Single molecule tools have begun to revolutionize the molecular sciences, from biophysics to chemistry to cell biology. They hold the promise to be able to directly observe previously unseen molecular heterogeneities, quantitatively dissect complex reaction kinetics, ultimately miniaturize enzyme assays, image components of spatially distributed samples, probe the mechanical properties of single molecules in their native environment, and "just look at the thing" as anticipated by the visionary Richard Feynman already half a century ago. Single Molecule Tools, Part A: Fluorescence Based Approaches captures a snapshot of this vibrant, rapidly expanding field, presenting articles from pioneers in the field intended to guide both the newcomer and the expert through the intricacies of getting single molecule tools.
- Includes time-tested core methods and new innovations applicable to any researcher employing single molecule tools
- Methods included are useful to both established researchers and newcomers to the field
- Relevant background and reference information given for procedures can be used as a guide to developing protocols in a number of disciplines
1. Watching single DNA replication loops under flow extension 2. Star polymer surface passivation for single molecule detection 3. Ultrahigh resolution detection of single active motor proteins in live cells 4. Molecules and Methods for Superresolution Imaging 5. Aqueous nanodroplets for studying single molecules 6. High-speed atomic force microscopy techniques for visualizing dynamic behavior of biological macromolecules 7. Single-Biomolecule Spectroscopy Using Microfluidic Platforms 8. DNA Looping Kinetics Analyzed by Tethered Particle Microscopy 9. Single molecule observation of proteins in vivo 10. DNA curtains as a high-throughput approach to single molecule imaging 11. Single-molecule enzymology of protein synthesis 12. Single molecule fluorescence studies of intrinsically disordered proteins 13. Nanovesicle trapping for studying transient protein-protein interactions by single molecule FRET 13. Tracking single motor proteins in the cytoplasm of mammalian cells 14. Conformational States of F1-ATPase by Single-Molecule Rotation 15. Single Molecule Sequencing by Fluorescence Imaging 16. Real-Time DNA Sequencing from Single Polymerase Molecules 17. Micropatterning and single molecule imaging for quantitative analysis of protein-protein interactions in living cells 18. Probing virus-receptor interactions by atomic force spectroscopy 19. Single-Molecule Fluorescence Spectroscopy of Cytochrome P450 in Nanodiscs 20. Analysis of complex single molecule FRET time traces 21. Application of super-resolution imaging to single particle tracking in nanotechnology 22. Scanning FCS for the characterization of protein dynamics in live cells 23. Single mRNA molecule tracking in live cells 24. Single-molecule high-resolution colocalization (SHREC) or Single-molecule optical-trap analyses of protein structure 25. Nanopore force Spectroscopy tools for analyzing single bio-complexes 26. Use of plasmon coupling to reveal DNA dynamics at the single molecule level 27. Fluorescence-force spectroscopy 28. Multiplexed single mRNA imaging in fixed cells 29. Size-Minimized Quantum Dots for Single-Molecule and Intracellular Imaging 30. The ABEL trap 31. An optical torque wrench 32. Determining the Stoichiometry of Protein Hetero-complexes in Living Cells with Fluorescence Fluctuation Spectroscopy 33. Fluorescent Visualization of Single Protein-DNA Complexes 34. Direct Measurement of Tertiary Contact Cooperativity in RNA Folding by single molecule FRET 35. Nanometer-localized multiple single-molecule (NALMS) 36. Multiparameter single molecule fluorescence detection with applications to FRET 37. Single-particle tracking-photoactivated localization microscopy (sptPALM) within live cells 38. Site-specific incoporation of fluorescent probes into RNA polymerase 39.Quantitative single-molecule imaging by confocal laser scanning microscopy 40. Studies of DNA-replication at the single molecule level using magnetic tweezers 41. RNA labeled for single molecule FRET analysis from ligation with T4 RNA ligases 42. Combining optical tweezers, single-molecule fluorescence microscopy and microfluidics for studying reversible protein-DNA interactions 43. How dwell time distributions and other such observables in single molecule analysis can be used to extract information from molecular systems