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Spie Press Book

Confocal Microscopy and Multiphoton Excitation Microscopy: The Genesis of Live Cell Imaging
Author(s): Barry R. Masters
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Book Description

This text guides you through the principles and practical techniques of confocal and multiphoton microscopy. It also describes the historical connections and parallel inventions that resulted in modern techniques of live cell imaging and their use in biology and medicine. You will find comparisons of different types of confocal and multiphoton microscopes, solutions to the problems one would encounter when using various microscopic techniques, tips on selecting equipment, and an extensive annotated bibliography of additional resources.

Book Details

Date Published: 9 February 2006
Pages: 230
ISBN: 9780819461186
Volume: PM161

Table of Contents
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List of Abbreviations
Part I. Optical Microscopy
Chapter 1: A Brief History of the Microscope and its Significance in the Advancement of Biology and Medicine
1.1 Timeline of Optical Microscope Development
1.2 Key Developments of Fluorescence Microscopy and its Limitations, Genesis, and Some Applications
1.3 Key Advances in Biology and Medicine Made Possible with the Microscope
1.4 Summary
Chapter 2: The Optical Microscope: Its Principles, Components, and Limitations
2.1 What Is an Optical Microscope?
2.2 Image Fidelity: Mapping the Object into the Image
2.3 Optical Aberrations
2.4 The Compound Microscope
2.5 Chief Components of an Optical Microscope
2.6 Microscope Objectives
2.7 Sets of Conjugate Planes in the Optical Microscope
2.8 Epi-Illumination Fluorescence Microscope
2.9 Summary
Chapter 3: Abbe Theory of Image Formation and Diffraction of Light in Transmitted Light Microscopes
3.1 The Contributions of Abbe
3.2 Abbe Diffraction Theory of Image Formation and Optical Resolution in the Light Microscope
3.3 Summary
Chapter 4: Optical Resolution and Resolving Power: What It Is, How to Measure It, and What Limits It
4.1 Criteria for Two-Point Resolution
4.2 The Role of Depth Discrimination
4.3 Point Spread Functions Characterize Microscope Performance
4.4 Summary
Chapter 5: Techniques that Provide Contrast
5.1 Nonoptical Techniques
5.2 Optical Techniques
5.2.1 Phase contrast microscopy
5.2.2 Differential Interference Contrast (DIC) Microscopy
5.2.3 Video-Enhanced Contrast Microscopy
5.3 Summary
Part II. Confocal Microscopy
Chapter 6: Early Antecedents of Confocal Microscopy
6.1 The Problem with Thick Specimens in Light Microscopy
6.2 Some Early Attempts to Solve These Problems
6.3 Scanning Optical Microscopes: How Scanning the Illumination Reduces Light Scatter and Increases Contrast
6.4 Some Early Developments of Scanning Optical Microscopy
6.5 Summary
Chapter 7: Optical Sectioning (Depth Discrimination) with Different Scanning Techniques: The Beginnings of Confocal Microscopy
7.1 The Confocal Microscope: The Problem and Its Solution
7.2 Stage-Scanning Confocal Microscope Invented by Marvin Minsky
7.3 Mojmir Petran, Milan Hadravsky, and Coworkers Invent the Tandem-Scanning Light Microscope
7.4 Guoqing Xiao and Gordon Kino Invent the One-Sided Confocal Scanning Light Microscope
7.5 Effect of Pinhole Size and Spacing on the Performance of Nipkow Disk Confocal Microscopes
7.6 Akira Ichihara and Coworkers at Yokogawa Institute Corporation Invent a Microlens Nipkow Disk Confocal Microscope
7.7 Svishchev Invents An Oscillating Mirror Scanning-Slit Confocal Microscope
7.8 Laser-Scanning Confocal Microscope Designs
7.9 Analytical Expression of Resolution in a Confocal Microscope
7.10 Comparison of Different Confocal Microscope Designs: Which One Should You Purchase?
7.11 Limitations of the Confocal Microscope
7.12 Summary
Chapter 8: The Development of Scanning-Slit Confocal Systems for Imaging Live Cells, Tissues, and Organs
8.1 Scanning-Slit Confocal Microscope
8.2 Statement of the Problem: Slit Width Versus Field of View
8.3 Goldmann's Wide-Field Microscope
8.4 Maurice Invents Several Types of Specular Microscopes
8.5 Svishchev's Invention of a Scanning-Slit Confocal Microscope
8.6 Baer Invents a Tandem-Scanning-Slit Confocal Microscope with an Oscillating Moving Mirror-Slit Assembly
8.7 Maurice Invents a Scanning-Slit Wide-Field Specular Microscope
8.8 Koester Invents a Wide-Field Confocal (Specular) Microscope for In Vivo Imaging
8.9 Masters Develops a Confocal Microscope Based on the Maurice Design with an Axial Scanning Microscope Objective
8.10 Thaer Real-Time Scanning-Slit Clinical Confocal Microscope
8.11 Summary
Chapter 9: The Components of a Confocal Microscope
9.1 Light Sources
9.2 Scanning Systems
9.3 Dichroic Mirrors and Filters
9.4 Pinholes
9.5 Detectors
9.6 Microscope Objectives 147
9.7 Summary 149
Part III. Nonlinear Microscopy
Chapter 10: The Development of Nonlinear Spectroscopy and Microscopy
10.1 Nonlinear Optical Processes in Spectroscopy and Microscopy
10.2 The Nonlinear, Scanning, Harmonic Optical Microscope Is Invented at Oxford University
10.3 The Role of Lasers in the Development of Nonlinear Microscopy
10.4 Summary
Chapter 11: Multiphoton Excitation Microscopy
11.1 Goppert-Mayer's Theory of Two-Photon Absorption
11.2 The Denk, Strickler, and Webb 1990 Science Publication and 1991 Patent
11.3 Comparison of Multiphoton Excitation Microscopy and Confocal Microscopy
11.4 Summary
Chapter 12: Theory and Instrumentation of Multiphoton Excitation Microscopy
12.1 Theory
12.2 Instrumentation
12.2.1 Laser sources
12.2.2 Laser beam diagnostic instrumentation
12.2.3 Laser pulse spreading due to dispersion
12.2.4 Microscope objectives
12.2.5 Scanners
12.2.6 Detectors
12.3 Summary
Part IV. The Path to Imaging Live Cells, Tissues, and Organs
Chapter 13: Remaining Problems, Limitations, and their Partial Solutions
Chapter 14: Speculation on Future Directions for Confocal and Multiphoton Excitation Microscopy
14.1 Correlative Microscopy
14.2 Multimodal Microscopes
14.3 In-Vivo Microscopy or Live Cell and Tissue Imaging
14.4 Instrument Development
14.5 Summary
Chapter 15: Safety and Cleanliness Considerations
15.1 Laser Safety
15.2 How to Clean Optics
Appendix: Reference Materials and Resources


This text explains the fundamentals of confocal microscopy and multiphoton excitation microscopy. It presents the big picture of technological development in optical microscopy and provides insight into the origins, development, modification, and application of confocal and multiphoton excitation microscopes and their use in biology and medicine. This insight is presented in light of the key problems that each new invention attempted to solve, the various paths to the solution, the myriad interactions of various inventors and their associated technologies, and the practical limitations of each step of discovery and technological development. The human side of these technological developments is also revealed by describing the individual motivations that drove different scientists to their inventions, as well as the parallel developments that preceded each stage of technological development.

The repeated convergence of disparate techniques, instruments, theoretical studies, inventions, and reinventions from a wide variety of disciplines partially solved a series of problems in the field of microscopy and produced the current renaissance in modern opticalmicroscopy. Innovative ideas and technical developments came from many individuals living and working in several countries around the world. Innovation evolves from a broad knowledge base, an awareness of advances in disparate fields of science, the courage to radically depart from mainstream thinking, and a clear understanding and statement of the problem to be solved. In many cases, innovations arise from technology transfer and not true invention.

Only recently have technical developments in many separate fields (for example, medical imaging and cell biology) spread across disciplines. There are many more examples of advances in different fields finding applications in optical microscopy. The field of digital image processing was first developed for air and space imaging applications. The field of adaptive optics, now being developed into optical microscopes and medical laser imaging devices, was first developed in the fields of astronomy and military laser weapons. Finally, the emerging medical imaging field of optical low-coherence reflectometry and tomography was first developed for the telecommunications industry as devices for checking fiber optics and integrated optical devices.

The biomedical applications of optical microscopy constitute an emerging field driven by spectacular advances in the field of in vivo microscopy. Advances in confocal microscopy are providing new and important technical solutions in the fields of endoscopy, minimally invasive surgery, dermatology, and ophthalmology. New technical advances in the fields of neurobiology and developmental biology build on the instruments described in this book. In many cases, the solutions to these problems required the optimization of one or more other solutions; typically, designs compromised one or more parameters (resolution, contrast, time for image acquisition) to serve a specific purpose. Optical microscopy began with the observation of living specimens, and recently there has been a revolution to return to the observation of in vivo specimens. The combination of spectroscopic techniques and optical microscopy has resulted in important advances in the field of "optical biopsy." Furthermore, these promising new diagnostic techniques are transitioning from the laboratory to the clinic.

There is an advantage to staying aware of the theoretical and technical advances of disparate fields of science. Such awareness may prove to be useful in the development of techniques that seem far from the problem at hand. Being aware of current and interesting problems in the biomedical area as well as advances in modern techniques of imaging, signal processing, nanotechnology, and integrated optics creates the conditions for success in interdisciplinary research. I hope the reader will find these themes useful for stimulating developments in new instrumentation as well as innovative, clinically useful applications in the exciting field of optical microscopy.

Since many excellent books, courses, and Internet resources are available that describe many aspects of modern microscopy, is there anything new to write on this subject? I delayed the completion of this book over a period of years while I attempted to answer this question myself. I believe the answer is a definite yes. In this book, I present several new ways of approaching these two topics. First, I present the recent developments as partial solutions to existing long-term problems. Second, I show thatmany developments are advances on previous instruments and techniques; there was an intellectual lineage in the development of modern microscopes. Third, I connect the developments of unique types of microscopes in disparate fields of science and medicine, and demonstrate their similarities. Fourth, I indicate the problems, limitations, artifacts, and experimental difficulties with modern microscopes. And fifth, I describe the techniques that use optical microscopes for studies on living tissue and organs and explain why the new types of microscopes are emerging as important clinical tools for medical diagnostics. In vivo microscopy and optical biopsy are active fields of research. This is evident from the exciting research in developmentalbiology, ophthalmology, dermatology, oncology, and brain imaging.

Another unique feature of this text is the discussion of the historical developments of optical microscopy and the technology's critical impact on the fields of biology and medicine. The reader may ask why this is necessary for an understanding of the modern instruments and their applications. There are several reasons. First, an appreciation of the chain of invention may serve to correct some incorrect attributions of priority and rediscovery of previous inventions. Second, an understanding of the historical development of both instruments and techniques has an important educational value in demonstrating serial and parallel approaches to problem solving in opticalmicroscopy. Third, the study of the antecedents to various technical developments can put each invention and advance in its proper perspective and perhaps stimulate innovation. So many excellent books focus on applications that I decided it would be redundant to present extensive reviews of applications. Application notes are available online from the companies that manufacture confocal and multiphoton microscopes.

The projected audience for this text includes those who wish to gain insight into confocal microscopy and multiphoton excitation microscopy, and who intend to apply these techniques to biology and medicine. Therefore, it cannot be stated that this book was written for a single group of individuals. The projected audience includes undergraduate students who seek a global insight into the field of modern optical microscopy, graduate and postdoctoral students who will work with these instruments, and physicians who work with engineers and scientists to design and develop new, noninvasive, diagnostic instruments based on confocal or multiphoton excitation microscopy.

Optical microscopy is a nexus of theory, techniques, and devices from a wide variety of sources and disciplines, and the organization of this book reflects this fact. The text is divided into four parts. The largest part is devoted to confocal microscopy, with an introduction and a part devoted to multiphoton excitation microscopy. The emphasis is not on the main types of optical microscopes, but on how various technical developments served to solve the common problems of optical microscopy. However, throughout the book there are common themes, connections, and technical solutions to the problems of light microscopy that necessitated the deliberate repetition of some central concepts and ideas.

Each chapter of this text begins by introducing the materials to be covered and explaining their role in the book. A summary of key points at the end of each chapter reinforces those critical points. Hopefully the text contains everything that is essential and excludes those topics and details that are not critical for an understanding of the principles and their applications in microscopy. Further insight into the theory and practice of optical microscopy may be garnered by perusal of the printed and electronic resources that I have recommended in the appendix.

Part I covers the background, significance, and principles of the optical microscope. Chapter 1 presents a history of the microscope and the development of fluorescent microscopy, and describes the role of microscopy in the advancement of biology and medicine. Chapter 2 introduces the reader to the optical microscope by describing its chief components and limitations. Chapter 3 describes the contributions of Abbe, including the Abbe theory of image formation in an optical microscope. Chapter 4 discusses optical resolution in a microscope. When the major problems of optical resolution and optical aberrations were solved, the new primary problems concerned the development of techniques to provide contrast. These techniques resulted in the emergence of live cell imaging in optical microscopy. Chapter 5 explains both the nonoptical and optical techniques (phase contrast and differential interference contrast microscopy) that provide contrast.

Part II describes the partial solutions to the following problem: how to image thick, highly scattering specimens with an optical microscope. The invention of the confocal microscope, with its many technical variants, provided one partial solution. Confocal microscopy improves the resolution, contrast, and optical sectioning capability of the light microscope. The connecting theme in Part II is that a variety of techniques were invented and reinvented to solve the same problem: how to construct an optical microscope that has depth discrimination, and thus provide a microscope with the capability to �optically section� thick, scattering specimens. Chapter 6 formulates this problem and then describes several early antecedents to the development of confocal microscopy. Chapter 7 analyzes the myriad solutions to the problem of depth discrimination: the various types of confocal microscopes and their limitations.

Chapter 8 describes the development of scanning-slit confocal microscopes, which were developed in disparate fields: ophthalmology, neurobiology, and cell biology. Chapter 8 also plays a special pedagogical role in this book. While superficially it may seem that the theme is of interest only to ophthalmologists because the applications are predominantly imaging of the in vivo eye, there is a much deeper motivation to include these topics. This chapter demonstrates the linkages, connections, and technology transfers from numerous sources in the progression of technological development of the confocal microscope. For example, the inventions of Baer were motivated by the desire to develop a confocal microscope for cell biology, and the inventions of Svishchev were motivated by the desire to develop a confocal microscope to study neurobiology.

The primary message contained in Chapter 8 is that technical problems are solved by building on the previous and parallel work of others. The insights exposed in this chapterwere derived not only from reading the published papers and patent literature, but also from personal conversations with Maurice, Svishchev, Petran, Hadravsky, Baer, Koester, Kino, and Thaer. I also gained insight from working in the laboratory with Kino, Maurice, and Thaer. This chapter also provides an important lesson: teachers should teach not only those techniques that are popular; they must have a larger objective to teach how to solve problems by devising creative solutions. Many of the technical advances developed in Chapter 8 have found their applications in modern biomedical confocal instruments: scanning-slit confocal microscopes to investigate the cochlea, study in vivo human skin, and study the normal and pathological eye. Confocal microscopes based on slits are also being developed to image large embryos and study their development.

Chapter 9 describes the components of a confocal microscope. Even with the invention and development of the many types of confocal microscopes, problems remain. First, the ultraviolet excitation light used to excite many fluorescent dyes in molecular biology, ion indicator dyes, and endogenous molecules such as NAD(P)H and neurotransmitters, with absorption bands in the ultraviolet, is toxic to live cells, tissues, and organisms. Second, the depth of penetration of thick, scattering specimens is a few hundred microns and therefore precludes the imaging of thicker specimens. Third, the highly intense visible and short wavelength light causes photobleaching of the specimens during observation. The partial solution to these problems came with the invention and development of nonlinear microscopy. Part III describes nonlinear optical microscopy with an emphasis on multiphoton excitation microscopy. Chapter 10 presents the development of nonlinear spectroscopy and microscopy - in particular, the seminal role played by the invention of the laser. Chapter 11 presents a detailed description of multiphoton excitation microscopy, from the Goppert-Mayer theory (Maria Goppert, 1929) to the Denk, Strickler, and Webb 1990 Science publication. Chapter 12 summarizes the theory behind and describes the instrumentation of multiphoton excitation microscopy.

Part IV discusses the path to imaging live cells, tissues, and organs. Chapter 13 sets out the remaining problems and describes the limitations of nonlinear microscopy. Chapter 14 presents future directions for confocal and multiphoton excitation microscopy. Chapter 15 addresses the important topic of laser safety and includes a section on how to clean optics. An epilogue discusses humans as tool makers and tool users.

The book concludes with an appendix containing an annotated listing of carefully selected reference materials and resources. They present applications in great detail as well as experimental protocols. The appendix also contains a partial listing of the author's publications in ophthalmology and dermatology that illustrate the benefits of confocal and multiphoton microscopy in clinical medicine.

This book tells the story of the development of solutions to formidable problems in optical microscopy. It also tells the story of the limitations of optical microscopy: optical aberrations, optical artifacts, fundamental physical limitations of signal and noise, the quantum nature of light, stray light, background fluorescence, and light damage to the specimen. The information in this book will be an ongoing story - microscope development continues as an active field of progress toward the partial solution of the following problems: resolution, contrast, and optical microscopy of live cells, tissues, and organisms with minimal toxic and destructive effects. There is much work to be done, as we have only partial solutions to these problems. The state of the art is a moving target.

Finally, I gladly thank Margaret Thayer and Sharon Streams of SPIE for their help with the manuscript.

Confocally yours,

Barry R. Masters
November 2005

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