Optical Spectroscopic and Microscopic Techniques: Analysis of Biological Molecules

✍ Scribed by Harekrushna Sahoo (editor)

• Publisher: Springer
• Year: 2022
• Tongue: English
• Leaves: 260
• Edition: 1st ed. 2022
• Category: Library

✦ Synopsis

This book illustrates the significance of various optical spectroscopy and microscopy techniques, including absorption spectroscopy, fluorescence spectroscopy, infrared spectroscopy, and Raman spectroscopy for deciphering the nature of biological molecules. The content of this book chiefly focuses on (1) the principle, theory, and instrumentation used in different optical spectroscopy techniques, and (2) the application of these techniques in exploring the nature of different biomolecules (e.g., proteins, nucleic acids, enzymes, and carbohydrates). It emphasizes the structural, conformational and dynamic, and kinetic including the changes in biomolecules under a range of conditions. In closing, the book summarizes recent advances in the field of optical spectroscopic and microscopic techniques.

✦ Table of Contents

Contents
About the Editor
1: Absorption Spectroscopy: What Can We Learn About Conformational Changes of Biomolecules?
1.1 Introduction
1.2 Origin of UV/Vis Spectrum
1.3 Lambert-Beer´s Law
1.4 Instrumentation of UV Spectroscopy
1.5 Chromophores and Auxochromes
1.6 Factors Affecting Absorption Spectra
1.7 Nature of Shifts in the UV Spectrum
1.8 Fundamental Absorption Characteristics of Biomolecules
1.8.1 Characteristic UV-Vis Spectra of Nucleic Acid Bases
1.8.2 Electronic Spectroscopy of DNA and RNA
1.8.3 Nucleic Acid Denaturation
1.8.4 Electronic Spectroscopy of Proteins
1.8.5 Folding and Unfolding of Protein
1.9 Applications of UV-Vis Spectroscopy in Accessing the Conformational Changes of Biomolecules
1.9.1 Monitoring the Self-Association of Insulin [12]
1.9.2 Determination of Protein and Nucleic Acid Content of the Virus [13]
1.9.3 Location of Abnormal Tyrosines in Actin [14]
1.10 Conclusion
References
2: Circular Dichroism Spectroscopy: Principle and Application
2.1 Introduction
2.2 Instrumentation
2.2.1 Modulation Method
2.2.2 Direct Subtraction Method
2.2.3 Ellipsometric Method (Fig. 2.3)
2.3 Sample Preparation and Electronic CD Measurement
2.4 Application to Protein Structure
2.4.1 Protein-Lipid Interaction
2.4.2 Protein-Ligand Interactions
2.4.3 Denaturation Study of Proteins
2.4.3.1 Thermal Denaturation
2.4.3.2 Chemical Denaturation
2.4.4 Change in pH Induced Transition of Proteins
2.4.5 Thermal Stability
2.5 CD Study of Nucleic Acids
2.5.1 Denaturation Study of DNA
2.6 Conclusion
References
3: Steady-State Fluorescence Spectroscopy as a Tool to Monitor Protein/Ligand Interactions
3.1 Introduction
3.1.1 Basic Concepts
3.1.2 Intrinsic Protein Fluorescence
3.1.3 Extrinsic Fluorescent Probes
3.2 Steady-State Fluorescence Analysis
3.2.1 Fluorescence Analysis of Protein-Ligand/Drug Interactions
3.2.1.1 Chipman Analysis
3.2.1.2 Scatchard Plot Analysis
3.2.1.3 Quantification Using Labeled Ligand
3.2.1.4 Correction for Inner Filter Effect
3.2.1.5 Applications
3.2.2 Fluorescence Analysis of Protein-Lipid Interaction
3.2.2.1 Binding (Partitioning) Coefficient Analysis
3.3 Fluorescence Quenching Analysis
3.3.1 Collisional Quenching: The Stern-Volmer Plot
3.4 Red-Edge Excitation Shift (REES) Analysis
3.5 Synchronous Spectroscopy
References
4: Fluorescence Anisotropy: Probing Rotational Dynamics of Biomolecules
4.1 Fluorescence
4.2 Steady-State Fluorescence Anisotropy
4.2.1 Factors Affecting Fluorescence Anisotropy
4.2.2 Importance of Apparent Rotational Correlation Time
4.3 Time-Resolved Anisotropy Decays
4.4 Applications of Fluorescence Anisotropy Measurements
4.4.1 Determination of Phase Transition Temperature of Lipid
4.4.2 Determination of Microviscosity of the Environment
4.4.3 Study of Protein-Protein Interaction
4.4.4 Study of Protein Conformation and Misfolding
4.4.5 Study of Membrane Organization
4.4.6 Study of Drug-Protein Interaction
4.5 Concluding Remark and Future Perspectives
References
5: Fluorescence Lifetime: A Multifaceted Tool for Exploring Biological Systems
5.1 Introduction
5.2 Fluorescence Lifetime: Definition and Principle
5.2.1 Factors Affecting Fluorescence Lifetime
5.3 Time-Resolved Fluorometry: The Technique
5.3.1 Frequency-Domain or Phase-Modulation Method
5.3.2 Time-Domain or Pulse Fluorometry
5.3.2.1 Time-Correlated Single-Photon Counting (TCSPC)
5.4 Fluorescence Lifetime-Based Applications
5.4.1 Fluorescence Lifetime Assays
5.4.1.1 Interaction of Proteins with Small and Macromolecules
Protein-Surfactant Interaction
Protein-Drug Interaction
Protein-Ionic Liquid Interaction
Interaction of Proteins with Macromolecules
5.4.1.2 Investigation of the Conformational Changes of Proteins
5.4.1.3 Binding of DNA with Small Molecules
5.4.1.4 Elucidating the Self Assembly of Bile Salts and Their Interaction with Drugs
5.4.1.5 Understanding the Physical Properties of Lipid Bilayers
Determining Membrane Permeability Mechanisms
Determining Distances in Lipid Bilayers Using Time-Resolved FRET
Interaction of Liposomes with Small and Macromolecules
Interaction of Liposomes with Proteins and DNA
5.4.2 Fluorescence Lifetime Sensing
5.4.2.1 Sensing of pH
5.4.2.2 Sensing of Glucose
5.4.2.3 Sensing of Different Ions
5.4.3 Fluorescence Lifetime Imaging
5.4.3.1 FLIM for Mapping Viscosity
5.4.3.2 FLIM for Mapping Intracellular Temperature
5.4.3.3 FLIM to Map Ion Concentrations
5.4.3.4 FLIM for Mapping pH
5.4.3.5 FLIM for Mapping Glucose
5.4.3.6 FLIM for Mapping Oxygen
5.4.3.7 FLIM for Tissue Imaging and Medical Applications
5.4.3.8 FLIM for Tracking Drug Delivery and Release
5.5 Concluding Remarks and Future Perspectives
References
6: From Ensemble FRET to Single-Molecule Imaging: Monitoring Individual Cellular Machinery in Action
6.1 Introduction
6.2 Fluorescence
6.2.1 Fluorescence Quenching
6.2.2 Fluorescence Resonance Energy Transfer
6.2.3 FRET Efficiency
6.3 Single-Molecule FRET
6.3.1 Essentials for Experimental Designing
6.3.2 Selecting the Right Fluorophore Pair
6.3.3 Sample Design
6.3.4 Slide Preparation and Surface Immobilization
6.4 Single-Molecule Detection
6.4.1 Total Internal Reflection Microscopy
6.4.2 Confocal Microscopy
6.5 Data Analysis
6.6 Visualization of Dynamical Conformations of Biomolecules Using Single-Molecule Fluorescence Resonance Energy Transfer
6.6.1 Initiation and Reinitiation of DNA Unwinding by the Escherichia coli Rep Helicase
6.6.2 Involvement of G-triplex and G-hairpin in the Multipathway Folding of Human Telomeric G-quadruplex
6.6.3 A Four-Way Junction Accelerates Hairpin Ribozyme Folding via a Discrete Intermediate
6.6.4 Monitoring of Structural Dynamics of a Holliday Junction [28]
6.6.5 smFRET Reveals the Kinetics and Dynamics of a DNA Repair Protein MutL [29]
6.6.6 smFRET Analysis of Helicases Involved in DNA Replication [30]
6.7 Summary
References
7: Nanosecond Time-Resolved Fluorescence Assays
7.1 Introduction
7.1.1 Assay Development
7.1.2 Fluorescence Methods in Assay Development
7.2 Time-Resolved Fluorescence (TRF) Assays
7.3 TRF Probes with Nanosecond Lifetimes
7.3.1 Ru(II)-Based Nano-TRF Probes
7.3.1.1 Hydrolase Assays with Ru(II) Complexes
7.3.1.2 Ru(II)-Based Binary Probes for DNA Detection
7.3.1.3 Ru(II)-Based Immunoassays
7.3.2 Nano-TRF Probes Based on Pyrene
7.3.3 Nano-TRF Assays Based on the Fluorazophore DBO
7.3.3.1 Nano-TRF Protease Assays with DBO
7.3.3.2 Nano-TRF Kinase Assays with DBO
7.4 Multiple-Pulse Pumping with Nano-TRF Probes
7.5 Conclusion
References
8: Fluorescence Correlation Spectroscopy: A Highly Sensitive Tool for Probing Intracellular Molecular Dynamics and Disease Dia...
8.1 Introduction
8.2 General Principles, Instrumentation, and Evaluation of FCS Data
8.3 Developments in FCS and Related Techniques
8.3.1 Fluorescence Cross-Correlation Spectroscopy (FCCS)
8.3.2 Scanning Fluorescence Correlation Spectroscopy (sFCS)
8.3.3 Stimulated-Emission Depletion Microscopy-Fluorescence Correlation Spectroscopy (STED-FCS)
8.4 Intracellular Molecular Dynamics Measurements with FCS
8.5 FCS as a Diagnostic Tool for Disease Conditions
8.6 Conclusions and Perspectives
References
9: Principles and Applications of Fluorescence Microscopy
9.1 Introduction
9.2 Phenomena of Fluorescence
9.3 Major Developments
9.4 Fluorescent Molecules
9.4.1 Properties of Fluorescence Emission
9.4.2 Fluorescent Proteins
9.5 Principles of Fluorescence Microscopy
9.6 Resolution
9.6.1 Nyquist Criterion
9.7 Advanced Microscopic Techniques
9.8 Application of Fluorescence Microscopy in Biological and Biophysical Research
9.8.1 Immunofluorescence and Live Cell Imaging
9.8.2 Reconstituted Lipid Membranes
9.9 Conclusion and Future Perspective
References
10: Analysis of Biomolecular Dynamics Under Fourier Transform Infrared Spectroscopy
10.1 Introduction
10.2 Modified Techniques to Detect Biomolecular Dynamics Under FTIR
10.3 Principle and Methodology of Fourier Transform Infrared Spectroscopy
10.4 Instrumentation in FTIR Spectroscopy
10.4.1 IR Radiation Sources in Detail
10.4.2 Monochromator
10.4.3 Sample Cells and Preparation of a Sample for Analysis
10.4.4 Detectors Used in FTIR Spectroscopy
10.4.5 Optical Arrangement of Fourier Transform Infrared Spectroscopy
10.4.6 Advantage
10.4.7 Precaution Needs to Be Taken to Avoid Trouble while Using FTIR Spectroscopy for Analysis of Biomolecules
10.5 Biomedical Importance of FTIR Spectroscopy
10.5.1 Measurement of Lipid Content
10.5.2 Carbohydrate Analysis
10.5.3 Protein Dynamics Study
10.5.4 Monitoring the Mechanism of Action of Protein by Time-Resolved FTIR Spectroscopy
10.5.5 Analysis of Nucleic Acid in Aqueous Solution
10.5.6 To Understand the Bacterial Adhesion Mechanism at Metal Oxide Surface
10.5.7 Disease Diagnosis
10.6 Conclusion
References
11: Raman Spectroscopy in Biology: Perspectives and Emerging Frontiers
11.1 Introduction
11.1.1 Raman Effect and Its Discovery
11.1.2 Historical Perspective
11.1.3 Invention of Lasers and Other Technological Breakthroughs: Impact on the Growth and Development of Raman Spectroscopy
11.1.4 Scope of This Chapter
11.2 Principles: Basic Mechanisms of Photon-Molecule Interactions in Rayleigh Scattering Different Types of Raman Scattering a...
11.3 Instrumentation
11.3.1 Basic Set-Up Used in a Raman Spectrometer
11.3.1.1 Dispersive Raman Spectrometer
11.3.1.2 Fourier Transform Raman Spectrometer
11.3.2 Advanced Raman Techniques
11.3.2.1 Surface Enhanced Raman Spectroscopy (SERS)
11.3.2.2 Coherent Anti-Stokes Raman Spectroscopy (CARS)
11.4 Applications to Biomolecules: Overview
11.4.1 Proteins: Conformational and Related Studies
11.4.2 Nucleic Acids: Conformational and Drug-DNA Interaction Studies
11.4.3 Drug-DNA Interaction Probed via Raman Spectroscopy
11.5 Conclusions and Future Outlook
References