E-Book, Englisch, Band Volume 72, 544 Seiten
Reihe: Methods in Cell Biology
Sluder Digital Microscopy
2. Auflage 2003
ISBN: 978-0-08-054603-2
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: 6 - ePub Watermark
A second edition of "Video Microscopy"
E-Book, Englisch, Band Volume 72, 544 Seiten
Reihe: Methods in Cell Biology
ISBN: 978-0-08-054603-2
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: 6 - ePub Watermark
This updated second edition of the popular methods book 'Video Microscopy' shows how to track dynamic changes in the structure or architecture of living cells and in reconstituted preparations using video and digital imaging microscopy. Contains 10 new chapters addressing developments over the last several years. Basic information, principles, applications, and equipment are covered in the first half of the volume and more spcialized video microscopy techniques are covered in the second half. - Shows how to track dynamic changes in the structure or architecture of living cells and in reconstituted preparations using video and digital imaging microscopy - Contains 10 new chapters addressing developments over the last several years - Covers basic principles, applications, and equipment - Spcialized video microscopy techniques are covered
Autoren/Hrsg.
Weitere Infos & Material
1;Front Cover;1
2;Methods in Cell Biology;4
3;Copyright Page;5
4;Contents;6
5;Contributors;14
6;Preface;16
7;Chapter 1. Microscope Basics;20
7.1;I. Introduction;20
7.2;II. How Microscopes Work;21
7.3;III. Objective Basics;25
7.4;IV. Mounting Video Cameras on the Microscope;27
7.5;References;29
8;Chapter 2. Optics of The Microscope Image Formation;30
8.1;I. Introduction;31
8.2;II. Physical Optics—The Superposition of Waves;31
8.3;III. Huygens’ Principle;33
8.4;IV. Young’s Experiment—Two Slit Interference;34
8.5;V. Diffraction from Single Slit;37
8.6;VI. The Airy Disk and the Issue of Microscope Resolution;38
8.7;VII. Fourier or Reciprocal Space—The Concept of Spatial Frequencies;42
8.8;VIII. Resolution of the Microscope;46
8.9;IX. Resolution and Contrast;47
8.10;X. Conclusions;49
8.11;XI. Appendix I;50
8.12;XII. Appendix II;55
8.13;XIII. Appendix III;59
8.14;References;61
9;Chapter 3. Proper Alignment of the Microscope;64
9.1;I. Key Components of Every Light Microscope;65
9.2;II. Koehler Illumination;69
10;Chapter 4. Mating Cameras to Microscopes;76
10.1;I. Introduction;76
10.2;II. Optical Considerations;81
11;Chapter 5. ‘‘Do Not (Mis-) Adjust Your Set’’: Maintaining Specimen Detail in the Video Microscope;84
11.1;I. Introduction;84
11.2;II. The Black and White Video Signal;85
11.3;III. Adjusting the Camera and Video Monitor;87
11.4;IV. Practical Aspects of Coordinately Adjusting Camera and Monitor;97
11.5;V. Digital Imaging;100
11.6;References;104
12;Chapter 6. Cameras for Digital Microscopy;106
12.1;I. Overview;106
12.2;II. Basic Principles;107
12.3;III. Application of CCD Cameras in Fluorescence Microscopy;119
12.4;IV. Future Developments in Imaging Detectors;121
12.5;References;121
13;Chapter 7. Electronic Cameras for Low-Light Microscopy;122
13.1;I. Introduction;123
13.2;II. Parameters Characterizing Imaging Devices;126
13.3;III. Specific Imaging Detectors and Features;139
13.4;IV. Conclusions;149
13.5;References;150
14;Chapter 8. Cooled vs. Intensified vs. Electron Bombardment CCD Cameras—Applications and Relative Advantages;152
14.1;I. Introduction;153
14.2;II. Sensitivity;155
14.3;III. Dynamic Range (DR) and Detectable Signal (DS) Change;167
14.4;IV. Spatial Resolution Limits;170
14.5;V. Temporal Resolution;171
14.6;VI. Geometric Distortion;172
14.7;VII. Shading;172
14.8;VIII. Usability;172
14.9;IX. Advanced Technology;174
15;Chapter 9. Fundamentals of Fluorescence and Fluorescence Microscopy;176
15.1;I. Introduction;177
15.2;II. Light Absorption and Beer’s Law;177
15.3;III. Atomic Fluorescence;179
15.4;IV. Organic Molecular Fluorescence;181
15.5;V. Excited-State Lifetime and Fluorescence Quantum Efficiency;183
15.6;VI. Excited-State Saturation;184
15.7;VII. Nonradiative Decay Mechanisms;184
15.8;VIII. Fluorescence Resonance Energy;185
15.9;IX. Fluorescence Depolarization;187
15.10;X. Measuring Fluorescence in the Steady State;188
15.11;XI. Construction of a Monochromator;190
15.12;XII. Construction of a Photomultiplier Tube;190
15.13;XIII. Measuring Fluorescence in the Time Domain;191
15.14;XIV. Filters for the Selection of Wavelength;199
15.15;XV. The Fluorescence Microscope;201
15.16;XVI. The Power of Fluorescence Microscopy;202
15.17;References;203
16;Chapter 10. A High-Resolution Multimode Digital Microscope System;204
16.1;I. Introduction;205
16.2;II. Design Criteria;206
16.3;III. Microscopy Design;210
16.4;IV. Cooled CCD Camera;218
16.5;V. Digital Imaging System;226
16.6;VI. Example Applications;228
16.7;References;234
17;Chapter 11. Fundamentals of Image Processing in Light Microscopy;236
17.1;I. Introduction;236
17.2;II. Digitization of Images;237
17.3;III. Using Gray Values to Quantify Intensity in the Microscope;239
17.4;IV. Noise Reduction;240
17.5;V. Contrast Enhancement;244
17.6;VI. Transforms, Convolutions, and Further Uses for Digital Masks;248
17.7;VII. Conclusions;260
17.8;References;260
18;Chapter 12. Techniques for Optimizing Microscopy and Analysis through Digital Image Processing;262
18.1;I. Fundamentals of Biological Image Processing;262
18.2;II. Analog and Digital Processing in Image Processing and Analysis;268
18.3;III. Under the Hood—How an Image Processor Works;272
18.4;IV. Acquiring and Analyzing Images—Photography Goes Digital;281
18.5;References;289
19;Chapter 13. The Use and Manipulation of Digital Image Files in Light Microscopy;290
19.1;I. introduction;290
19.2;II. What is an Image File?;291
19.3;III. Sampling and Resolution;293
19.4;IV. Bit Depth;295
19.5;V. File Formats;296
19.6;VI. Color;297
19.7;VII. Converting RGB to CMYK;299
19.8;VIII. Compression;301
19.9;IX. Video Files;302
19.10;X. Video CODECs;303
19.11;XI. Choosing a Compression/Decompression Routine (CODEC);303
19.12;XII. Conclusions;306
19.13;References;307
20;Chapter 14. High-Resolution Video-Enhanced Differential Interference Contrast Light Microscopy;308
20.1;I. Introduction;309
20.2;II. Basics of DIC Image Formation and Microscope Alignment;311
20.3;III. Basics of Video-Enhanced Contrast;316
20.4;IV. Selection of Microscope and Video Components;320
20.5;V. Test Specimens for Microscope and Video Performance;333
20.6;References;336
21;Chapter 15. Quantitative Digital and Video Microscopy;338
21.1;I. What Is an Image?;339
21.2;II. What Kind of Quantitative Information Do You Want?;340
21.3;III. Applications Requiring Spatial Corrections;340
21.4;IV. Two-Camera and Two-Color Imaging;346
21.5;V. A Warning About Transformations—Don’t Transform Away What You Are Trying to Measure!;346
21.6;VI. The Point-Spread Function;347
21.7;VII. Positional Information beyond the Resolution Limit of the Microscope;348
21.8;VIII. Intensity Changes with Time;354
21.9;IX. Summary;354
21.10;References;354
22;Chapter 16. Computational Restoration of Fluorescence Images: Noise Reduction, Deconvolution, and Pattern Recognition;356
22.1;I. Introduction;356
22.2;II. Adaptive Noise Filtration;357
22.3;III. Deconvolution;360
22.4;IV. Pattern Recognition-Based Image Restoration;363
22.5;V. Prospectus;366
22.6;References;366
23;Chapter 17. Quantitative Fluorescence Microscopy and Image Deconvolution;368
23.1;I. Introduction;369
23.2;II. Quantitative Imaging of Biological Samples Using Fluorescence Microscopy;369
23.3;III. Image Blurring in Biological Samples;377
23.4;IV. Applications for Image Deconvolution;383
23.5;V. Concluding Remarks;384
23.6;References;384
24;Chapter 18. Ratio Imaging: Measuring Intracellular Ca++ and pH in Living Cells;388
24.1;I. Introduction;388
24.2;II. Why Ratio Imaging?;389
24.3;III. Properties of the Indicators BCECF and Fura-2;390
24.4;IV. Calibration of the Fluorescence Signal;396
24.5;V. Components of an Imaging Workstation;402
24.6;VI. Experimental Chamber and Perfusion System—A Simple Approach;404
24.7;VII. Conclusion;405
24.8;References;406
25;Chapter 19. Ratio Imaging Instrumentation;408
25.1;I. Introduction;409
25.2;II. Choosing an Instrument for Fluorescence Measurements;411
25.3;III. Different Methodological Approaches to Ratio Imaging;414
25.4;IV. Optical Considerations for Ratio Microscopy;419
25.5;V. Illumination and Emission Control;424
25.6;VI. Detector Systems;425
25.7;VII. Digital Image Processing;428
25.8;VIII. Summary;429
25.9;References;429
26;Chapter 20. Fluorescence Resonance Energy Transfer Microscopy: Theory and Instrumentation;434
26.1;I. Introduction;434
26.2;II. Principles and Basic Methods of FRET;435
26.3;III. FRET Microscopy;441
26.4;IV. Conclusions;448
26.5;References;449
27;Chapter 21. Fluorescence-Lifetime Imaging Techniques for Microscopy;450
27.1;I. Introduction;451
27.2;II. Time-Resolved Fluorescence Methods;453
27.3;III. Fluorescence-Lifetime-Resolved Camera;460
27.4;IV. Two-Photon Fluorescence Lifetime Microscopy;466
27.5;V. Pump-Probe Microscopy;472
27.6;VI. Conclusion;479
27.7;References;481
28;Chapter 22. Fluorescence Correlation Spectroscopy: Molecular Complexing in Solution and in Living Cells;484
28.1;I. Introduction;485
28.2;II. Studying Biological Systems with FCS;485
28.3;III. Designing and Building an FCS Instrument;503
28.4;IV. What are the Current Commercial Sources of FCS?;514
28.5;V. Summary;514
28.6;References;515
29;Index;518
30;Volume in Series;536
31;Color Plate Section;543
Preface In our introduction to Video Microscopy, the forerunner to this volume, we commented upon the marvelous images then coming in from the Mars Pathfinder in the summer of 1997: “The opening of a previously inaccessible world to our view is what we have come to expect from video imaging. In the past three decades, video imaging has taken us to the outer planets, to the edge of the universe, to the bottom of the ocean, into the depths of the human body, and to the interior of living cells. It can reveal not only the forces that created and altered other planets but also the forces of life on earth.’ This continues to reflect what must be the ever sanguine view of scientists about their world, their work, and the use of technology to promote the pursuit of pure knowledge. That said, we recognize that the world has changed enormously in the intervening six years. Our choice of the title Digital Microscopy, as opposed to the earlier volume's Video Microscopy, reflects the profound change that the past six years have brought. For the most part, true video detectors are gone from the arsenal of microscopy and are replaced by a new generation of digital cameras. This is more than a vogue; it represents how microscopy, by becoming more and more specialized, is constantly evolving in the nature of the information that it can provide us. This book follows a similar organization of material to that of Video Microscopy, with the notable caveat that we now focus on issues related to digital microscopy. The organization of this book is loosely tripartite. The first group of chapters presents some of the optical fundamentals needed to provide a quality image to the digital camera. Specifically, it covers the fundamental geometric optics of finite- and infinite-corrected microscopes, develops the concepts of physical optics and Abbe's theory of the microscope, presents the principles of Kohler illumination, and finally reviews the fundamentals of fluorescence and fluorescence microscopy. The second group of chapters deals with digital and video fundamentals: how digital and video cameras work, how to coordinate cameras with microscopes, how to deal with digital data, the fundamentals of image processing, and low light level cameras. The third group of chapters address some specialized areas of microscopy. Since quantitative microscopy is at the heart of these topics, we begin with a discussion of some critical issues of quantitative microscopy and then move on to ratio imaging of cellular analytes, fluorescence resonance energy transfer, high-resolution differential interference microscopy, lifetime imaging, and fluorescence correlation spectroscopy. It is in this last category that the field of microscopy is now most dynamic. We have chosen here to confine our discussion to wide-field microscopy. This is because the area of confocal microscopy merits a volume onto itself and, indeed, has been covered extensively elsewhere. That said, our discussions of the critical issues involved in quantitative microscopy are directly applicable and extremely germane to laser scanning confocal microscopy. As we put the final touches on this book, we have just completed the twenty-third Analytical and Quantitative Light Microscopy (AQLM) course, started by Shinya Inoué, which we now direct at the Marine Biological Laboratories at Woods Hole, Massachusetts. Beyond a doubt, this book is a direct outgrowth of AQLM and differences between this book and its forerunner, Video Microscopy, published in 1998, reflect not only changes in the field, but changes in the course as well. In beginning to write and to involve (or was it to coerce?) others to help write this book, we were keenly aware that we were fast approaching the twenty-fifth anniversary of AQLM. We therefore asked Shinya Inoue to comment on the first AQLM course: On April 27–May 3, 1980, at the suggestion of Mort Maser, Associate Director in charge of education and research at the Marine Biological Laboratory, we offered the first course on “Analytical and Quantitative Light Microscopy” (AQLM). I was joined by Gordon Ellis and Ted Salmon from the University of Pennsylvania Program in Biophysical Cytology, by Lance Taylor, a past student with Bob Allen and then at Harvard, and by several others, including commercial faculty that Mort Maser and I had recruited from Zeiss, Leitz, Nikon, Olympus, Eastman Kodak, Hamamatsu Photonics, Colorado Video, Venus Scientific, Crimson Tech, and Sony. These manufacturers also provided the needed equipment, supplies, and technical personnel as they had for Bob's course. For the early offerings of the AQLM course, we concentrated two days on polarized light microscopy, including the interaction of polarized light with matter, after an introduction to the principles of microscope image formation and the phase contrast principle. We reasoned that, early in the course, we should deal thoroughly with the basic nature of the probe used in light microscopy—the light wave—and how it interacts with the optical components, as well as the electronic structure of the molecules that make up the specimen. Then, after a day and a half exploring further details of image formation and other modes of contrast generation, we spent the final day and a half examining the principles and application of fluorescence microscopy. Throughout the course, interspersed with discussions on microscope optics, image interpretation, and analysis, we reviewed some advanced applications of microscopy in cellular and molecular biology. As has pretty much become the tradition for our course, from 9 a.m. to noon each day we held lectures and demonstrations on the subject matters, with afternoons and evenings mostly devoted to laboratory experiences, sometimes lasting until past midnight. As a major innovation for this course, every pair of students was provided with an up-to-date research microscope together with video and digital processing equipment and received personal attention from both commercial and academic faculty. The intensive, hands-on laboratory gave the participants an opportunity for total immersion, with the students, academic faculty, and commercial representatives all interacting with each other to try out well-tested, as well as never-tried-before, novel ideas and observations. One early finding in both of the MBL microscopy courses was that the video devices that were attached to the microscopes, mainly to allow a large group of participants to watch the events taking place under the microscope, behaved, in fact, incredibly better than had been anticipated. The cameras and monitors would enhance the image contrast beyond expectation, and simple digital processors would allow the unwanted background to be subtracted away, or noise to be integrated so effectively that a totally new world appeared under the microscope. For example, images that were totally invisible or undetectable before, including faint DIC diffraction images of molecular filaments (whose diameters were far below the microscope's limit of resolution) and weakly birefringent or fluorescent minute specimen regions, could now be recorded or displayed on the video monitor with incredible clarity. As has often happened at the MBL courses, the combination of (a) students and faculty with varied backgrounds but a strong, shared interest in making pioneering contributions, (b) total immersion in the course and laboratory experience away from the daily distractions at their home institutions, and (c) hands-on availability of contemporary equipment and specimens (thanks to the generosity of the participating vendors) led to synergy and interactions that opened up new technology and new fields of study. Not only was the new field of video microscopy born at the early MBL microscopy courses, but soon after attending the AQLM course, some of the students began to develop even more powerful approaches, including those that finally allowed the tracking of specific individual molecules directly under the microscope. At the same time, the friendships developed at the courses opened up new channels of communication between users of microscopes, between users and manufacturers, and among manufacturers themselves. In fact, several improvements that we see today in microscopes and their novel uses arose from discussions, interactions, or insights gained in these courses. Owing in part to the success and impact of the MBL microscopy courses, light microscopy has undergone a virtual renaissance in the last two decades. We must, at the outset, express the enormous debt that we and the field of microscopy owe to Shinya Inoué, Lans Taylor, Ted Salmon, and Gordon Ellis. We would be remiss if we did not also mention Bob and Nina Allen's parallel and simultaneous endeavours with the fall course, Optical Microscopy. These people set the standard for AQLM and established its dual essence. It is at once a course for students in the basics of light microscopy and also a meeting for faculty, both academic and commercial, to develop new methods and applications. We owe a tremendous debt to our faculty, many of whom have participated in the course from its beginning and contributed to this volume. Most of all, we owe a tremendous debt to our students, who each year bring this course alive, bring us out of our offices, and rekindle in us the love of microscopy. Together, these people make AQLM what it is—an extremely exciting environment in which to study and explore microscopy. A lot may be said about the genesis of AQLM and, in parallel, about the metamorphosis of the field of optical microscopy. Shinya, we know, laments the fact that the need to cover ever more advanced topics...