapter 15 Electrophysiology

497

Coauthored with Christian A. Lindensmith
and Thomas Knöpfel
Chapter 16 Spectroscopy Tools and Techniques

553

Chapter 17 Introduction to Nanofabrication

623

Orad Reshef


Contents

Glossary643
Appendix A: Common Solutions

683

Appendix B: Common Media

689

Appendix C: Restriction Endonucleases

693

Appendix D: Common Enzymes

721

Appendix E: Fluorescent Dyes and Quenchers

723

Appendix F: Fluorescent Proteins

729

Index731

vii





ix

Detailed Contents

Series Preface
xvii
Prefacexix
Acknowledgmentsxxi
Authorxxiii
Contributorsxxv
Chapter 1
Introduction and Background1
1.1 BASIC BIOCHEMISTRY1
Molecules important to molecular biophysics
1
Making use of functional groups
7
1.2 ENERGIES AND POTENTIALS8
Biologically relevant energy scales
8
Ionic bonds
8

Ion–dipole interactions
10
Dipole–dipole interactions
11
Hydrogen bonds
12
The (strept)avidin/biotin interaction
14

1.6 TRANSLATION AND THE GENETIC CODE26
1.7 PROTEIN FOLDING AND TRAFFICKING28
1.8 ALTERNATIVE GENETICS33
1.9 WHAT IS CLONING?34
1.10 DESIGN OF A MOLECULAR BIOLOGY
EXPERIMENT AND HOW TO USE THIS BOOK35
BACKGROUND READING

40

Chapter 2
Basic Molecular Cloning of DNA and RNA43
2.1INTRODUCTION43
2.2 OBTAINING AND STORING PLASMIDS45

1.4CELLS19

2.3 SELECTION OF AN APPROPRIATE E. COLI
AMPLIFICATION STRAIN; TRANSFORMATION
OF E. COLI WITH PLASMID47
Transformation47

Selection48
Transformation efficiency
49

1.5 DNA, RNA, REPLICATION, AND TRANSCRIPTION21
The structure and function of DNA and RNA
21
Replication23
Transcription25

2.4 PLASMID AMPLIFICATION AND PURIFICATION49
Amplification49
Purification49
Measuring concentration and purity of extracted DNA 51

1.3 PRINCIPLES OF SPECTROSCOPY17
What can be measured
17
How transitions are measured
18


x

Detailed Contents

2.5 PLASMID RESTRICTION MAPPING
AND AGAROSE GEL ELECTROPHORESIS52
Restriction enzymes
52

Screening purified DNA
53
Separation of restriction fragments for ligation
54
2.6 AN EXAMPLE CLONING EXPERIMENT56
Determining a cloning strategy
56
Digestion and purification of fragments
57
Determination of parameters for optimal ligation
57
2.7 CLONING BY THE POLYMERASE CHAIN
REACTION60

Electroporation of cell cultures
105
Microinjection of DNA and RNA: For a few select cells
or constructs that are difficult to transfect
105
3.5 GENE DELIVERY USING VIRUSES108
Lentivirus114
Some other types of viruses used as vectors
118
3.6SUMMARY121
BACKGROUND READING

123

2.8SEQUENCING62

Chapter 4
Advanced Topics in Molecular Biology129

2.9 RNA METHODS63

4.1INTRODUCTION129

2.10 SOUTHERN AND NORTHERN BLOTS65

4.2 CLONING TECHNIQUES FOR LARGE
CLONING PROBLEMS AND MULTIPLE
INSERTS129
Phage vectors
129
Cosmids132
Bacterial artificial chromosomes
and yeast artificial chromosomes
132

2.11 SITE-DIRECTED MUTAGENESIS66
2.12SUMMARY68
BACKGROUND READING

71

Chapter 3
Expression of Genes in Bacteria, Yeast,
and Cultured Mammalian Cells75
3.1INTRODUCTION75
3.2 EXPRESSING GENES IN MICROORGANISMS76

E. coli76
Other bacterial strains
76
Yeast cells
79
3.3 MAMMALIAN CELL CULTURE84
Introduction to immortalized cell lines
84
Primary cultures
90
3.4 TRANSFECTION OF MAMMALIAN CELLS I:
STANDARD TECHNIQUES97
Introduction97
Cationic liposomes: Easy, transient expression
in 40–90% of dividing cells
98
Stable transfection: For long-term and/or inducible
expression of entire cultures of dividing cells
100
Example experiment: Transfecting CHO cells with LacZ
and GFP
103

4.3 MULTIPLE MUTAGENESIS: WHEN POINT
MUTATIONS ARE NOT ENOUGH133
4.4 REVERSE TRANSCRIPTASE PCR
AND QUANTITATIVE REAL-TIME PCR134
Reverse transcriptase PCR
134
Quantitative real-time PCR

135
4.5MICROARRAYS136
4.6 SMALL INTERFERING RNA138
General principles
138
Example experiment: Mechanisms of drug
resistance139
Data analysis
141
Secondary screening
146
4.7CRISPR146
General principles
146
Practical considerations
148
Caveats150
Validating CRISPR
150
BACKGROUND READING

151


Detailed Contents

xi

Chapter 5
Protein Expression Methods157

Joshua A. Maurer

Chapter 6
Protein Crystallization187
Oliver M. Baettig and Albert M. Berghuis

5.1INTRODUCTION157

6.1INTRODUCTION187

5.2 EXPRESSION SYSTEMS157

6.2 CRYSTALLIZATION OF MACROMOLECULES188
General concerns and motivations
188
Vapor diffusion
190
Interface diffusion
193
Microbatch193
Dialysis193

5.3 IDENTIFICATION OF A DNA SOURCE158
5.4 SELECTING AN EXPRESSION VECTOR159
Promoters159
Protein tags
161
Vector selection
162
5.5 SUBCLONING INTO AN EXPRESSION VECTOR163

5.6 SELECTION OF AN EXPRESSION STRAIN
OR CELL LINE163
Bacterial strains
163
Yeast164
Insect cells
164
Mammalian cells
164
5.7 PROTEIN EXPRESSION164
5.8 CHECKING PROTEIN EXPRESSION
(AND PURITY) USING SDS-PAGE166
Protein separation
167
Protein visualization
168
5.9 PROTEIN ISOLATION AND PURIFICATION170
Native versus nonnative purification
170
Preparation of protein lysate
171
5.10CHROMATOGRAPHY172
Chromatography systems
172
Affinity chromatography
173
Size exclusion chromatography
174
5.11 BUFFER EXCHANGE AND CONCENTRATION175
Buffer exchange

175
Protein concentration
176
5.12 EXAMPLE EXPERIMENT: EXPRESSION AND
PURIFICATION OF FLUORESCENT PROTEIN
DRONPA177
5.13 CONCLUSIONS AND FINAL REMARKS180
BACKGROUND READING

183

6.3 PREPARATION OF PROTEINS
FOR CRYSTALLIZATION194
Protein purity
194
Monodispersity195
Protein quantity
198
Protein variability
198
6.4 COMPONENTS OF CRYSTALLIZATION
SOLUTIONS199
Precipitant199
Buffer200
Salt200
6.5 OTHER FACTORS AFFECTING
CRYSTALLIZATION200
Protein concentration
202
Diffusion rate

202
Temperature203
Vibrations203
Mechanical contaminants
203
Solution quality
203
6.6 CRYSTALLIZATION STRATEGIES204
Initial screening
204
Pitfalls208
Fine-screening208
Additive screens
209
Seeding211
Improving the protein
211
Obtaining different crystal forms of the same protein 212
6.7 EXAMPLE EXPERIMENT: LYSOZYME212
6.8 DATA COLLECTION AND STRUCTURE
DETERMINATION USING X-RAY
CRYSTALLOGRAPHY215
Where to do x-ray crystallography
215
Protecting crystals from radiation damage
216


xii

Detailed Contents

6.9 TROUBLESHOOTING Q AND A217
6.10 CONCLUSIONS AND FINAL REMARKS221
BACKGROUND READING

222

Chapter 7
Introduction to Biological Light
Microscopy225
Coauthored with Michael W. Davidson
7.1INTRODUCTION225
7.2 PHYSICS OF MICROSCOPY: MAGNIFICATION
AND RESOLUTION225
7.3 ANATOMY OF A BIOLOGICAL MICROSCOPE229
Hardware229
Imaging cells on an inverted microscope
231
7.4 BRIGHTFIELD IMAGING TECHNIQUES232
Köhler illumination
232
Brightfield and darkfield
233
Phase contrast
238
Polarization and DIC
240
7.5 BASIC FLUORESCENCE MICROSCOPY243
Physics of fluorescent molecules

243
Epifluorescence microscopy
247
Confocal laser scanning microscopy
252
7.6 FLUOROPHORES FOR CELL LABELING257
Autofluorescence257
Traditional organic dyes
258
New-generation fluorescent dyes
261
Attaching dyes to cell-targeting molecules
262
Organelle probes
266
Environmental probes
267
7.7 FLUORESCENT PROTEINS269
7.8 SUMMARY AND REMARKS273
BACKGROUND READING

275

Chapter 8
Advanced Light Microscopy Techniques279
Coauthored with Lina Carlini

8.3 FLUORESCENCE RESONANCE ENERGY
TRANSFER MICROSCOPY284
8.4 TWO-PHOTON MICROSCOPY284

8.5 TOTAL INTERNAL REFLECTANCE
MICROSCOPY286
8.6 FLUORESCENCE LIFETIME IMAGING (FLIM)287
General principles and use
287
Example experiment: Measuring lifetimes
of QDs inside cells
289
8.7 FOUR PI MICROSCOPY293
8.8 PHOTOACTIVATED LOCALIZATION
MICROSCOPY (PALM) AND STOCHASTIC
OPTICAL RECONSTRUCTION MICROSCOPY
(STORM)294
Principles of photoactivated localization microscopy/
stochastic optical reconstruction microscopy
294
Probe requirements
295
8.9 SUMMARY AND CONCLUSION296
BACKGROUND READING

298

Chapter 9
Advanced Topics in Microscopy II:
Holographic Microscopy305
Coauthored with Manuel Bedrossian
9.1INTRODUCTION305
9.2 PHYSICS OF HOLOGRAPHY306
9.3 RECONSTRUCTING HOLOGRAMS306

9.4 SOURCES OF NOISE309
9.5 INSTRUMENT DESIGNS311
Mach–Zehnder311
Common path
312
In-line314
Incoherent DHM
315

8.1INTRODUCTION279

9.6 BUILDING A LOW-COST DHM317
Hardware317
Gradient index lens common mode
317
Reconstruction and analysis software
320

8.2 MULTISPECTRAL TECHNIQUES279

BACKGROUND READING

321


Detailed Contents

Chapter 10
Quantitative Cell Culture Techniques325
10.1INTRODUCTION325

10.2 QUANTIFYING BACTERIAL GROWTH
AND DEATH325
Quantifying bacterial concentrations
325
Bacterial growth curves
326
Bacterial inhibition curves and modeling
328
IC50 and minimum inhibitory concentration
329
10.3 QUANTIFYING MAMMALIAN CELLS331
Counting mammalian cells
331
End-point methods for mammalian cells: The
sulforhodamine B assay and other colorimetric
methods333
10.4 FLOW CYTOMETRY341
10.5 EXAMPLE EXPERIMENT: DETERMINING
LEUKEMIC B CELLS AND T CELLS BY FLOW
CYTOMETRY345
10.6 QUANTIFYING VIRUSES349
Titering viral vectors
349
Titering phage by plaque assay
350
Titering adenovirus by plaque assay
350
Titering adenovirus by optical density
353
Titering lentiviral vectors by flow cytometry

353
Titering retroviruses expressing a selectable
marker354
Titering lentivirus using p24
355
10.7 SUMMARY AND FINAL REMARKS355
BACKGROUND READING

358

Chapter 11
Semiconductor Nanoparticles
(Quantum Dots)361
11.1INTRODUCTION361
11.2 QUANTUM DOT PROPERTIES
AND SYNTHESIS361
Physics of quantum dots
361
Synthesis of QDs
365
Determination of QD size and concentration
367
Solubilization and biofunctionalization of QDs
370
Commercial QDs
373

xiii

11.3 QD APPLICATIONS375

Single-particle tracking
375
QD delivery to living cells
376
Multicolor labeling and avoidance
of autofluorescence
380
Correlated fluorescence and electron microscopy 381
QDs as biosensors
383
11.4 EXAMPLE EXPERIMENT: CONJUGATION
OF QDs TO DOPAMINE AND QUANTIFYING
THE EFFECTS ON FLUORESCENCE PER
MOLECULE BOUND387
11.5 SUMMARY AND REMARKS390
BACKGROUND READING

391

Chapter 12
Gold Nanoparticles395
Edward S. Allgeyer, Gary Craig,
Sanjeev Kumar Kandpal, Jeremy Grant,
and Michael D. Mason
12.1INTRODUCTION395
12.2 THE PHYSICS OF SCATTERING AND
SPHERICAL METAL NANOPARTICLES396
General theory for all particles
396
Simplifications for nanosized particles

398
12.3 SYNTHESIS OF GOLD NANOPARTICLES403
12.4 CHARACTERIZATION AND SURFACE
MODIFICATION OF GOLD NANOPARTICLES408
Recommended characterization techniques
408
Surface stabilization and biocompatibility
409
Targeting schemes
411
12.5 APPLICATIONS FOR COLORIMETRIC
DETECTION AND MICROSCOPY411
Metal nanoparticles as local sensors
411
Darkfield microscopy
412
Prospects for high-speed imaging
413
Confocal microscopy
414
12.6 SAMPLE EXPERIMENT: LABELING
CELLS WITH LECTIN-TAGGED GOLD
NANOPARTICLES415


xiv

Detailed Contents

12.7 APPLICATIONS IN SURFACE-ENHANCED

RAMAN SCATTERING416
Introduction to Raman scattering
416
Protected Raman-active nanospheres
419
SERS nanoparticles: Beyond spheres
420
12.8 GOLD NANOPARTICLES AS
PHOTOTHERMAL TRANSDUCERS422
12.9CONCLUSION423
BACKGROUND READING

424

Chapter 13
Advanced Topics in Gold Nanoparticles:
Biomedical Applications429
13.1INTRODUCTION429
13.2 THE USE OF GOLD IN MEDICINE429
13.3 ACTIVE AND PASSIVE TARGETING OF AU
NANOPARTICLES430
13.4 THE USE OF GOLD IN PHOTOTHERMAL
THERAPY432
13.5 THE USE OF GOLD IN RADIATION THERAPY432
Principles of radiation therapy
432
Gold nanoparticle–assisted radiation therapy
435
Improving GNRT by targeting
435

Improving GNRT by addition of photothermal
therapy439
13.6 EXAMPLE: HOW TO MAKE A
NANOMEDICINE—THE CASE OF AU–DOX439
Why Au–Dox?
440
Physical characterization
440
Efficacy against cultured cancer cells
442
In vivo studies
446
The nanotechnology characterization laboratory
assay cascade
449
Good laboratory practice, good manufacturing
practice, and scale-up
449
Steps toward approval: The investigational new drug 450
BACKGROUND READING

450

Chapter 14
Surface Functionalization Techniques453
14.1INTRODUCTION453
14.2 PREPARING MONOLAYERS USING
FUNCTIONAL SILANES OR THIOLS454
Silanes454
Alkanethiol self-assembled monolayers

457
Some special considerations
461
14.3 TECHNIQUES FOR CHARACTERIZING
SURFACE MONOLAYERS462
Interaction with reactive dyes
463
Ellipsometry463
Contact angle
464
X-ray photoelectron spectroscopy
465
Scanning probe microscopy
466
Other methods
470
14.4 FUNCTIONALIZATION OF MODIFIED
SURFACES USING CROSS-LINKERS470
Types of cross-linkers
470
Controlling protein orientation
473
14.5 EXAMPLE EXPERIMENT: PREPARING A
SILANE–BIOTIN–STREPTAVIDIN SANDWICH
ON SIO2 FEATURES ON A SI CHIP477
Observing and cleaning the substrate
477
Silanization479
Biotinylation and blocking
479

Assembling streptavidin, final characterization,
and using the sensor
480
Variations on a theme
481
Micropatterning482
14.6 PREVENTING NONSPECIFIC BINDING
OF BIOMOLECULES483
14.7 TESTING THE FUNCTION OF IMMOBILIZED
PROTEINS484
Specific binding: Quantity and kinetics
484
Enzymatic function
485
Electrochemistry485
Ion channel function
485


Detailed Contents

14.8 CONCLUSION AND FINAL REMARKS491
BACKGROUND READING

492

Chapter 15
Electrophysiology497
Coauthored with Christian A. Lindensmith
and Thomas Knöpfel

15.1INTRODUCTION497
15.2 PHYSICAL BASIS AND CIRCUIT MODELS499
Cell circuit models
499
Types of recording: Bilayers, single-channel patches,
whole cell
502
Voltage clamp and current clamp
504
Issues of space clamp
505
15.3 SOLUTIONS AND BLOCKERS506
Internal and external solutions
506
Junction potential
508
Blockers, agonists, antagonists
509
15.4INSTRUMENTATION510
Amplifiers510
Grounding and shielding
514
Micromanipulators515
15.5 LIPID BILAYER SETUP516
General principles and use
516
Making the lipid bilayer
517
Monitoring bilayer formation electrically
520

Adding ion channels
521
15.6 CELL PATCH-CLAMP SETUP: WHAT
IS NEEDED523
15.7 THE ART AND MAGIC OF PIPETTE PULLING527
Pullers and glass
527
Making patch pipettes
528
Sylgard529
Recording artifacts caused by pipette materials
530
15.8 STEP-BY-STEP GUIDE TO PERFORMING
A WHOLE-CELL RECORDING530
15.9 EXAMPLE EXPERIMENT: WHOLE-CELL
RECORDING ON CELLS TRANSFECTED
WITH K+ CHANNELS AND GFP532
15.10 BRIEF INTRODUCTION TO SINGLE-CHANNEL
MODELING AND DATA ANALYSIS535

Why do single-channel measurements?
Analyzing data
Interpreting single-channel data

xv

535
535
539

15.11NETWORKS539
15.12 CONCLUSIONS AND FINAL REMARKS539
BACKGROUND READING

548

Chapter 16
Spectroscopy Tools and Techniques553
16.1INTRODUCTION553
16.2 GUIDING PRINCIPLES553
16.3 UV–VISIBLE ABSORBANCE SPECTROSCOPY554
16.4 FLUORESCENCE SPECTROSCOPY557
Instrumentation557
Caveats and sources of error
560
Applications of fluorescence spectroscopy:
Quenching561
Applications of fluorescence spectroscopy:
Anisotropy562
Applications of fluorescence spectroscopy:
Energy transfer
567
16.5 TIME-RESOLVED EMISSION570
16.6 TIME-RESOLVED ABSORPTION575
16.7 INFRARED SPECTROSCOPY577
16.8 NUCLEAR MAGNETIC RESONANCE582
Introduction582
Example: Examining QD surfaces with liquid-phase
NMR584
Solid-state NMR

586
Pulse techniques and MRI
587
Paramagnetic nanoparticles as MR contrast agents 589
16.9 ELECTRON PARAMAGNETIC RESONANCE
SPECTROSCOPY592
Basic principles
592
Spin probes and spin traps
596
Instrumentation601
16.10 X-RAY SPECTROSCOPY602


xvi

Detailed Contents

16.11 EXAMPLE EXPERIMENT: CHARACTERIZATION
OF CDSE/ZNS NANOPARTICLE
BIOCONJUGATE USING UV–VIS,
FLUORESCENCE EMISSION, TIME-RESOLVED
EMISSION, FTIR, AND EPR SPECTROSCOPY606
UV–Vis and fluorescence emission
606
FTIR607
TCSPC609
EPR610
16.12 FINAL COMMENTS610
BACKGROUND READING AND RESOURCES

612

Chapter 17
Introduction to Nanofabrication623
Orad Reshef
17.1INTRODUCTION623
The planar process
623
17.2PATTERNING624
Resist624
Lithography625
Nanoimprint lithography
626
Focused ion beam
626
17.3 PATTERN TRANSFER626
Wet etching
626
Dry etching
627
Lift-off629
Template stripping
630
17.4 MATERIAL DEPOSITION631
Spin coating
631
Sputter deposition
632
Thermal and electron beam evaporation

633
Chemical vapor deposition
633
Atomic layer deposition
633
17.5METROLOGY634
Scanning electron microscopy
634
Profilometry635
Atomic force microscopy
635

17.6 THIN-FILM CHARACTERIZATION636
Ellipsometry636
X-ray photoelectron spectroscopy
636
Raman spectroscopy
637
X-ray diffraction
637
Four-point probe
637
17.7 KEEPING A SAMPLE CLEAN638
Yield yield yield
638
Minimum feature sizes
638
Mind your tolerances
638
Dedicated labware

638
“Nothing goes in the bottle”
639
AMI wash, RCA clean, piranha etch
639
Descumming639
Take your time
639
Be nice to the cleanroom staff
639
Before experimenting with
a new recipe, recreate something
you KNOW will work
640
Keep your toolbox properly outfitted
640
Spinning resist on small samples
640
Keep your surroundings clean
640
Double check
641
17.8 FINAL COMMENTS641
BACKGROUND READING

641

Glossary643
Appendix A: Common Solutions

683

Appendix B: Common Media

689

Appendix C: Restriction Endonucleases

693

Appendix D: Common Enzymes

721

Appendix E: Fluorescent Dyes
and Quenchers

723

Appendix F: Fluorescent Proteins

729

Index731


xvii

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
molecules. 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
pharmaceutical sciences and medicine.
These two highly interconnected fields are the focus of this book series. It covers
both the use of traditional tools from physical chemistry, such as nuclear magnetic
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, senior publishing
editor at CRC Press, Taylor & Francis Group ().





xix

Preface

T

he second edition has been revised and updated to reflect changes in the
fields between 2010 and 2016, with references, suppliers, and software all
brought up to date. The study questions at the back of each chapter have been
thoroughly revised and expanded, and a solutions manual is available.
The book has also been restructured to make a clear distinction between the basic
techniques and more advanced approaches that are usually not accessible to an
undergraduate laboratory. The book can be used on two levels: as an introductory
course with only the basic techniques covered or as a more advanced course that
requires access to more sophisticated equipment. The advanced material may
also be used for self-study.
The advanced material is included within selected chapters as callouts, as well
as forming the basis of five entirely new chapters: advanced molecular biology
techniques (Chapter 4), advanced light microscopy (Chapter 8), holographic
microscopy (Chapter 9), biomedical applications of gold nanoparticles
(Chapter 13), and microfabrication techniques (Chapter 17).
A large fraction of the basic course material provides the basis for a one-semester
or summer course on introductory molecular biology techniques.
This textbook is bundled with a laboratory companion guide. It is structured
according to the chapters in the book, although it refers to the first edition of this
book. The series of 14 experiments presents a wide variety of techniques that may
be performed during a semester-long, three-credit course or during a 1-month
intensive.





xxi

Acknowledgments

I

thank all of the people who made this book possible. The biggest thanks are
to the chapter authors, who provided years of firsthand experience on how
to do things right (or wrong). Sections of some chapters were also contributed
by colleagues. I am grateful to Chris Ratcliffe of the National Research Council,
who wrote the section on solid-phase nuclear magnetic resonance (NMR) in
Chapter  16, and to Jonathan Saari of McGill, who contributed the section on
time-resolved absorption spectroscopy in Chapter 16.
A special mention also goes to Jenna Blumenthal, who was a senior undergraduate
in physics/physiology when she helped to proofread the first edition and prepare
the first version of the glossary. Ildiko Horvath of McGill drew some of the
illustrations in Chapter 1. Thanks also to my former graduate students Samuel
Clarke, Xuan Zhang, and Daniel Cooper, whose thesis material is incorporated
into several of the chapters.
Another thanks goes to all of the people who provided figures, both published
and unpublished, to help illustrate this work. When approached out of the blue,
they responded with data, micrographs, and other material that allowed the
illustrations to be as beautiful, relevant, and practical as I hoped they might be.
Thanks to those who helped to proofread, and special hugs to Susan Foster, the
world’s best copy editor.
Finally, this book would not have been possible without my editor, Lu Han, who
helped develop the book’s idea, encouraged me throughout its evolution, and
solicited the second edition long before I had started to think about it.

xxiii

Author

Jay L. Nadeau is an associate professor of physics at Portland State University (PSU).
Prior to PSU, she was a research professor in the Graduate Aerospace Laboratories
(GALCIT) at the California Institute of Technology (2015–2017) and an associate
professor of biomedical engineering and physics at McGill University (2004–
2015). Her research interests include nanoparticles, fluorescence imaging, and
development of instrumentation for the detection of life elsewhere in the solar
system.
She has published over 70 papers on topics ranging from theoretical condensed
matter physics to experimental neurobiology to the development of anticancer
drugs and, in the process, has used almost every technique described in this book.
Her work has been featured in New Scientist, Highlights in Chemical Biology, Radio
Canada’s Les Années Lumière, Le Guide des Tendances, and educational displays
in schools and museums. Her research group features chemists, microbiologists,
roboticists, physicists, and physician–scientists, all learning from each other and
hoping to speak each other’s language. A believer in bringing biology to physicists
as well as physics to biologists, she has created two graduate-level courses:
methods in molecular biology for physical scientists and mathematical cellular
physiology. She has also taught pharmacology in the medical school and was
one of the pioneers in the establishment of multiple mini-interviews for medical
school admission.
She retains adjunct positions at McGill and Caltech and has collaborators in
industry and academia in the United States, Europe, Australia, and Japan. She has
given several dozen invited talks at meetings of the American Chemical Society, the
American Geophysical Union, the International Society for Optics and Photonics
(SPIE), the Committee on Space Research, the American Association of Physics
Teachers (AAPT), and many others. Before her time at McGill, she was a member

of the Jet Propulsion Laboratory’s Center for Life Detection, and previous to that,
a Burroughs Wellcome postdoctoral scholar in the laboratory of Henry A. Lester
at Caltech. She earned a PhD in physics at the University of Minnesota in 1996.