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E-raamat: FLIM Microscopy in Biology and Medicine

Edited by (University of Virginia, Charlottesville, USA), Edited by (University of Illinois, Urbana, USA)
  • Formaat: 472 pages
  • Ilmumisaeg: 06-Jul-2009
  • Kirjastus: Chapman & Hall/CRC
  • Keel: eng
  • ISBN-13: 9781040220023
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  • Formaat: 472 pages
  • Ilmumisaeg: 06-Jul-2009
  • Kirjastus: Chapman & Hall/CRC
  • Keel: eng
  • ISBN-13: 9781040220023

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Detecting Signals at the Single Molecule Level: Pioneering Achievements in Microscopy

Recent advances have led to such remarkable improvements in fluorescence lifetime imaging microscopys (FLIM) capacity for contrast and sensitivity that researchers can now employ it to detect signals at the single molecule level. FLIM also offers the additional benefit of independence from fluorophore concentration and excitation intensity. Moreover, its unique sensitivity makes it an excellent reporter of conformational changes and of variations in the molecular surroundings of biological molecules.

Most of this improvement and discovery have occurred during the past decade, and, to date, information that would benefit a broad range of researchers remains scattered in the literature. Edited by two of the top pioneers in the field, FLIM Microscopy in Biology and Medicine presents the fundamentals of FLIM along with a number of advanced considerations so that a wider audience can appreciate recent and potential improvements that make it such a valuable tool.

New Opportunities for Biomedical Researchers New Challenges for Microscopy Researchers

Discussion sections in all the chapters clearly show the challenges for implementing FLIM for various applications. Certain chapters discuss limits on the number of photons required for highly accurate lifetime determinations, as well as the accuracy with which multiple, closely associated lifetime components can reliably be determined. Such considerations are important for the user when he or she is selecting the most advantageous method of FLIM to use for a particular application.

While this book provides an introduction for those new to FLIM, it gathers a wealth of material to enhance the work of experts involved in pioneering technological improvements, as well as those research opportunities in this unique and promising area of microscopy.

Arvustused

"this book represents a clear presentation of the theory, instrumentation, and data analysis of FLI microscopy and is enhanced by the critical discussions of assumptions, limitations and confounding elements ." Barry R. Masters, MIT, Microscopy and Microanalysis

"an excellent and comprehensive treatment of FRET in biology investigation. It has a good balance of basic theory and cutting-edge development in both techniques and applications." Peter So, Ph.D., MIT

"An amazingly comprehensive referenceI cant imagine anyone looking for information there and not finding what they need. It is quite essential for any lab doing FLIM." Guy Cox, University of Sydney, Australia

Preface xix
Acknowledgments xxiii
The Editors xxv
Contributors xxvii
Section 1 Introduction, Microscopy, Fluorophores
Fluorescence Lifetime-Resolved Imaging: What, Why, How---A Prologue
3(32)
Robert M. Clegg
Introduction
3(1)
Goal of This
Chapter
4(1)
Why Measure Fluorescence Lifetimes?
4(3)
Why Measure Lifetime-Resolved Images?
7(1)
Specific Features of the Different Pathways and Rates of De-Excitation
8(10)
Intrinsic Rate of Emission (Fluorescence)
8(2)
Thermal Relaxation (Internal Conversion)
10(1)
Molecular Relaxation of the Solvent or Molecular Matrix Environment
11(1)
Quenchers (Dynamic)
12(1)
Excited-State Reactions
12(1)
Forster Resonance Energy Transfer (FRET)
13(2)
Intersystem Crossing and Delayed Emission
15(1)
Slow Luminescence without Intersystem Crossing
16(1)
Photolysis (Process and Interpretation of Its Measurement)
17(1)
The Unifying Feature of Extracting Information from Excited-State Pathways
18(1)
Other Parameters Related to Lifetime-Resolved Fluorescence---Dynamic and Steady-State Measurements
18(3)
Anisotropy Decay
18(3)
Steady-State Quenching (Dynamic) Measurement
21(1)
Data Acquisition
21(6)
Scanning and Full Field
21(1)
Scanning Modes
22(1)
Full-Field Modes
23(1)
Time and Frequency Domains
23(1)
Time Domain
24(1)
Frequency Domain
25(1)
Equivalence of Time and Frequency Domains
26(1)
Performance Goals and Comparisons
26(1)
Data Analysis
27(1)
Display of Lifetime-Resolved Images
28(1)
Summary
28(1)
References
29(6)
Principles of Fluorescence for Quantitative Fluorescence Microscopy
35(30)
Neil Anthony
Peng Guo
Keith Berland
Introduction
35(1)
What is Fluorescence?
35(1)
Absorption
36(9)
Molecular Excitation Rates
38(1)
One-Photon Excitation
39(2)
Two-Photon Excitation
41(4)
Fluorescence and Molecular Relaxation Pathways
45(9)
Internal Conversion
45(1)
Fluorescence Emission
45(2)
Quantum Yield
47(1)
Fluorescence Lifetimes
47(2)
Fluorescence Emission Spectra
49(1)
Nonradiative Relaxation Pathways
50(1)
Basics of FRET
51(1)
Intersystem Crossing and Phosphorescence
52(1)
Photoselection and Anisotropy
52(2)
Measuring Fluorescence in the Microscope
54(5)
Sensitivity of Fluorescence Measurements
54(2)
Fluorescence Signals
56(1)
Observation Volumes and Molecular Brightness
56(1)
Saturation
57(1)
Photobleaching
58(1)
Summary
59(1)
References
59(6)
Visible Fluorescent Proteins for FRET-FLIM
65(28)
Richard N. Day
Introduction
65(1)
Background
66(6)
Overview of the Fluorescent Proteins
66(2)
Spectral Variants from the Aequorea GFP
68(2)
Aequorea Fluorescent Proteins and Dimer Formation
70(1)
New Fluorescent Proteins from Corals
70(2)
Methods
72(9)
Visible Fluorescent Proteins for FRET Measurements
72(1)
Standards for Live-Cell FRET Imaging
73(2)
Using FRET-FLIM to Detect Protein Interactions in Living Cells
75(2)
Verifying Protein Interactions Using Acceptor Photobleaching FRET
77(1)
Alternative Fluorophore Pairs for FRET-FLIM
77(2)
Fluorescent Proteins Designed Specifically for FLIM Applications
79(2)
Critical Discussion
81(3)
General Considerations and Limitations
81(1)
Overexpression Artifacts
82(1)
Factors Limiting FRET-FLIM
82(1)
False Positives and False Negatives
83(1)
Analysis in the Cell Population
83(1)
Summary
84(1)
Future Perspective
84(1)
References
84(9)
Section 2 Instrumentation
Wide-Field Fluorescence Lifetime Imaging Microscopy Using a Gated Image Intensifier Camera
93(22)
Yuansheng Sun
James N. Demas
Ammasi Periasamy
Introduction
93(2)
Background
95(1)
Methods
95(11)
Theory behind the RLD Method
95(1)
Single-Exponential Decay
96(1)
Double-Exponential Decay
97(2)
Components Required for RLD-Based Lifetime Imaging
99(1)
How Data Were Acquired Using the RLD Method
100(1)
Calibration of the System with a Known Fluorophore (Single-Exponential Decay)
100(2)
Double-Exponential Decays: Biological Examples
102(4)
Critical Discussion
106(1)
Pitfalls
107(3)
Summary
110(1)
Monte Carlo Simulation
110(1)
Preparation of Cells
111(1)
References
112(3)
Frequency-Domain FLIM
115(28)
Bryan Q. Spring
Robert M. Clegg
Introduction to Frequency-Domain Methods
115(2)
Overview
115(1)
Heterodyne and Homodyne Methods for Measuring Fluorescence Lifetimes
116(1)
A Few Preliminary Comments
117(1)
Relationship Between Observables and Fluorescence Lifetimes
117(9)
A Primer in Complex Analysis
117(1)
A General Expression for the Fluorescence Signal
117(4)
A General Expression for the Measured Homo-/Heterodyne Signal
121(1)
Single- Versus Multifrequency FLIM
122(1)
Single-Frequency FLIM
122(1)
Multifrequency FLIM
123(1)
Homodyne Multifrequency FLIM
124(1)
Heterodyne Multifrequency FLIM
125(1)
Extracting the Demodulation and Phase Shift Values Using a Digital Fourier Transform
126(1)
Video-Rate Flim
127(6)
Overview
127(1)
Instrumentation
127(1)
Illumination Sources and Electro-optics for Modulated Excitation Light
127(2)
Gain-Modulated Image Intensifiers
129(1)
Optical Setup and Electronics
130(1)
Corrections for Random Noise and Systematic Errors
131(1)
Correcting for Laser Fluctuations and Dark Current
131(1)
Gain-Modulated Image Intensifier Performance
131(2)
Enhanced Flim Modes
133(3)
Video-Rate Confocal FLIM
133(1)
Rapid Spectral FLIM
134(2)
Data Display
136(3)
Dual-Layer FLIM Images
136(1)
Dual-Layer Fractional Concentration Images
137(2)
Summary
139(1)
References
139(4)
Laser Scanning Confocal FLIM Microscopy
143(22)
Hans C. Gerritsen
Arjen Bader
Sasha Agronskaia
Introduction
143(3)
Historical Background
145(1)
Lifetime Detection Methods In Scanning Microscopy
146(5)
Time-Correlated Single-Photon Counting (TCSPC)
146(3)
Time Gating
149(2)
Detectors and Electronics
151(3)
Detectors
151(2)
Front-End Electronics
153(1)
Count Rate and Acquisition Time
154(4)
Detector and Electronics Limitations
155(1)
Efficiency of Time-Domain Lifetime Detection Methods
156(2)
Example
158(2)
Summary
160(1)
Future Perspective
160(1)
References
161(4)
Multiphoton Fluorescence Lifetime Imaging at the Dawn of Clinical Application
165(24)
Karsten Kong
Aisada Uchugonova
Introduction
165(2)
Principle of Multiphoton Imaging
167(2)
Clinical Multiphoton Tomography
169(1)
Multiphoton Flim Technique
170(4)
Applications
174(9)
Multiphoton Skin Imaging
174(3)
Two-Photon FLIM Imaging of Stem Cells
177(6)
Conclusion
183(1)
Acknowledgment
184(1)
References
184(5)
Flim Microscopy with a Streak Camera: Monitoring Metabolic Processes in Living Cells and Tissues
189(22)
Krishnan Ramanujan
Javier A. Jo
Ravi Ranjan
Brian A. Herman
Introduction
189(1)
Streakflim: System Integration
190(5)
Step-by-Step Demonstration of StreakFLIM System Application
193(1)
Data Acquisition
193(2)
Data Analysis
195(1)
Critical Discussion
195(5)
Further Applications
200(7)
Imaging Cancer Cells in Three-Dimensional Architecture
200(3)
Kinetic Imaging of pH Transients during Glucose Metabolism
203(2)
FLIM-Based Enzyme Activity Assays In Vivo
205(2)
Summary and Future Perspective
207(1)
References
208(3)
Spectrally Resolved Fluorescence Lifetime Imaging Microscopy: SLIM/mwFLIM
211(34)
Christoph Biskup
Birgit Hoffmann
Klaus Benndorf
Angelika Ruck
Introduction
211(3)
Background
214(10)
The Spectral Axis of the Fluorescence Decay Surface
214(2)
The Time Axis of the Fluorescence Decay Surface
216(1)
Global Analysis
217(1)
A Special Case: Global Analysis of FRET Measurements
218(6)
Methods
224(15)
The Setup
224(2)
Operation Principle of the Streak Camera
226(1)
Operation Principle of the mwFLIM/SLIM Setup
226(2)
Benefits of the Techniques
228(1)
Calibration
229(1)
Calibration of the Spectral Axis
229(1)
Calibration of the Time Axis
230(1)
Calibration of the Intensity Axis
230(1)
Data Analysis
231(1)
The Instrument Response Function
231(1)
Deconvolution and Data Fitting
232(2)
Applications
234(1)
Functional Staining of Cell Structures
234(1)
Photodynamic Therapy (PDT)
234(3)
Forster Resonance Energy Transfer
237(2)
Critical Discussion
239(2)
Summary
241(1)
References
241(4)
Time-Resolved Fluorescence Anisotropy
245(46)
Steven S. Vogel
Christopher Thaler
Paul S. Blank
Srinagesh V. Koushik
Introduction
245(1)
Underlying Concepts
246(1)
Light Has An Orientation
247(1)
Photoselection
248(7)
Photoselection of a Randomly Oriented Population of Fluorophores
251(4)
How Do We Detect Polarized Emissions?
255(6)
How Do We Quantify Polarized Emissions?
261(3)
The Anisotropy of Randomly Oriented Populations of Fluorophores
264(1)
Depolarization Factors and Soleillet's Rule
265(17)
Instrumental Depolarization
268(2)
Depolarization Caused by Absorption and Emission Dipole Orientation
270(1)
Timescale of Depolarization
270(1)
Depolarization Caused by Molecular Rotation
271(4)
Depolarization Caused by FRET
275(7)
Fluorescence Anisotropy Applications
282(3)
Phosphorylation Assay
283(1)
Putting Limits on the Value of κ2
284(1)
Differentiating between Directly Excited Acceptors and FRET
285(1)
Conclusion
285(1)
Acknowledgments
286(1)
References
286(5)
Section 3 Data Analysis
General Concerns of FLIM Data Representation and Analysis: Frequency-Domain Model-Free Analysis
291(50)
Yi-Chun Chen
Bryan Q. Spring
Chittanon Buranachai
Bianca Tong
George Malachowski
Robert M. Clegg
Introduction
291(2)
Time Domain Assuming Very Short Excitation Pulses
293(3)
Frequency Domain
296(6)
Calculating F(t)meas Directly from the Convolution Integral
296(3)
Calculating F(t)meas from the Finite Fourier Transform of the Repetitive δ-Pulse Result
299(2)
Calculating the Frequency Response from the Convolution Theorem of Fourier Transforms
301(1)
Analysis of the Measured Data, F(T)meas, At Every Pixel
302(1)
Remarks About Signal-To-Noise Characteristics of Time-And Frequency-Domain Signals: Comparison To Single-Channel Experiments
303(2)
Flim Experiments: Challenges, Advantages, and Solutions
305(1)
How Flim Circumvents The Data Deluge
306(14)
Polar Plots of Frequency-Domain Data (Model-Free Analysis)
307(1)
Polar Plot Description of Fluorescence Directly Excited by Light Pulses
307(4)
Polar Plot of Fluorescence from a Product Species of an Excited-State Reaction
311(3)
Combining Spectra and Polar Plots
314(1)
Two Different Noninteracting Fluorophores
315(3)
FRET: Observing Donor and Acceptor Fluorescence Simultaneously
318(2)
Wavelets and Denoising
320(10)
Why Use This Image Analysis?
320(1)
Wavelet Transforms for Discriminating Fluorescence Lifetimes Based on Spatial Morphology
321(1)
What Is a Wavelet Transform?
321(3)
Applications of Wavelets to Homodyne FLIM
324(1)
Denoising Homodyne FLIM Data
325(1)
Sources of Noise for Homodyne FLIM
325(1)
Removal of Signal-Dependent Noise: TI-Haar Denoising
325(1)
TI-Haar Denoising Improves Homodyne FLIM Accuracy
326(2)
The Future of Wavelet and Denoising Image Analysis for Homodyne FLIM
328(2)
Noniterative Data Regression (Chebyshev and Laguerre Polynomials)
330(5)
Noniterative Data Regression
330(1)
Convexity in Modeling and Multiple Solutions
330(2)
Formulation of Modeling as a Dynamic System
332(1)
Solution to Convexity in a Hilbert Space
332(2)
Error Evaluation
334(1)
References
335(6)
Nonlinear Curve-Fitting Methods for Time-Resolved Data Analysis
341(30)
Ignacy Gryczynski
Rafal Luchowski
Shashank Bharill
Julian Borejdo
Zygmunt Gryczynski
Introduction
341(1)
Background
342(1)
Methods
343(8)
Basic Terminology and Assumptions
343(2)
Least-Squares Analysis
345(1)
Time Domain
346(2)
Frequency Domain
348(1)
Least-Squares Parameter Estimation
349(1)
Diagnostics for Quality of Curve-Fitting Results
350(1)
Uncertainty of Curve-Fitting Procedures
350(1)
Examples
351(16)
How to Analyze Experimental Data
351(1)
Systematic Errors
352(1)
Light Delay
352(1)
Color Effect in the Detector
353(2)
Polarization Effect
355(1)
Pileup Effect
356(1)
Solvent Effect
357(1)
Analysis of Multiexponential Decays
358(1)
Effect of the Signal Level
359(2)
Two and Three Components of Intensity Decays
361(3)
Fluorescence Lifetime Distribution: Biological Examples
364(3)
Summary
367(1)
References
368(3)
Global Analysis of Frequency Domain FLIM Data
371(14)
Hernan E. Grecco
Peter J. Verveer
Introduction
371(1)
Fourier Description of Flim Data
372(2)
Global Analysis of Flim Data
374(1)
Application To Fret-Flim Data
375(1)
Discussion and Outlook
375(5)
Summary
380(1)
Methods
380(1)
Cell Preparation
380(1)
Fluorescence Lifetime Imaging Microscopy
381(1)
References
381(4)
Section 4 Applications
FLIM Applications in the Biomedical Sciences
385(16)
Ammasi Periasamy
Robert M. Clegg
Introduction
385(1)
A Brief Historical Journey Through The Development of Lifetime-Resolved Imaging
386(2)
Autofluorescence Lifetime Imaging of Cells
388(2)
Pap Smear Detection Using Time-Gated Lifetime Imaging Microscopy
390(4)
FLIM in Alzheimer's Disease
394(1)
Optical Projection of Flim Images of Mouse Embryo
394(1)
Full-Field Flim With Quadrant Detector
395(1)
Conclusion
396(2)
References
398(3)
Index 401
Ammasi Periasamy, University of Virginia, Charlottesville, USA

Robert M. Clegg, University of Illinois, Urbana-Champaign, USA