Topics in Fluorescence Spectroscopy BGU

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Fluorescence Spectroscopy,Techniques,Topics in Fluorescence Spectroscopy. Edited by JOSEPH R LAKOWICZ,Volume 1 Techniques,Volume 2 Principles. Volume 3 Biochemical Applications,Fluorescence,Spectroscopy. Techniques,JOSEPH R LAKOWICZ,Center for Fluorescence Spectroscopy. Department of Biological Chemistry,University of Maryland School of Medicine.
Baltimore Maryland,KLUWER ACADEMIC PUBLISHERS,NEW YORK BOSTON DORDRECHT LONDON MOSCOW. eBook ISBN 0 306 47057 8,Print ISBN 0 306 43874 7,2002 Kluwer Academic Publishers. New York Boston Dordrecht London Moscow,Print 1999 Kluwer Academic Plenum Publishers. All rights reserved, No part of this eBook may be reproduced or transmitted in any form or by any means electronic. mechanical recording or otherwise without written consent from the Publisher. Created in the United States of America,Visit Kluwer Online at http kluweronline com.
and Kluwer s eBookstore at http ebooks kluweronline com. Contributors, David J S Birch Department of Physics and Applied Physics. Strathclyde University Glascow G4 ONG Scotland, Michael Edidin Department of Biology The Johns Hopkins University. Baltimore Maryland 21218, Ignacy Gryczynski Center for Fluorescence Spectroscopy Department of. Biological Chemistry University of Maryland School of Medicine Baltimore. Maryland 21201, Robert E Imhof Department of Physics and Applied Physics Strathclyde. University Glascow G4 ONG Scotland, Karl J Kutz Georgia Instruments Inc Atlanta Georgia 30366.
Joseph R Lakowicz Center for Fluorescence Spectroscopy Department. of Biological Chemistry University of Maryland School of Medicine. Baltimore Maryland 21201, Margaret M Martin SERC Daresbury Laboratory Warrington WA4 4AD. L szl M tyus Department of Biology The Johns Hopkins University. Baltimore Maryland 21218 permanent address Department of Biophysics. University Medical School of Debrecen H 4012 Debrecen Hungary. Ian H Munro SERC Daresbury Laboratory Warrington WA4 4AD. Thomas M Nordlund Department of Biophysics and Department of. Physics and Astronomy University of Rochester Rochester New York. 14642 present address Department of Physics University of Alabama at. Birmingham Birmingham Alabama 35294, Enoch W Small Department of Biochemistry and Biophysics Oregon. State University Corvallis Oregon 97331 6503,vi Contributors. Nancy L Thompson Department of Chemistry University of North. Carolina at Chapel Hill Chapel Hill North Carolina 27599 3290. John E Wampler Department of Biochemistry University of Georgia. Athens Georgia 30602, Robert A White Department of Biochemistry University of Georgia. Athens Georgia 30602, Fluorescence spectroscopy and its applications to the physical and life sciences.
have evolved rapidly during the past decade The increased interest in. fluorescence appears to be due to advances in time resolution methods of. data analysis and improved instrumentation With these advances it is now. practical to perform time resolved measurements with enough resolution to. compare the results with the structural and dynamic features of macro. molecules to probe the structure of proteins membranes and nucleic acids. and to acquire two dimensional microscopic images of chemical or protein. distributions in cell cultures Advances in laser and detector technology have. also resulted in renewed interest in fluorescence for clinical and analytical. Because of these numerous developments and the rapid appearance of. new methods it has become difficult to remain current on the science of. fluorescence and its many applications Consequently I have asked the. experts in particular areas of fluorescence to summarize their knowledge and. the current state of the art This has resulted in the initial two volumes of. Topics in Fluorescence Spectroscopy which is intended to be an ongoing series. which summarizes in one location the vast literature on fluorescence. spectroscopy The third volume will appear shortly, The first three volumes are designed to serve as an advanced text These. volumes describe the more recent techniques and technologies Volume 1. the principles governing fluorescence and the experimental observables. Volume 2 and applications in biochemistry and biophysics Volume 3. Additional volumes will be published as warranted by further advances in. this field I welcome your suggestions for future topics or volumes offers to. contribute chapters on specific topics or comments on the present volumes. Finally I thank all the authors for their patience with the delays incurred. in release of the first three volumes,Joseph R Lakowicz. Baltimore Maryland,This page intentionally left blank. 1 Time Domain Fluorescence Spectroscopy Using Time Correlated. Single Photon Counting,David J S Birch and Robert E Imhof. 1 1 Introduction 1,1 1 1 What Is a Fluorescence L i f e t i m e 2.
1 1 2 Methods of Measuring Fluorescence Lifetimes 4. 1 1 3 Analytical Applications 6,1 2 The Single Photon Technique 8. 1 2 1 Basic Principles 9,1 2 2 Statistics and Pileup 11. 1 2 3 Instrumental Response and Convolution 14,1 3 Data A n a l y s i s 16. 1 3 1 Formulation of the Problem 17,1 3 2 Least Squares Fitting 19. 1 3 3 Fitting Algorithms 22,1 3 4 Anisotropy Analysis 26.
1 4 Instrumentation 28,1 4 1 Pulsed Light Sources 28. 1 4 2 Optical Components 36,1 4 3 Detectors 41,1 4 4 Electronics and Computer Hardware 47. 1 5 Applications and Performance 52,1 5 1 Exponential Decay 52. 1 5 2 Nonexponential Decay 59,1 5 3 Anisotropy 64,1 5 4 Time Resolved Spectra 71. 1 6 Multiplexing Techniques 73,1 6 1 Differential Pulse Fluorometry 75.
1 6 2 T Format Studies 81,1 6 3 Array Fluorometry 83. References 88,x Contents, 2 Laser Sources and Microchannel Plate Detectors for Pulse Fluorometry. Enoch W Small,2 1 Introduction 97,2 2 Laser S o u r c e s 99. 2 2 1 Laser Fundamentals 100, 2 2 2 The Argon Ion Nd YAG and Dye Lasers Energy Levels. and Physical Layout 123,2 2 3 Laser Pulse Generation 132.
2 3 The MicroChannel Plate MCP Photomultiplier 146. 2 3 1 How an MCP Photomultiplier W o r k s 147,2 3 2 Photomultiplier Performance 155. 2 4 Tuning a Monophoton Decay Fluorometer for High Performance 168. 2 4 1 Judging the Quality of Data 168,2 4 2 Tuning 170. 2 4 3 Performance of a Tuned Instrument 174,2 5 Conclusion 178. References 180,3 Streak Cameras for Time Domain Fluorescence. Thomas M Nordlund,3 1 Introduction 183,3 1 1 What Is a Streak Camera 183.
3 1 2 Scope of the Chapter 185, 3 1 3 Basic Features and Abilities of a Photoelectronic Streak. Camera 186,3 1 4 History 187,3 2 System Building Blocks 190. 3 2 1 Modes of Operation 190,3 2 2 Image Converter Tube 194. 3 2 3 Image Intensifiers 205,3 2 4 Signal Readout and Storage 208. 3 2 5 Computer and Data Manipulation 212,3 3 Streak Camera Capabilities 213.
3 3 1 Detector Characteristics 213,3 3 2 Signal Averaging 222. 3 3 3 Streak Cameras and Time Resolved Single Photon. Counting 223,3 4 Time Dependence of Fluorescence 225. 3 4 1 Decay Times 225,3 4 2 Rise Times 231, 3 4 3 Physical Processes and Characteristic Times 233. Contents xi,3 5 Wavelength Dependence of Fluorescence 233. 3 5 1 Measurement of Time and Wavelength Dependence of. Signals Additional Time Dispersion 234,3 5 2 Time Dependence of Emission Spectra 237.
3 6 Two Dimensional Detection Space and Time Coordinates 239. 3 6 1 Position and Time Detection 239,3 6 2 Two Dimensional Spatial Detection 240. 3 7 Fluorescence Anisotropy 241, 3 7 1 Simultaneous versus Separate Measurements 242. 3 7 2 Signal to Noise Ratio 244,3 8 Applications 245. 3 8 1 Laser Diagnostics 245,3 8 2 Solid State S y s t e m s 246. 3 8 3 Solutions Molecular Relaxation in Solvents 247. 3 8 4 Biological Systems 249,3 8 5 X Ray Streak Cameras Fusion Reactions 252.
3 9 The Future 254,3 9 1 The 0 1 20 ps Time Regime 254. 3 9 2 Integrated Detection Systems 255, 3 9 3 Time Resolved Photon Counting Streak Cameras. TRPCSC 255,References 256, 4 Time Resolved Fluorescence Spectroscopy Using Synchrotron. Ian H Munro and Margaret M Martin,4 1 Introduction 261. 4 2 Proteins and Peptides 262,4 2 1 Lumazine 263,4 2 2 Angiotensin II 266.
4 2 3 Lactate Dehydrogenase 270,4 3 Photosynthesis 272. 4 4 Membranes 276,4 4 1 Diphenylhexatriene 276,4 4 2 Triazine Dyes 279. 4 5 Excited State Processes 281,4 5 1 Host Guest Complexes 282. 4 5 2 Stilbene 283,4 5 3 The Vacancy in Diamond 286. References 289,xii Contents,5 Frequency Domain Fluorescence Spectroscopy.
Joseph R Lakowicz and Ignacy Gryczynski,5 1 Introduction 293. 5 2 Comparison of Time and Frequency Domain Fluorometry 294. 5 2 1 Intensity Decays 294,5 2 2 Anisotropy Decays 298. 5 3 Theory of Frequency Domain Fluorometry 302,5 3 1 Decays of Fluorescence Intensity 302. 5 3 2 Decays of Fluorescence Anisotropy 304,5 4 Instrumentation and Applications 305. 5 4 1 FD Instruments with Intensity Modulated Light. Sources 305,5 4 2 A 2 GHz Harmonic Content FD Instrument 309.
5 4 3 Resolution of Anisotropy Decays 316,5 5 Future Developments 326. 5 6 Summary 331,References 331,6 Fluorescence Correlation Spectroscopy. Nancy L Thompson,6 1 Introduction 337, 6 2 Conceptual Basis and Theoretical Background 337. 6 3 Experimental Apparatus and Methods 343, 6 4 Analysis of Autocorrelation Function Magnitudes 351. 6 4 1 Number Densities 351,6 4 2 Molecular Weights 352.
6 4 3 Molecular Aggregation and Polydispersity 353. 6 5 Analysis of Autocorrelation Function Temporal Decays 354. 6 5 1 Translational Diffusion 354,6 5 2 Flow and Sample Translation 357. 6 5 3 Kinetic Rate Constants 359,6 6 Special Versions of FCS 361. 6 6 1 Nonideal Solutions 361,6 6 2 Rotational Diffusion 362. 6 6 3 Total Internal Reflection Illumination 364,6 6 4 High Order Autocorrelation 369. 6 6 5 Cross Correlation 373,6 7 Summary and Future Directions 374.
References 374,Contents xiii,7 Fundamentals of Fluorescence Microscopy. Robert A White Karl J Kutz and John E Wampler,7 1 Introduction 379. 7 1 1 Sensitivity and Its Limitations 382,7 1 2 Visualization versus Quantitation 382. 7 2 The Illumination Light Path of the Fluorescence Microscope 384. 7 2 1 Lamps 386,7 2 2 Lamp Housing 387,7 2 3 Auxiliary and Alternative Optics 389. 7 2 4 The Objective Lens as Condenser 397,7 2 5 Specimen and Mount 397.
7 3 The Imaging Light Path in Fluorescence Microscopy 398. 7 3 1 The Specimen as an Optical Component 398,7 3 2 The Objective 399. 7 3 3 Optics between Objective and Eyepiece 402,7 3 4 Role and Position of Ocular 403. 7 3 5 Detector Placement for Electronic Imaging and. Photometry 403,References 407,8 Flow Cytometry and Cell Sorting. L szl M tyus and Michael Edidin,8 1 Introduction History 411. 8 2 Operation of Flow Cytometers 413,8 2 1 Sample Handling and Delivery Systems 416.
8 2 2 The Nozzle and the Sheath Fluid 416,8 2 3 Light Sources 417. 8 2 4 The Intersection Point 418,8 2 5 Detectors 420. 8 2 6 Electronics 423,8 2 7 Data Analysis and Storage 424. 8 2 8 Slit Scan Flow Cytometry 425,8 2 9 Cell Sorting 427. 8 3 Parameters of Flow Cytometry 428,8 3 1 Light Scatter 428.
8 3 2 Fluorescence 428,8 4 Conclusion 439,References 440. Topics in Fluorescence Spectroscopy which is intended to be an ongoing series which summarizes in one location the vast literature on fluorescence spectroscopy The third volume will appear shortly The first three volumes are designed to serve as an advanced text These volumes describe the more recent techniques and technologies Volume 1 the principles governing fluorescence and the

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