Cell Biological Applications of Confocal Microscopy (eBook)

Brian Matsumoto (Herausgeber)

eBook Download: PDF | EPUB

2003 | 2. Auflage
507 Seiten
Elsevier Science (Verlag)
978-0-08-049658-0 (ISBN)

Infos

This volume of the acclaimed Methods in Cell Biology series provides specific examples of applications of confocal microscopy to cell biological problems. It is an essential guide for students and scientists in cell biology, neuroscience, and many other areas of biological and biomedical research, as well as research directors and technical staff of microscopy and imaging facilities.

An integrated and up-to-date coverage on the many various techniques and uses of the confocal microscope (CM).

Key Features
* Includes detailed protocols accessible to new users
* Details how to set up and run a 'Confocal Microscope Core Facility'
* Contains over 170 figures

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This volume of the acclaimed Methods in Cell Biology series provides specific examples of applications of confocal microscopy to cell biological problems. It is an essential guide for students and scientists in cell biology, neuroscience, and many other areas of biological and biomedical research, as well as research directors and technical staff of microscopy and imaging facilities. An integrated and up-to-date coverage on the many various techniques and uses of the confocal microscope (CM). Includes detailed protocols accessible to new users Details how to set up and run a "e;Confocal Microscope Core Facility"e; Contains over 170 figures

Inhalt

Cover 1
Contents 6
Contributors 10
Preface 12
Chapter 1. Introduction to Confocal Microscopy 14
I. Introduction 15
II. Principle of Confocal Microscopy 20
III. Major Components of a Confocal microscope 27
IV. Designs of Confocal Microscopes 36
V. Choosing and Setting Up a Confocal Microscope System 62
VI. Specimen Preparation 65
VII. Applications of Confocal Microscopy 65
VIII. Alternatives to Confocal Microscopy 81
IX. Conclusions and Future Directions 86
X. Resources for Confocal Microscopy 87
References 94
Chapter 2. Direct-View High-Speed Confocal Scanner: The CSU-10 100
I. Introduction 101
II. Overview of Confocal Microscopes 103
III. Design of the CSU-10 104
IV. Sample Biological Applications 106
V. Low Bleaching of Fluorescence Observed with the CSU-10 114
VI. Very-High-Speed Full-Frame Confocal Imaging with the CSU-10 117
VII. Haze Removal, Image Sharpening, and Dynamic Stereo Image Generation by Digital Signal Processing 118
VIII. Mechanical and Optical Performance of the CSU-10 130
IX. Potentials of the CSU-10 131
X. Addendum A (April, 2002) 135
XI. Addendum B (personal communication from Dr. Kenneth R. Spring, June, 2002) 137
References 138
Chapter 3. Introduction to Multiphoton Excitation Imaging for the Biological Sciences 142
I. Introduction 142
II. Principles of Multiphoton Imaging 144
III. Advantages of Multiphoton Imaging 146
IV. Imaging Parameters 150
V. Applications of Multiphoton Imaging 154
VI. New Development in Nonlinear Optics 157
VII. Conclusion 158
References 158
Chapter 4. Confocal Microscopy: Important Considerations for Accurate Imaging 162
I. Introduction 162
II. Factors Affecting Confocal Imaging 163
III. Conclusions 176
References 177
Chapter 5. Multicolor Laser Scanning Confocal Immunofluorescence Microscopy: Practical Application and Limitations 178
I. Introduction 179
II. Immunofluorescence Histochemistry 180
III. Instrumentation 198
IV. Approaches to Multicolor Laser Scanning Confocal Microscopy 204
V. Practical Aspects of Multicolor Laser Scanning Confocal Microscopy 220
VI. Multicolor Laser Scanning Confocal Microscopy Considerations 246
VII. Conclusions 253
References 254
Chapter 6. Practical Aspects of Objective Lens Selection for Confocal and Multiphoton Digital Imaging Techniques 258
I. Introduction 260
II. Confocal Microscopy Resolution 262
III. Infinity versus Finite Lens Systems 267
IV. Objective Lens Nomenclature and Features 269
V. Objective Lens Aberrations 274
VI. Dry (Nonimmersion) and Immersion Objective Lenses 276
VII. Epi (Reflected Light) Objectives 279
VIII. Live Cell and Fixed Tissue Confocal Applications and Objective Lens Selection 281
IX. Possible Solutions for Potential Problematic Applications 291
X. Optimum Objective Lenses for Multiphoton Applications 301
XI. Transmission Data for Confocal and Multiphoton Objectives 302
XII. Conclusions 303
References 306
Chapter 7. Resolution of Subcellular Detail in Thick Tissue Sections: Immunohistochemical Preparation and Fluorescence Confocal Microscopy 314
I. Introduction: Development of Fluorescence Microscopy Techniques for Resolution of Subcellular Detail 315
II. Immunohistochemical Preparation of Thick Tissue Sections 321
III. Confocal Imaging Parameters for Maximum Resolution 334
IV. Applications 339
V. Appendix: Procedures for Immunohistochemical Preparation 342
References 345
Chapter 8. Confocal Fluorescence Microscopy for Tetrahymena thermophila 350
I. Introduction 351
II. Indirect Immunofluorescence and Confocal Microscopy 351
III. Fluorescence in Situ Hybridization Detection of Tetrahymena thermophila rDNA for Confocal Microscopy 357
IV. Live Fluorescence Microscopy 360
References 371
Chapter 9. Confocal Imaging of Drosophila Embryos 374
I. Introduction 374
II. Preparation of the Embryos 376
III. Imaging Embryos 378
IV. Image Presentation 383
V. Multiple Labels 385
VI. Conclusions 388
References 389
Chapter 10. Confocal Fluorescence Microscopy of the Cytoskeleton of Amphibian Oocytes and Embryos 392
I. Introduction 393
II. Fixation of Xenopus Oocytes and Eggs for Confocal Fluorescence Microscopy of the Cytoskeleton 396
III. Processing Xenopus Oocytes and Eggs for Whole-MountŽ Confocal Microscopy 403
IV. An Introduction to Confocal Microscopy and 3-D Reconstruction of the Cytoskeleton of Xenopus Oocytes 410
V. Conclusions 421
VI. Recipes and Reagents 422
VII. Cytoskeletal and Nuclear Probes for Confocal Microscopy of Xenopus Oocytes 423
References 426
Chapter 11. Confocal Fluorescence Microscopy Measurements of pH and Calcium in Living Cells 430
I. Introduction 430
II. Measurement of pHi with BCECF 431
III. Design of a Dual-Excitation Laser Scanning Confocal Fluorescence Microscope for Measuring pHi with BCECF 433
IV. Measurement of pHi in the Cortical Collecting Tubule 435
V. Measurement of Intracellular Ca2+ in the Perfused Afferent Arteriole 436
VI. Future Development 438
References 439
Chapter 12. Confocal and Nonlinear Optical Imaging of Potentiometric Dyes 442
I. Introduction 442
II. Overview of Fluorescent Methods for Measuring Membrane Potential 443
III. Application of Laser Scanning Microscopy to Quantitative Imaging of Membrane Potential 447
IV. Perspectives 461
References 462
Chapter 13. Measurement of Intracellular Ca2+ Concentration 466
I. Introduction 466
II. Ratio Measurement of pCai with Visible-Light [Ca2+] Indicators 468
III. Measurement of Organellar pCai with Genetically Engineered [Ca2+] Indicators 473
References 483
Chapter 14. Running and Setting Up a Confocal Microscope Core Facility 488
I. Introduction 488
II. Planning Your Facility 489
III. Scheduling 490
IV. Training Operators and Users 491
V. Instrument Care 494
VI. Data Storage, Analysis, and Handling 495
VII. Core Issues 496
VIII. Ancillary Services 498
IX. Resources 498
X. Summary 498
Index 500
Volumes in Series 514

Textauszug

Chapter 2

Direct-View High-Speed Confocal Scanner: The CSU-10

Shinya Inoué*; Ted Inoué    Marine Biological Laboratory, Woods Hole, Massachusetts
Universal Imaging Corporation, West Chester, Pennsylvania
*This author is a consultant to Yokogawa Electric Corporation.

I Introduction

Over the past decade, confocal microscopes have dramatically improved our ability to examine the structural and functional detail of biological tissues and cells. The same characteristics that made these advances possible, namely the ability of confocal microscopes to provide exceptionally clean serial optical sections that are free of out-of-focus flare, have also made it possible to directly generate tilted sections, as well as to generate striking three-dimensional (3-D) images.

Alternative methods, such as the application of computationally intensive deconvolution algorithms to serial sections (obtained with nonconfocal, or wide-field, fluorescence microscopes) can also yield clean optical sections and reconstructed 3-D images (e.g., Agard, 1984; Holmes et al., 1995).

More recently, two- (and multi-) photon optics have been used to further improve optical sectioning, especially for deep, living tissues. With only the fluorophores lying in the focal plane being exposed to the coincident action of the double- (multiple-) wavelength excitation wave, fluorescence is strictly limited to that plane. Meanwhile, the longer wavelength excitation wave dramatically reduces light scattering in general and photobleaching of the fluorophore that lie outside of the focal plane.

Although many of these confocal and deconvolution approaches yield exquisite optical sections and their composites, it may take from many seconds to considerably longer for each two-dimensional image covering a reasonable area to become available.

The CSU-10 described in this chapter is a disk-scanning, direct-view confocal scanning unit.1 As detailed in the next section, the CSU-10 incorporates, in addition to the main Nipkow disk, a second disk with some 20,000 microlenses that are each aligned with a corresponding pinhole on the main Nipkow disk, thus substantially improving the transmission of the confocal illuminating beam. Thus, one can view confocal fluorescence images in real time, i.e., at video rate or faster, through the eyepiece or captured through a video camera.

The compact unit, attached to an upright or inverted microscope, transforms a research microscope into an exceptionally easy-to-use and effective, direct-view epifluorescence confocal microscope (Fig. 1). Thus, optical sections of fluorescent specimens show with high resolution, and in true color, directly through the ocular as one focuses through the specimen. Sparsely distributed, weakly fluorescent objects are readily brought to view.

Fig. 1 The CSU-10 confocal scanning unit attached to the C-mount video port on top of an upright microscope (Nikon E-800). The scanner can also be attached to the video port of an inverted microscope. Illumination is conducted through a polarization-maintaining optical fiber from the laser source (not shown). (Figure courtesy of Yokogawa Electric Corporation.)

In addition to viewing the image through the ocular in real time, the confocal image generated by the CSU-10 can be directly captured by a photographic camera or a video or CCD camera attached to the C-mount on top of the unit.

Since images of the microscope field are scanned at 360 frames/s by the multiple arrays of pinholes on its Nipkow disk, the CSU-10 not only provides very clean, full-frame video-rate images but, by use of high-speed intensified cameras, can even capture frames in as short an interval as 3 ms or less.

In this paper prepared in 1999, we describe the basic design of the CSU-10; some sample applications including video-rate and higher speed confocal imaging; the low rate of fluorescence bleaching observed using the CSU-10; rapid digital processing for further haze removal and image sharpening and for generation of dynamic stereo images; mechanical and optical performance of the CSU-10; and potential future applications. Addendum A provides some recent updates.

II Overview of Confocal Microscopes

In a point-scanning confocal microscope, the specimen is illuminated through a well-corrected objective lens by an intense, reduced image of a point source that is located in the image plane (or its conjugate) of the objective lens. Light emitted by the focused point on the specimen traces the light path back through the objective lens and (after deviation through a dichromatic mirror) progresses to an exit pinhole. The small exit pinhole, which is also placed in the image plane of the objective lens, effectively excludes light emanating from planes in the specimen other than those in focus. Thus, confocal imaging selectively collects signals from the focused spot in the specimen with dramatic reduction of signals from out-of-focus planes.

To achieve an image of the specimen with a point-scanning confocal system, either the specimen itself is moved in a raster pattern or the confocal spot and exit pinhole together are made to scan the specimen. In other words, either the specimen is precisely raster scanned in the x–y plane, or the illuminating and return imaging beam are made to raster scan a stationary specimen by tilting these beams at the aperture plane of the objective lens (commonly with galvanometer-driven mirrors) (see, e.g., this volume, Chapter 1, and Pawley, 1995). The very rapidly changing intensity of light passing the exit pinhole is detected by a photomultiplier tube and captured in a digital frame buffer. The output of the frame buffer, the confocal image, is displayed on a computer monitor.

For a number of reasons, it generally requires a few seconds to generate a low-noise, full-screen confocal fluorescent image with a point-scanning confocal system (see, e.g., Amos and White, 1995). One way of overcoming this speed limitation is to use many points to scan the specimen in parallel, such as by the use of a Nipkow disk.

In Nipkow-disk-type confocal microscopes, the disk with multiple sets of spirally arranged pinholes is placed in the image plane of the objective lens. The pinholes are illuminated from the rear, and their highly reduced images are focused by the objective lens onto the specimen. As the Nipkow disk spins, the specimen is thus raster scanned by successive sets of reduced images of the pinholes.

The light emitted by each illuminated point on the specimen is focused back, by the same objective lens, onto a corresponding pinhole on the Nipkow disk. This exit pinhole may be the same pinhole that provided the scanning spot, as in the Kino-type confocal system (Kino, 1995), or may be one located on the diametrically opposite side of the Nipkow disk as in the Petráň-type confocal microscope (Petráň et al., 1968; see this volume, Chapter 1, Figs. 8 and 9).

With any Nipkow-type confocal system, one needs to maintain a moderately large separation between adjacent pinholes relative to their diameters in order to minimize crosstalk (i.e., leakage) of the return beam through neighboring pinholes. On the other hand, with the pinholes separated by, say, 10 times their diameter, only 1% of the incoming beam is transmitted by the Nipkow disk since the pinholes occupy only that fraction of the disk area. Nipkow disk systems, therefore, generally tend to suffer from low levels of transmission of the beam that illuminates the specimen.

In addition, a major fraction of the illuminating beam can be backscattered by the Nipkow disk and contribute to unwanted background light in the Kino-type arrangement. The Kino system thus includes several design features to minimize this source of unwanted light (see, e.g., Kino, 1995). The Petráň, tandem-scanning-type does not suffer from backscatter of the illuminating beam but does require very high precision of pin-hole placement on the Nipkow disk as well as stability of rotational axis since the entrance and exit pinholes are located on opposite sides of the axis of rotation of the disk.

III Design of the CSU-10

In order to overcome these difficulties encountered with the past Nipkow disk systems, the Yokogawa CSU-10 uses the same pinholes for the entrance and exit beams but is equipped with a second, coaxially aligned Nipkow disk that contains some 20,000 microlenses. Each microlens is precisely aligned with its corresponding pinhole on the main Nipkow disk onto which the illuminating beam is focused. Thus, instead of the 1% or so found with conventional Nipkow disk systems, some 40% to 60% of the light impinging on the disk containing the microlenses becomes transmitted through the pinholes to illuminate the specimen (Fig. 2).

Fig. 2 Schematic of optics in the CSU-10. The expanded and collimated laser beam illuminates the upper Nipkow disk containing some 20,000 microlenses. Each microlens focuses the laser beam onto its corresponding pinhole, thus significantly raising the fraction of the illuminating beam that is transmitted by the main Nipkow disk containing the pinhole array. From the pinholes, the beams progress down to fill the aperture of the objective lens. The objective lens generates a reduced image of the pinholes into the specimen focal plane. Fluorescence given off by the illuminated regions in...

Produkt-Details

• Erscheint lt. Verlag: 4.1.2003
• Sprache: englisch
• Themenwelt: Naturwissenschaften ► Biologie ► Genetik / Molekularbiologie
• Themenwelt: Naturwissenschaften ► Biologie ► Zellbiologie
• Themenwelt: Naturwissenschaften ► Physik / Astronomie ► Angewandte Physik
• Themenwelt: Technik
• ISBN-10: 0-08-049658-X / 008049658X
• ISBN-13: 978-0-08-049658-0 / 9780080496580