As molecular and cellular biologists move toward nano–techniques for performing experiments on single molecules rather than on populations of molecules, a comprehensive manual on how (and why) to carry out such experiments is needed. Single-Molecule Techniques: A Laboratory Manual fills this requirement—it is the first to take researchers who know nothing about single–molecule analyses to the point where they can successfully design and execute appropriate experiments. Geared toward research scientists in structural and molecular biology, biochemistry, and biophysics, the manual will be useful to all who are interested in observing, manipulating, and elucidating the molecular mechanisms and discrete properties of macromolecules. Techniques range from in vivo and in vitro fluorescent–based methods to the use of atomic force microscopy, optical and magnetic tweezers, and nanopores. The book is edited by Paul R. Selvin and Taekjip Ha, two pioneers in the field of experimental biophysics who have made significant contributions to the development and application of single–molecule technologies.
Contents
1. The New Era of Biology In Singulo
T. Ha and P.R. Selvin
IN VITRO FLUORESCENCE
2. Single-Molecule FRET with Total Internal Reflection Microscopy
C. Joo and T. Ha
3. In Vitro and In Vivo FIONA and Other Acronyms for Watching Molecular Motors Walk
P.R. Selvin, T. Lougheed, M.T. Hoffman, H. Park, H. Balci, B.H. Blehm, and E. Toprak
4. Colocalization of Fluorescent Probes: Accurate and Precise Registration with Nanometer Resolution
L.S. Churchman and J.A. Spudich
5. Alternating-Laser Excitation of Single Molecules
A.N. Kapanidis, M. Heilemann, E. Margeat, X. Kong, E. Nir, and S. Weiss
6. Orientation and Rotational Motions of Single Molecules by Polarized Total Internal Reflection Fluorescence Microscopy
J.F. Beausang, Y. Sun, M.E. Quinlan, J.N. Forkey, and Y.E. Goldman
FLUORESCENCE IN LIVING CELLS
7. Imaging Gene Expression in Living Cells at the Single-Molecule Level
J. Xiao, J. Elf, G.-W. Li, J. Yu, and X.S. Xie
8. Single-Virus Tracking in Live Cells
M.J. Rust, M. Lakadamyali, B. Brandenburg, and X. Zhuang
9. Ultrasensitive Imaging in Live Cells Using Fluorescent Quantum Dots
S. Courty and M. Dahan
10. Imaging Real-Time Gene Expression in Living Systems
A.L. Wells, J.S. Condeelis, R.H. Singer, and D. Zenklusen
11. Single-Molecule Imaging of Stochastic Signaling Events in Living Cells
S. Matsuoka, Y. Miyanaga, T. Yanagida, and M. Ueda
12. Fluorescence Correlation Spectroscopy In Vitro and In Vivo
E. Haustein and P. Schwille
OPTICAL TRAPS
13. Optical Traps to Study Properties of Molecular Motors
J.A. Spudich, S.E. Rice, R.S. Rock, T.J. Purcell, and H.M. Warrick
14. High-Resolution Dual-Trap Optical Tweezers with Differential Detection
C. Bustamante, Y.R. Chemla, and J.R. Moffitt
15. Imaging and Nanomanipulation of an Actomyosin Motor
S. Nishikawa, T. Komori, T. Ariga, T. Okada, M. Morimatsu, Y. Ishii, and T. Yanagida
MAGNETIC TRAPS
16. Single-Molecule Studies Using Magnetic Traps
T. Lionnet, J.-F. Allemand, A. Revyakin, T.R. Strick, O.A. Saleh, D. Bensimon, and V. Croquette
FORCE PROBES/ATOMIC FORCE MICROSCOPY
17. Probing Polysaccharide and Protein Mechanics by Atomic Force Microscopy
M. Rabbi and P.E. Marszalek
CHANNELS
18. Single-Molecule Analysis of Nucleic Acids and DNA–Protein Interactions Using Nanopores
M. Wanunu and A. Meller
OTHER TECHNIQUES
19. Single-Molecule Gold-Nanoparticle Tracking with High Temporal and Spatial Resolution and without an Applied Load
A.R. Dunn and J. A. Spudich
20. Advances in Surface-based Assays for Single Molecules
P.M. Fordyce, M.T. Valentine, and S.M. Block
21. Hydrodynamic Flow-stretching Assay for Single-Molecule Studies of Nucleic Acid–Protein Interactions
C.M. Schroeder, P.C. Blainey, S. Kim, and X.S. Xie
APPENDIX: Cautions
INDEX