Preface
Contents
1: The Physical Principles of Light Propagation and Light-Matter Interactions
1.1 Definitions of Light
1.2 Particle and Wave Properties of Light
1.3 Polarization
1.4 Refraction
1.5 Reflection
1.6 Light Scattering
1.7 Diffraction and Its Limits
1.8 Photon Absorption, Fluorescence, and Stimulated Emission
1.9 Filtering Light
References
Further Reading
2: Design, Alignment, and Usage of Infinity-Corrected Microscope
2.1 Development of Compound Microscope with Infinity Optics
2.2 Design Parameters: Resolution Limit, Numerical Aperture, Magnification, Depth of Field, and Field of View
2.3 Optical Aberrations and Their Corrections
2.4 Design Specifications of the Infinity-Corrected Microscope Objective
2.5 Critical and KΓΆhler Illuminations
2.6 Components of Infinity-Corrected Microscope System
2.7 Alignment for Designing the Infinity-Corrected Bright Field Microscopy
2.8 Label-Free and Quantitative Phase Microscopy
2.8.1 Dark Field Microscopy
2.8.2 Zernike Phase Contrast Microscopy
2.8.3 Differential Interference Contrast (DIC) Microscope
2.8.4 Digital Holographic Microscopy for Quantitative Phase Measurement
References
3: Epifluorescence Microscopy
3.1 Fluorescence
3.1.1 Jablonski Diagram
3.1.2 Fluorescent Markers
3.2 Fluorescence Microscope Setup
3.2.1 Excitation Module
3.2.2 Objective
3.2.3 Fluorescence Filters
3.2.4 Emission Module
3.3 Basics of Sample Preparation for Fluorescence Microscopy
3.4 Main Applications of Fluorescence Microscopy
3.4.1 Structural Imaging
3.4.2 Functional Imaging
References
Further Reading
4: Basic Digital Image Acquisition, Design, Processing, Analysis, Management, and Presentation
4.1 Image Acquisition and Analysis: Why they Are Needed
4.1.1 Image Relevance in Science from Biology to Astronomy
4.1.2 Need for Quantitative Methods Is Extended to Light Microscopy
4.2 Representation of Reality: Image Acquisition
4.2.1 What Is the Difference Between a Material Object and a Representation of It?
4.2.2 Light Microscopy Key Concepts
4.2.2.1 What Is Light Microscopy?
4.2.2.2 Optical Resolution
4.2.2.3 Microscope Elements
4.2.2.4 Fluorescence
4.2.2.5 Transmitted Light
4.2.2.6 Detectors: Cameras and PMT/Hybrid
4.2.2.7 Digitization
4.2.3 Examples of Microscope Systems: Simplified Light Path for Wide-Field and Confocal Laser Scanning Microscopy
4.2.4 Experiment Design
4.2.5 Questions and Answers
4.3 Image Visualization Methods
4.4 Image Analysis
4.4.1 Questions and Answers
4.4.2 Questions and Answers
4.5 Publication of Images and Data
4.5.1 Questions and Answers
References
Additional References
5: Confocal Microscopy
5.1 Introduction
5.2 Fluorescence Blurring
5.2.1 The Advantages and Disadvantages of Fluorescence
5.2.2 Why Blurring Occurs
5.2.3 Optical Sectioning
5.2.4 Structural Illumination Microscopy (SIM)
5.2.5 Deconvolution
5.3 The Confocal Microscope
5.3.1 How the Confocal Microscope Works
5.3.2 Resolving Power of the Confocal Microscope
5.3.3 Advantages and Disadvantages of the Single-Beam Confocal Scanning Microscope
5.3.4 Line-Scanners and Array-Scanning Confocal Microscopes
5.4 Step by Step Protocol
5.4.1 Acquiring 2-D Sections and 3-D Z-Stack Images
5.4.2 Spectral Unmixing
5.4.3 Quantitative Confocal Microscopy and Quality Control
5.4.4 Towards Super-Resolution: Image-Scanning and Pixel Re-assignment
References
6: Live-Cell Imaging: A Balancing Act Between Speed, Sensitivity, and Resolution
6.1 Essentials in Live-Cell Imaging
6.1.1 The Problem of Phototoxicity
6.1.2 The Problem of Creating a Stable Environment
6.1.2.1 Laboratory Environment Conditions
6.1.2.2 Stable Live-Cell Incubation Conditions on the Microscope
6.1.2.3 Temperature
6.1.2.4 Osmolarity
6.1.3 The Problem of Focal Drift
6.1.3.1 Focus Drift
Experimental Box 6.1 Monitoring Cellular Events Under the Microscope: Timeline and Cell Health
6.2 Microscope Components and Key Requirements for Live-Cell Imaging
6.2.1 Upright or Inverted Microscopes
6.2.2 Microscope Components and Key Requirements for Live-Cell Imaging
6.2.2.1 Motorized Stage
6.2.2.2 Z-axis Control
6.2.2.3 Hardware-Based Autofocus
6.2.2.4 Illumination Device
6.2.2.5 Filters and Condenser Turret
6.2.2.6 Shutter
6.2.2.7 Objective Lens
6.2.2.8 Detector
6.2.2.9 Image Acquisition Software
Experimental Box 6.2 Re-scanning Confocal Microscopy (RCM) long-Term, High-Resolution Live-Cell Imaging: Laser Excitation Ener...
6.3 Fluorescent Proteins, Probes, and Labelling Techniques for Live-Cell Imaging
6.3.1 Protein Labelling Methods
6.3.2 DNA Labelling Methods
6.3.3 RNA Labelling Methods
6.3.4 Fluorescence Resonance Energy Transfer (FRET)
Experimental Box 6.3 Label-Free Holographic Imaging
6.4 Live-Cell Imaging in 3D Cell Cultures: Spheroids and Organoids
6.4.1 Challenges
6.4.1.1 Multiphoton Imaging of Spheroids
6.4.1.2 Confocal and Spinning-Disk Live-Cell Imaging of Organoids
6.4.1.3 Light-Sheet Imaging of Spheroids and Organoids
Experimental Box 6.4 Microscopy-Based High-Content Screening (HCS)
Appendix: Microscope Company and Resources List with Internet-Links
References
7: Structured Illumination Microscopy
7.1 What You Should Already Know
7.2 Fourier Decomposition of Images
7.3 Optics in Reciprocal Space
7.4 Principles of SIM
7.4.1 Reconstructing a SIM Image
7.5 Strengths and Limitations of SIM
References
Further Reading
Prior Knowledge
Mathematical Derivations of 2D and 3D SIM
8: Stimulated Emission Depletion Microscopy
8.1 Introduction
8.2 Basic Principle of the STED Microscope
8.3 Basic Design of the STED Microscope
8.4 Microscope Performance Tests
8.4.1 Laser Power Measurements
8.4.2 Gold Beads
8.4.3 Fluorescent Beads, Nanorulers, and Immunostained Cell Samples
8.4.4 Time Alignment and Gating
8.5 Application Examples
8.5.1 STED Imaging in Cells
8.5.2 STED Imaging in Deep Tissue
References
9: Two-Photon Imaging
9.1 Introduction
9.2 Two-Photon Excitation Process
9.2.1 Historical Perspectives
9.2.2 Principles of Two-Photon Excitation
9.2.3 Optical Sectioning and 3D Microscopy
9.2.4 Scattering and Photobleaching Reduction due to Fluorescence Excitation in the NIR Window
9.2.5 The Two-Photon Microscope
9.3 Advanced Analysis and Applications
9.3.1 Second Harmonic Generation (SHG)
9.3.2 Fluorescence Lifetime Imaging Microscopy (FLIM)
9.3.3 Tissue Imaging
9.3.4 Functional Brain Imaging
9.4 Step-by-Step Protocol
References
10: Modern Microscopy Image Analysis: Quantifying Colocalization on a Mobile Device
10.1 Introduction
10.1.1 The Benefits of Using Tablet Computers for Analysis of Light Microscopy Images
10.1.1.1 Ease of Use
10.1.1.2 Superior Engagement and Improved Work Efficiency
10.1.1.3 More Comfortable Analysis
10.1.1.4 Better Affordability
10.1.1.5 New Possibilities
10.2 Performing Microscopy Image Analysis on a Tablet Computer
10.2.1 Analyzing Colocalization of Fluorescence Markers
10.2.2 Setting Up Microscopy Image Analysis on a Mobile Device
10.2.3 Steps of the Workflow
10.2.4 The Importance of Image Resolution
10.2.4.1 Using a Machine Learning Model to Transform Conventional Fluorescence Images to Super-Resolution
10.2.5 Selecting a Region of Interest (ROI)
10.2.6 The Importance of Noise Reduction
10.2.6.1 Using an ML Model to Reduce Background Image Noise
10.2.7 Analyzing Colocalization
10.2.7.1 Calculating Colocalization Coefficients
10.2.7.2 Revealing Areas with Colocalization
10.2.8 Exporting Results
10.2.9 Documentation
10.2.10 Current Limitations
10.3 Interpretation of Results
10.4 Benchmark Datasets
References
11: Spinning Disk Microscopy
11.1 Overview
11.2 History
11.3 Sample Preparation
11.4 Fundamental Microscope Design
11.5 Imaging Resolution
11.6 Super Resolution via Optical Reassignment Imaging (a.k.a. SoRa)
11.7 Spinning Disk Confocal Microscopy vs. Laser Scanning Confocal Microscopy
11.8 General Questions
11.9 Chapter Summary
References
12: Light-Sheet Fluorescence Microscopy
12.1 Introduction
12.1.1 Issues with Conventional Fluorescence Microscopy Techniques
12.1.2 Introducing Light-Sheet Microscopy
12.2 Principles of Light-Sheet Microscopy
12.2.1 Creating and Using a Light Sheet
12.2.2 Capturing an Image
12.2.3 Imaging a Whole Sample
12.2.4 Image Properties and Analysis
12.3 Preparation of a Sample for Light-Sheet Fluorescence Microscopy
12.3.1 Dyeing Your Sample
12.3.2 Clearing Your Sample
12.3.3 Mounting Your Sample
12.4 Trade-Offs and Light-Sheet Microscopy: Development of Novel Systems to Meet Demands
12.4.1 Cost and Commercial Systems
12.4.2 Light Penetration
12.4.3 Resolution Limitations
12.4.4 Sample Size
12.4.5 Data Deluge
References
Further Reading
History
Reviews
13: Localization Microscopy: A Review of the Progress in Methods and Applications
13.1 The Optical Resolution Limit
13.2 Super-Resolved Localization in 2D
13.3 STORM and PALM
13.3.1 STORM
13.3.2 PALM
13.3.3 Pros and Cons
13.3.4 Techniques Using Modified Point Spread Functions that Use Localization Microscopy at Some Level
13.4 Choosing a Fluorophore
13.5 Ideal Properties of a Super-Resolution Microscope Relevant to Localization Microscopy
13.6 3D Localization
13.7 Analyzing 2- and 3D Localization Data
13.8 Applications of Localization Microscopy
13.9 Conclusion and Future Perspectives
References
Correction to: Live-Cell Imaging: A Balancing Act Between Speed, Sensitivity, and Resolution
Correction to Chapter 6 in: V. Nechyporuk-Zloy (ed.), Principles of Light Microscopy: From Basic to Advanced, https://doi.org/...