Fluorescence Spectroscopy and Microscopy in Biology

Aims and Scope
Preface
Contents
Part I: Fluorescence Spectroscopy: Basics and Advanced Approaches
Choosing the Right Fluorescent Probe
1 Intrinsic and Extrinsic Fluorescent Probes
2 Organic Dyes
2.1 Ideal Properties of Fluorescent Probes
2.1.1 Molecular Brightness
2.1.2 Extinction Coefficient
2.1.3 Quantum Yield
2.1.4 Photostability
2.1.5 Aggregation and Solubility
2.1.6 Fluorescence Emission Spectra
2.1.7 Stokes Shift
2.1.8 Fluorescence Lifetime
2.1.9 pH Sensitivity
2.1.10 Overview of Fluorescent Probe Classes
2.2 Methods for Fluorescent Protein Labelling
2.3 Membrane Probes
3 Fluorescent Proteins
4 Fluorescent Probes for Super-Resolution Microscopy
4.1 Synthetic Probes
4.2 Fluorescent Proteins
5 Perspectives
References
Fluorescence Kinetics and Time-Resolved Measurement
1 Basics and a Bit of History
1.1 Nobel Prizes Related to Fluorescence
1.2 Fluorescence as a Tool of Growing Importance
1.2.1 Jabloński Diagram and Stokes Shift
1.2.2 Fluorescence as a Reporter of Molecular Properties and Interactions
1.3 Basic Characteristics of Excitation and Emission
1.4 Kinetics of Fluorescence
1.5 Time-Resolved Fluorescence Anisotropy
2 Time-Resolved Spectroscopy and Microscopy
2.1 Time-Resolved Techniques and Their Development
2.2 State-of-the-Art Techniques for ps-fs Time Resolution
2.2.1 Time-Resolved Photon Counting
2.2.2 Fluorescence Up-Conversion (Decay Sampling)
3 Some Nonlinear Techniques and New Trends
3.1 Multi-photon Excitation Techniques
3.2 Emerging New Techniques: Excitation by Structured Photons
3.3 Combined Nonlinear Optical and Fluorescence Spectroscopies and Microscopy
4 Conclusions
References
A Quantitative Approach to Applications of Electronic Energy Transfer (EET)
1 Background to Förster´s Theory
2 Observing EET
2.1 Analyses of EET Data Within Pairs of Chromophores
2.2 Analyses of EET Data of Donors in Spatial Distributions
2.3 Analyses of EET Data Among Donors Within Periodic Spatial Distributions
3 Closing Comments
References
Single-Molecule FRET: Principles and Analysis
1 Introduction
2 Principles of Ensemble FRET
3 Principles of smFRET: Beyond the Ensemble Average
3.1 Biomolecular Conformations and Dynamics
3.2 Quantitative FRET
4 Experimental Considerations for smFRET
4.1 Microscope Configurations: Confocal and Widefield Microscopy
4.2 Excitation Schemes and Multiplexed Detection
4.3 Data Analysis of Conformational Distributions and Dynamics
5 smFRET Application Examples
6 Concluding Remarks
References
Principles of Fluorescence Correlation and Dual-Color Cross-Correlation Spectroscopy
1 Fluorescence Intensity
2 Fluorescence Correlation
3 Brightness Correlation Function
4 Position Correlation Function
References
Part II: Fluorescence Microscopy: Basics and Advanced Approaches
Introduction to Fluorescence Microscopy
1 Introduction
2 Epifluorescence Microscope
2.1 Light Sources
2.2 Beamsplitters and Filters
2.2.1 Linear Spectral Unmixing
2.3 Image Formation
2.4 (Widefield) Image Detection
3 Objective Lenses and Image Resolution
3.1 Resolution of a Fluorescence Microscope
3.2 Resolution and Optical Aberrations
3.3 Objective Lens Numerical Aperture
3.4 Resolution and Image Pixel Size
4 Confocal Microscopy
4.1 Laser Scanning Microscopy
4.2 Image Sampling and Magnification in Point-Scanning Microscopy Modalities
4.3 Spinning Disk Confocal Microscopy
4.4 Chromatic Aberration in Confocal Microscopy
4.5 Optical Sectioning and Volumetric Confocal Imaging
5 Other Modalities with Optical Sectioning Capabilities
5.1 Multi-Photon Fluorescence Microscopy
5.2 Lightsheet Microscopy
5.3 Total Internal Reflection Fluorescence Microscopy
5.4 Structured Illumination
5.5 Computational Approaches
6 Resolution Beyond the Diffraction Limit
6.1 Near-Field Fluorescence Microscopy
6.2 Single Molecule Localisation Microscopy (SMLM) and Fluorescence Fluctuation-Based Techniques
6.3 Structured Illumination Microscopy (SIM)
6.4 Confocal Microscopy and Related Approaches
6.5 Computational Approaches
7 Concluding Remarks and Outlook
References
STED and RESOLFT Fluorescent Nanoscopy
1 Introduction
2 Light-Induced Reversible State Transitions
2.1 Reversible Electronic Transitions: Stimulated Emission
2.2 Reversible Molecular Transitions
3 Adaptive and Smart Scanning of the Illumination
4 Optimized Patterns and Adaptive Detection
5 Parallelization
5.1 Parallelized STED
5.2 Parallelized RESOLFT
5.2.1 Widefield-RESOLFT
5.2.2 MoNaLISA or Multifoci Parallelized RESOLFT
5.2.3 3D Parallelized RESOLFT
5.3 Digital Pinholing of Camera Data
6 STED and RESOLFT for Live-Cell Imaging
7 Challenges and Outlooks
References
Fluorescence Correlation Spectroscopy in Space and Time
1 Introduction
2 Image-Based Correlation Spectroscopy
2.1 Image Correlation Spectroscopy: ICS
2.2 Temporal Image Correlation Spectroscopy: TICS
2.3 Spatiotemporal Image Correlation Spectroscopy: STICS
2.4 k-Space Image Correlation Spectroscopy: kICS
2.5 Image Mean Squared Displacement: iMSD
2.6 Pair Correlation Function: pCF
3 Scanning FCS
3.1 Scanning FCS in the Presence of Immobile Particles
3.2 Scanning FCS in the Presence of Flow
3.3 Scanning FCS as a Tool for Multiplexing and Avoidance of Artefacts
3.4 Raster Image Correlation Spectroscopy: RICS
4 Multipoint and Imaging FCS
5 Concluding Remarks
References
Part III: Applications of Fluorescence Spectroscopy and Microscopy to Biological Membranes
Determination of Biomolecular Oligomerization in the Live Cell Plasma Membrane via Single-Molecule Brightness and Co-localizat...
1 Biomolecular Interactions on the Plasma Membrane
2 Detecting Biomolecular Assemblies Directly on the Plasma Membrane
2.1 How About Decreasing the Label Density?
2.2 Would One of the Single-Molecule Localization Microscopy (SMLM) Modalities Work for Detecting Molecular Dimers?
2.3 How about Using Stimulated Emission Depletion (STED) Imaging?
2.4 Thinning Out Clusters While Conserving Stoichiometry of Labeling (TOCCSL)
3 The TOCCSL Concept
3.1 Principle
3.2 Imaging Protocol
3.3 Brightness Analysis
3.4 Two-Color TOCCSL
3.5 Co-localization Analysis
4 Microscopy Setup
5 Choice of Parameters
5.1 Choice of Recovery Time, Search-Radius and Analysis Region
5.2 Choice of Photobleaching Time
5.3 Fluorescent Labels
5.3.1 No Unspecific Binding/Detection of Fluorophores
5.3.2 Fast and Efficient Photobleaching While Keeping a Good Signal-to-Noise Ratio
5.3.3 Minimal-Invasive Conjugation to Biomolecule of Interest Possible
5.3.4 Labeling Stoichiometry
6 TOCCSL Applications
References
Quantitative Photoactivated Localization Microscopy of Membrane Receptor Oligomers
1 Introduction
2 Theoretical Background of qPALM
2.1 Photophysics
2.2 Kinetic Models
3 qPALM Experiment
3.1 Labeling
3.2 Data Acquisition and Evaluation
3.3 Reference Structures for Calibration
4 Quantification of Membrane Protein Oligomers with qPALM
5 Discussion
References
Diffusion Measurements at the Nanoscale with STED-FCS
1 Introduction
2 A Brief Introduction to Super-Resolution Microscopy
3 STED-FCS as a Window to Nanoscale Dynamics
4 Practical Considerations and Implementations of STED-FCS
5 Variations of STED-FCS
6 Alternative Ways to Investigate Nanoscale
7 Future Developments
References
Single-Molecule Microscopy Methods to Study Mitochondrial Processes
1 Introduction
2 Single-Molecule Localization Microscopy
2.1 PALM and STORM of Mitochondrial Proteins
2.2 DNA-Based Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT)
3 Single-Particle Tracking (SPT)
4 Summary
References
Transient State (TRAST) Spectroscopy and Imaging: Exploiting the Rich Information Source of Fluorophore Dark State Transitions...
1 Introduction
2 Transient State Monitoring by FCS and Other Methods
3 Transient State (TRAST) Spectroscopy/Imaging: Basic Concept
4 TRAST: Some Experimental Realizations and Applications
5 Concluding Remarks
References
The Analysis of In-Membrane Nanoscopic Aggregation of Lipids and Proteins by MC-FRET
1 Introduction
2 FRET Between Homogeneously Distributed Donors and Acceptors
3 FRET Between Heterogeneously Distributed Donors and Acceptors
3.1 Heterogeneous Probe Distributions Induced by Lipid Nanodomain Formation or Protein Oligomerization
3.2 The Estimation of Lipid Nanodomain Sizes by MC-FRET
3.2.1 Workflow of MC-FRET for the Nanoscopic Characterization of Nanodomains
3.2.2 What Nanodomain Sizes Can Be Resolved by MC-FRET? [18]
3.3 Resolving Inter-Leaflet Coupled from Inter-Leaflet Independent Nanodomains by MC-FRET
3.4 Quantifying Protein Dimerization by MC-FRET
3.4.1 Workflow of MC-FRET for the Quantification of Protein Dimerization
3.4.2 Determination of Donor and Acceptor Surface Concentrations
3.4.3 Dealing with Kappa Squared (κ2)
3.4.4 Dimerizing and Non-dimerizing Membrane Proteins
4 Conclusions
References
Part IV: Applications of Fluorescence Spectroscopy to Protein Studies
Single-Molecule Fluorescence Spectroscopy of Intrinsically Disordered Proteins
1 Introduction
2 Technical Aspects of smFRET Experiments
2.1 Confocal smFRET Experiments
2.2 Correction Parameters in smFRET Experiments
3 Single-Molecule FRET: A Blurry Window into Molecular Disorder
3.1 Compaction and Expansion of Unfolded and Disordered Proteins Probed with smFRET
3.2 Mean-Field Homopolymer Theory
3.3 More Accurate Polymer Models: Combining smFRET with Molecular Simulations
4 Probing and Modeling Sub-microsecond Dynamics of Disordered Proteins
4.1 Nanosecond Fluorescence Correlation Spectroscopy (nsFCS) Coupled with FRET
4.2 Polymer Models for IDP Dynamics and Their Limitations
References
Insights into the Conformational Dynamics of Potassium Channels Using Homo-FRET Approaches
1 Introduction
2 FRET Toolbox for Studying Multimeric Symmetric Proteins
2.1 Hetero-FRET Measurements
2.1.1 Single Donor-Acceptor Pair
2.1.2 Homo-Tetrameric Proteins
2.2 Homo-FRET Measurements
2.3 General Applications of Homo-FRET Measurements
3 Homo-FRET Studies on the Potassium Channel KcsA: A Practical Guide from a Symmetric Tetrameric Membrane Protein
3.1 Functional and Structural Characteristics of the KcsA Channel
3.2 Fluorescence Properties of the KcsA Channel
3.3 Time-Resolved Anisotropy Measurements and Homo-FRET Analytical Framework to Characterize the Conformational Dynamics of th...
3.4 An Efficient Homo-FRET Process Among W67 Residues at the Pore-Helices Detects the Conformational Plasticity of the SF at p...
3.5 The Conformational Dynamics of the Outer Vestibule Is Hindered by the Presence of a Monoclonal Fab Fragment
3.6 The Conformational Dynamics of the Selectivity Filter Depends on the Conformational State of the Activation Gate of KcsA
3.7 Examining the Bidirectional Crosstalk Between the Inner and Outer Gates of KcsA Through Homo-FRET Monitoring of Two Indepe...
3.8 Consequences of Incomplete Labelling of G116C W67 KcsA with an Extrinsic Probe
3.9 Allosteric Coupling Between the Activation Gate and the SF
4 Concluding Remarks
References
Intrinsic Fluorescence Kinetics in Proteins
1 Introduction
1.1 Fluorescent Residues in Protein as Potential Fluorescence Sensors
2 Modelling the Kinetics of Intrinsic Fluorescence
2.1 Exponential and Multi-exponential Decays
2.2 Modelling Microheterogeneity and Dielectric Relaxation in Protein Fluorescence Kinetics
2.3 Time-Resolved Emission Spectra (TRES)
3 Applications of Intrinsic Fluorescence Studies of Protein
3.1 Non-Debye Kinetics of Trp in HSA
3.2 Oligomerisation/Glycation of Beta-Amyloid Aβ1-40
3.2.1 Free Aβ1-40 in a Buffer Solution
3.2.2 Aβ1-40 with Cu+2 Ions
3.2.3 Aβ1-40 with Glucose
3.3 Glycation of Collagen
4 Conclusion
References
Dynamics and Hydration of Proteins Viewed by Fluorescence Methods: Investigations for Protein Engineering and Synthetic Biology
1 Introduction
2 Hydration and Mobility Monitored by Time-Dependent Fluorescence Shift
2.1 Role of Protein Hydration and Mobility at the Tunnel Mouth of HLDs
2.1.1 TDFS as a Detector of Site-Specific Hydration and Mobility
2.1.2 TDFS and Structure-Function Relationship of HLDs: Enantioselectivity Study
3 Hydration Assay Based on Steady-State Fluorescence of HMC
3.1 Hydration at the Tunnel Mouth of HLDs: Site-Specific HMC Hydration Assay in Proteins Using Unnatural Amino Acid
4 Protein Dynamics Probed by Photoinduced Electron Transfer: Fluorescence Correlation Spectroscopy (PET-FCS)
4.1 Real-Time Capture of Molecular Gating in HLDs
5 Conclusions and Perspectives
References