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E-raamat: Fluorescence Microscopy: from Principles to Biological Applications [Wiley Online]

  • Formaat: 539 pages
  • Ilmumisaeg: 23-Apr-2013
  • Kirjastus: Wiley-VCH Verlag GmbH
  • ISBN-10: 3527671595
  • ISBN-13: 9783527671595
Teised raamatud teemal:
  • Wiley Online
  • Hind: 163,88 €*
  • * hind, mis tagab piiramatu üheaegsete kasutajate arvuga ligipääsu piiramatuks ajaks
Fluorescence Microscopy: from Principles to Biological ApplicationsSuurem pilt
  • Formaat: 539 pages
  • Ilmumisaeg: 23-Apr-2013
  • Kirjastus: Wiley-VCH Verlag GmbH
  • ISBN-10: 3527671595
  • ISBN-13: 9783527671595
Teised raamatud teemal:
Wiley Online e-raamatudRohkem infot Wiley Online kohta
Raamatu kodulehekülg: https://onlinelibrary.wiley.com/doi/book/10.1002/9783527671595
Physical and biological scientists, and engineers who work with optics, provide a textbook for graduate and advanced undergraduate students of the biological sciences and a reference for researchers on performing quantitative microscopy. Assuming a solid knowledge of physics and mathematics, they explain the theoretical foundations of light microscopy, the large variety of specialized microscopic techniques, and the quantitative utilization of microscopy data. The topics include optics and photophysics, fluorescence labeling, confocal microscopy, single-molecule microscopy in the life sciences, and inference and pattern recognition in super-resolution microscopy. Annotation ©2013 Book News, Inc., Portland, OR (booknews.com)

A comprehensive introduction to advanced fluorescence microscopy methods and their applications.
This is the first title on the topic designed specifically to allow students and researchers
with little background in physics to understand both microscopy basics and novel light microscopy techniques. The book is written by renowned experts and pioneers
in the field with a rather intuitive than formal approach. It always keeps the nonexpert reader in mind, making even unavoidable complex theoretical concepts readily accessible. All commonly used methods are covered.
A companion website with additional references, examples and video material makes
this a valuable teaching resource:
http://www.wiley-vch.de/home/fluorescence_microscopy/
Preface xiii
List of Contributors
xvii
1 Introduction to Optics and Photophysics
1(32)
Rainer Heintzmann
1.1 Interference: Light as a Wave
2(5)
1.2 Two Effects of Interference: Diffraction and Refraction
7(7)
1.3 Optical Elements
14(6)
1.3.1 Lenses
14(3)
1.3.2 Metallic Mirror
17(1)
1.3.3 Dielectric Mirror
18(1)
1.3.4 Pinholes
18(1)
1.3.5 Filters
19(1)
1.3.6 Chromatic Reflectors
20(1)
1.4 The Far-Field, Near-Field, and Evanescent Waves
20(3)
1.5 Optical Aberrations
23(1)
1.6 Physical Background of Fluorescence
24(6)
1.7 Photons, Poisson Statistics, and AntiBunching
30(3)
References
31(2)
2 Principles of Light Microscopy
33(64)
Ulrich Kubitscheck
2.1 Introduction
33(1)
2.2 Construction of Light Microscopes
33(9)
2.2.1 Components of Light Microscopes
33(1)
2.2.2 Imaging Path
34(2)
2.2.3 Magnification
36(2)
2.2.4 Angular and Numerical Aperture
38(1)
2.2.5 Field of View
38(1)
2.2.6 Illumination Beam Path
39(3)
2.3 Wave Optics and Resolution
42(19)
2.3.1 Wave Optical Description of the Imaging Process
43(4)
2.3.2 The Airy Function
47(3)
2.3.3 Point Spread Function and Optical Transfer Function
50(2)
2.3.4 Lateral and Axial Resolution
52(7)
2.3.5 Magnification and Resolution
59(1)
2.3.6 Depth of Field and Depth of Focus
60(1)
2.3.7 Over- and Under Sampling
61(1)
2.4 Apertures, Pupils, and Telecentricity
61(3)
2.5 Microscope Objectives
64(14)
2.5.1 Objective Lens Design
64(4)
2.5.2 Light Collection Efficiency and Image Brightness
68(5)
2.5.3 Objective Lens Classes
73(1)
2.5.4 Immersion Media
73(4)
2.5.5 Special Applications
77(1)
2.6 Contrast
78(16)
2.6.1 Dark Field
80(1)
2.6.2 Phase Contrast
81(5)
2.6.3 Interference Contrast
86(3)
2.6.4 Advanced Topic: Differential Interference Contrast
89(5)
2.7 Summary
94(3)
Acknowledgments
94(1)
References
95(2)
3 Fluorescence Microscopy
97(46)
Jurek W. Dobrucki
3.1 Features of Fluorescence Microscopy
98(5)
3.1.1 Image Contrast
98(3)
3.1.2 Specificity of Fluorescence Labeling
101(1)
3.1.3 Sensitivity of Detection
102(1)
3.2 A Fluorescence Microscope
103(20)
3.2.1 Principle of Operation
103(4)
3.2.2 Sources of Exciting Light
107(3)
3.2.3 Optical Filters in a Fluorescence Microscope
110(1)
3.2.4 Electronic Filters
111(1)
3.2.5 Photodetectors for Fluorescence Microscopy
112(1)
3.2.6 CCD-Charge-Coupled Device
113(3)
3.2.7 Intensified CCD (ICCD)
116(1)
3.2.8 Electron-Multiplying Charge-Coupled Device (EMCCD)
117(2)
3.2.9 CMOS
119(1)
3.2.10 Scientific CMOS (sCMOS)
120(1)
3.2.11 Features of CCD and CMOS Cameras
121(1)
3.2.12 Choosing a Digital Camera for Fluorescence Microscopy
121(1)
3.2.13 Photomultiplier Tube (PMT)
121(1)
3.2.14 Avalanche Photodiode (APD)
122(1)
3.3 Types of Noise in a Digital Microscopy Image
123(4)
3.4 Quantitative Fluorescence Microscopy
127(6)
3.4.1 Measurements of Fluorescence Intensity and Concentration of the Labeled Target
127(3)
3.4.2 Ratiometric Measurements (Ca++, pH)
130(1)
3.4.3 Measurements of Dimensions in 3D Fluorescence Microscopy
131(1)
3.4.4 Measurements of Exciting Light Intensity
132(1)
3.4.5 Technical Tips for Quantitative Fluorescence Microscopy
132(1)
3.5 Limitations of Fluorescence Microscopy
133(6)
3.5.1 Photobleaching
133(1)
3.5.2 Reversible Photobleaching under Oxidizing or Reducing Conditions
134(1)
3.5.3 Phototoxicity
135(1)
3.5.4 Optical Resolution
135(2)
3.5.5 Misrepresentation of Small Objects
137(2)
3.6 Current Avenues of Development
139(4)
References
139(2)
Further Reading
141(1)
Recommended Internet Resources
142(1)
Fluorescent Spectra Database
142(1)
4 Fluorescence Labeling
143(32)
Gerd Ulrich Nienhaus
Karin Nienhaus
4.1 Introduction
143(1)
4.2 Principles of Fluorescence
143(1)
4.3 Key Properties of Fluorescent Labels
144(5)
4.4 Synthetic Fluorophores
149(9)
4.4.1 Organic Dyes
149(1)
4.4.2 Fluorescent Nanoparticles
150(2)
4.4.3 Conjugation Strategies for Synthetic Fluorophores
152(3)
4.4.4 Nonnatural Amino Acids
155(1)
4.4.5 Bringing the Fluorophore to Its Target
156(2)
4.5 Genetically Encoded Labels
158(5)
4.5.1 Phycobiliproteins
158(1)
4.5.2 GFP-Like Proteins
159(4)
4.6 Label Selection for Particular Applications
163(5)
4.6.1 FRET to Monitor Intramolecular Conformational Dynamics
163(4)
4.6.2 Protein Expression in Cells
167(1)
4.6.3 Fluorophores as Sensors inside the Cell
167(1)
4.6.4 Live-Cell Dynamics
168(1)
4.7 Conclusions
168(7)
References
169(6)
5 Confocal Microscopy
175(40)
Nikolaus Naredi-Rainer
Jens Prescher
Achim Hartschuh
Don C. Lamb
5.1 Introduction
175(5)
5.1.1 Evolution and Limits of Conventional Widefield Microscopy
175(2)
5.1.2 History and Development of Confocal Microscopy
177(3)
5.2 The Theory of Confocal Microscopy
180(16)
5.2.1 The Principle of Confocal Microscopy
180(2)
5.2.2 Radial and Axial Resolution and the Impact of the Pinhole Size
182(7)
5.2.3 Scanning Confocal Imaging
189(5)
5.2.4 Confocal Deconvolution
194(2)
5.3 Applications of Confocal Microscopy
196(19)
5.3.1 Nonscanning Applications
196(4)
5.3.2 Advanced Correlation Techniques
200(5)
5.3.3 Scanning Applications Beyond Imaging
205(5)
Acknowledgments
210(1)
References
210(5)
6 Fluorescence Photobleaching and Photoactivation Techniques
215(30)
Reiner Peters
6.1 Introduction
215(1)
6.2 Basic Concepts and Procedures
216(5)
6.2.1 Putting Photobleaching to Work
216(3)
6.2.2 Setting Up an Instrument
219(1)
6.2.3 Approaching Complexity from Bottom Up
220(1)
6.3 Fluorescence Recovery after Photobleaching (FRAP)
221(7)
6.3.1 Evaluation of Diffusion Measurements
222(3)
6.3.2 Binding
225(1)
6.3.3 Membrane Transport
226(2)
6.4 Continuous Fluorescence Microphotolysis (CFM)
228(5)
6.4.1 Theoretical Background and Data Evaluation
229(2)
6.4.2 Combination of CFM with Other Techniques
231(1)
6.4.3 CFM Variants
232(1)
6.5 Confocal Photobleaching
233(5)
6.5.1 Use of Laser Scanning Microscopes (LSMs) in Photobleaching Experiments
233(1)
6.5.2 New Possibilities Provided by Confocal Photobleaching
234(3)
6.5.3 Artifacts and Remedies
237(1)
6.6 Fluorescence Photoactivation and Dissipation
238(3)
6.6.1 Basic Aspects
238(1)
6.6.2 Theory and Instrumentation
239(1)
6.6.3 Reversible Flux Measurements
239(2)
6.7 Summary and Outlook
241(4)
References
241(4)
7 Forster Resonance Energy Transfer and Fluorescence Lifetime Imaging
245(48)
Fred S. Wouters
7.1 General Introduction
245(1)
7.2 FRET
246(19)
7.2.1 Historical Development of FRET
246(8)
7.2.2 Requirements
254(4)
7.2.3 FRET as a Molecular Ruler
258(4)
7.2.4 Special FRET Conditions
262(3)
7.3 Measuring FRET
265(15)
7.3.1 Spectral Changes
266(6)
7.3.2 Decay Kinetics
272(8)
7.4 FLIM
280(5)
7.4.1 Frequency-Domain FLIM
282(1)
7.4.2 Time-Domain FLIM
283(2)
7.5 Analysis and Pitfalls
285(8)
7.5.1 Average Lifetime, Multiple Lifetime Fitting
285(1)
7.5.2 From FRET/Lifetime to Species
286(1)
Summary
287(1)
References
288(5)
8 Single-Molecule Microscopy in the Life Sciences
293(52)
Markus Axmann
Josef Modl
Gerhard J. Schutz
8.1 Encircling the Problem
293(2)
8.2 What Is the Unique Information?
295(6)
8.2.1 Kinetics Can Be Directly Resolved
295(1)
8.2.2 Full Probability Distributions Can Be Measured
296(1)
8.2.3 Structures Can Be Related to Functional States
297(1)
8.2.4 Structures Can Be Imaged at Superresolution
298(2)
8.2.5 Bioanalysis Can Be Extended Down to the Single-Molecule Level
300(1)
8.3 Building a Single-Molecule Microscope
301(15)
8.3.1 Microscopes/Objectives
301(3)
8.3.2 Light Source
304(6)
8.3.3 Detector
310(6)
8.4 Analyzing Single-Molecule Signals: Position, Orientation, Color, and Brightness
316(7)
8.4.1 Localizing in Two Dimensions
316(2)
8.4.2 Localizing along the Optical Axis
318(2)
8.4.3 Brightness
320(1)
8.4.4 Orientation
321(1)
8.4.5 Color
322(1)
8.5 Learning from Single-Molecule Signals
323(22)
8.5.1 Determination of Molecular Associations
323(2)
8.5.2 Determination of Molecular Conformations via FRET
325(4)
8.5.3 Superresolution Single-Molecule Microscopy
329(3)
8.5.4 Single-Molecule Tracking
332(1)
8.5.5 Detecting Transitions
332(2)
Acknowledgments
334(1)
References
334(11)
9 Super-Resolution Microscopy: Interference and Pattern Techniques
345(30)
Gerrit Best
Roman Amberger
Christoph Cremer
9.1 Introduction
345(2)
9.1.1 Review: The Resolution Limit
346(1)
9.2 Structured Illumination Microscopy (SIM)
347(15)
9.2.1 Image Generation in Structured Illumination Microscopy
349(3)
9.2.2 Extracting the High-Resolution Information
352(1)
9.2.3 Optical Sectioning by SIM
353(2)
9.2.4 How the Illumination Pattern is Generated
355(1)
9.2.5 Mathematical Derivation of the Interference Pattern
355(3)
9.2.6 Examples for SIM Setups
358(4)
9.3 Spatially Modulated Illumination (SMI) Microscopy
362(6)
9.3.1 Overview
362(1)
9.3.2 SMI Setup
363(1)
9.3.3 The Optical Path
364(2)
9.3.4 Size Estimation with SMI Microscopy
366(2)
9.4 Application of Patterned Techniques
368(4)
9.5 Conclusion
372(1)
9.6 Summary
372(3)
Acknowledgments
373(1)
References
373(2)
10 STED Microscopy
375(18)
Travis J. Gould
Patrina A. Pellett
Joerg Bewersdorf
10.1 Introduction
375(1)
10.2 The Concepts behind STED Microscopy
376(8)
10.2.1 Fundamental Concepts
376(4)
10.2.2 Key Parameters in STED Microscopy
380(4)
10.3 Experimental Setup
384(4)
10.3.1 Light Sources and Synchronization
384(1)
10.3.2 Scanning and Speed
385(1)
10.3.3 Multicolor STED Imaging
386(2)
10.3.4 Improving Axial Resolution in STED Microscopy
388(1)
10.4 Applications
388(5)
10.4.1 Choice of Fluorophore
388(1)
10.4.2 Labeling Strategies
389(1)
Summary
390(1)
References
391(2)
A Appendix: Practical Guide to Optical Alignment
393(10)
Rainer Heintzmann
A.1 How to Obtain a Widened Parallel Laser Beam
393(2)
A.2 Mirror Alignment
395(1)
A.3 Lens Alignment
396(1)
A.4 Autocollimation Telescope
396(1)
A.5 Aligning a Single Lens Using a Laser Beam
397(2)
A.6 How to Find the Focal Plane of a Lens
399(1)
A.7 How to Focus to the Back Focal Plane of an Objective Lens
400(3)
Index 403
Ulrich Kubitscheck is the department head of Biophysical Chemistry at the Institute of Physical and Theoretical Chemistry at the Rheinische Friedrich-Wilhelms-Universitat in Bonn, Germany. Having obtained his academic degrees from the University of Bremen, he spent his career working at The Weizmann Institute of Science and the Institute of Medical Physics and Biophysics of the Westfalische Wilhelms-Universitat Munster before taking up his present appointment at Bonn University. Professor Kubitscheck develops single molecule imaging techniques, has authored over 60 scientific publications and has extensive experience in teaching courses on physical chemistry, biophysics and quantitative microscopy.
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