Spectroscopy and Dynamics of Single Molecules: Methods and Applications

Description

Spectroscopy and Dynamics of Single Molecules: Methods and Applications reviews the most recent developments in spectroscopic methods and applications. Spectroscopic techniques are the chief experimental methods for testing theoretical models and research in this area plays an important role in stimulating new theoretical developments in physical chemistry. This book provides an authoritative insight into the latest advances in the field, highlighting new techniques, current applications, and potential future developments An ideal reference for chemists and physicists alike, Spectroscopy and Dynamics of Single Molecules: Methods and Applications is a useful guide for all those working in the research, design, or application of spectroscopic tools and techniques across a wide range of fields. Cover Spectroscopy and Dynamics of Single Molecules: Methods and Applications Copyright Contributors Section 1 Introduction Historical overview Beyond intensity: Expanding techniques New applications Topics covered in this book References Section 2 1 Photophysics of single-molecule probes Introduction Potential artifacts from surfaces and the use of extrinsic probes Potential artifacts due to the generation of dark states Photophysical processes and the generation of dark states Absorption of light Fluorescence Intersystem crossing and the triplet state Energy transfer Quenching The dynamic behavior of dark states Nanosecond dynamics of the S0/S1 system Dynamics of the S0/S1/T1 system Understanding the various contributions to the measured fluorescence intensities Photophysical considerations in the calculation of accurate FRET efficiencies Photophysical properties of common single-molecule fluorescent probes Rhodamines and carborhodamines Oxazines Carbocyanines Concluding remarks Appendix Acknowledgments References 2 Probing dynamics in single molecules Introduction and overview Single molecule FRET microscopy Fluorescence Förster resonance energy transfer (FRET) Widefield - Total internal reflection fluorescence (WF-TIRF) microscopy Total internal reflection Prism-based TIRF (pTIRF) microscopy Objective-based TIRF (oTIRF) microscopy Alternating laser excitation (ALEX) Camera-based detection Electron-multiplying CCD-based cameras CMOS-based cameras The quantum efficiency of emCCD and sCMOS cameras Surface and sample preparation for smFRET on surface-immobilized molecules Practical considerations regarding smTIRF microscopy Illumination Detection Sample channels for TIRF microscopy Experimental aspects Single molecule TIRF data evaluation Camera mapping Particle localization and pair finding Intensity trace extraction and background subtraction Trace evaluation and categorization FRET efficiency correction and distance calculation Direct excitation correction Spectral crosstalk or leakage correction Detection efficiency and quantum yield correction Summary FRET efficiency correction Frame- and trace-wise FRET histograms Analyzing dynamics using hidden Markov modeling (HMM) Markovian processes, Markovian chains and transition probability matrices Hidden Markov models, emission functions and likelihood The Baum-Welch forward-backward algorithm Viterbi algorithm Information theory and likelihood estimators HMM analysis of smTIRF FRET data Global and trace-wise HMM Transition density plots Dwell-time histograms and transition rate calculation HMM analysis of NC2-mediated TATA box-binding protein dynamics on DNA Future applications Summary References 3 Single molecule spectroscopy at interfaces Introduction Historical overview of single molecule spectroscopy at interfaces Instrumentation and analysis Sample considerations for single molecule fluorescence at interfaces Instrumentation - Total internal reflection fluorescence (TIRF) microscopy Instrumentation - Confocal microscopy Analysis - Single molecule localization methods for super-resolution imaging Analysis - Kinetic analysis of adsorption or turnover rates Analysis - Single molecule tracking to quantify diffusion Analysis - Correlation techniques for diffusion and super-resolution information Advancements in three-dimensional single molecule imaging techniques and analysis at interfaces Imaging single molecule interfacial dynamics with engineered point spread functions Biophysical interfaces Live cell lipid membranes Membraneless interfaces through liquid/liquid phase separation Improving single molecule fluorescent protein localization at biointerfaces Soft material interfaces used in separations Interfacial catalysis Sample considerations to achieve single molecule detection at heterogeneous catalytic interfaces Resolving spatiotemporal heterogeneity at catalytic solid/liquid interfaces Other applications of single molecule spectroscopy to interfaces Outlook Acknowledgements References 4 Quantum dots in single molecule spectroscopy Introduction Synthesis, shelling and functionalization of colloidal QDs Colloidal CdE (E=S, Se, Te) QD synthesis QD shelling QD functionalization Cd-free QDs Electronic structure and photophysics of single QDs Electronic structure of QDs Single QD photophysics QDs as single-molecule fluorescent probes and single photon sources Single particle tracking (SPT) Fluorescence/image correlation spectroscopy (FCS/ICS) Single molecule FRET Super-resolution microscopy Blinking dynamics as a probe of local environment and chemical reactions Fluorescence lifetime imaging and time-gated imaging (FLIM/TGI) Single photon sources Acknowledgments References Further reading 5 Three-dimensional single-molecule tracking in living cells Historical background Uses of 3D single-molecule tracking Alternatives to 3D molecular tracking Theoretical background Single-particle localization Experimental design PSF engineering Multi-plane imaging Confocal tracking Light-sheet microscopy Interference methods Labeling strategies Precautions Data analysis Applications Location Stoichiometry Kinetics and dynamics Future prospects Acknowledgments References Further reading 6 Multiparameter fluorescence spectroscopy of single molecules Outline Multiparameter fluorescence spectroscopy Data acquisition with time-correlated single-photon counting Burst-integrated fluorescence lifetime Spectral information Quantum yield Stoichiometry and brightness Fluorescence lifetime Inter-fluorophore distance and Förster resonance energy transfer Steady-state FRET Time-resolved FRET FRET network design Time-resolved anisotropy Fluorescence correlation spectroscopy Accuracy of MFS with FRET Spatial accuracy Temporal accuracy Temporal accuracy of FCS Sample preparation for MFS with FRET Summary and workflow of MFS Applications of MFS: Examples Benchmark and DNA standards Monitoring transient conformational states along enzymatic reactions Monitoring weak interdomain interactions Discussion and conclusion Acknowledgments References 7 Single molecule analysis in nanofluidic devices Introduction Unique physical phenomena in confined environments with nanometer critical dimensions Nanoslits (1D), nanochannels (2D) and nanopores (3D) Sense-of-scale Surface charge effects Electroosmotic flow (EOF) and the electric double layer (EDL) Electrokinetic versus hydrodynamic pumping at the nanometer scale Nanometer confinement and stretching of single DNA molecules Shaping electrical fields Concentration polarization Fabrication of nanochannel devices Fabrication techniques of nanofluidic devices Bonding strategies for nanofluidic devices Fabrication of thermoplastic nanofluidic devices Applications of single molecule detection in nanofluidic devices DNA stretching Effects of channel dimensions Effects of ionic strength Optical mapping in nanochannels Enzymatic labeling Affinity labeling DNA methylation detection using nanochannels Genomic mapping for sequence variation maps using nanofluidic devices Nanoscale electrophoresis Conclusions References Index A B C D E F G H I J K L M N O P Q R S T U V W Y Z Back Cover