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E-raamat: Imaging and Spectroscopic Analysis of Living Cells: Live Cell Imaging of Cellular Elements and Functions

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  • Sari: Methods in Enzymology
  • Ilmumisaeg: 15-Mar-2012
  • Kirjastus: Academic Press Inc
  • Keel: eng
  • ISBN-13: 9780123919076
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  • Formaat: EPUB+DRM
  • Sari: Methods in Enzymology
  • Ilmumisaeg: 15-Mar-2012
  • Kirjastus: Academic Press Inc
  • Keel: eng
  • ISBN-13: 9780123919076
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This volume of Methods in Enzymology is the second of 3 parts looking at current methodology for the imaging and spectroscopic analysis of live cells. The chapters provide hints and tricks not available in primary research publications. It is an invaluable resource for academics, researchers and students alike.

Expert authors who are leaders in the field

Extensively referenced and useful figures and tables

Provides hints and tricks to facilitate reproduction of methods



This volume of Methods in Enzymology is the second of 3 parts looking at current methodology for the imaging and spectroscopic analysis of live cells. The chapters provide hints and tricks not available in primary research publications. It is an invaluable resource for academics, researchers and students alike.

Expert authors who are leaders in the field

Extensively referenced and useful figures and tables

Provides hints and tricks to facilitate reproduction of methods

Muu info

This volume of Methods in Enzymology is the second of 3 parts looking at current methodology for the imaging and spectroscopic analysis of live cells
Contributors xiii
Preface xix
Volumes in Series xxi
Section I Organelles and Cellular Structure
1(254)
1 Monitoring Cytoskeletal Dynamics in Living Neurons Using Fluorescence Photoactivation
3(20)
Anne Gauthier-Kemper
Carina Weissmann
Hans-Jurgen Reyher
Roland Brandt
1 Introduction
4(1)
2 Generation and Expression of PAGFP Fusion Constructs with Cytoskeletal Proteins
5(7)
3 Fluorescence Photoactivation, Live Cell Imaging, and Data Analysis
12(11)
Acknowledgments
20(1)
References
20(3)
2 Red/Far-Red Fluorescing DNA-Specific Anthraquinones for Nucl: Cyto Segmentation and Viability Reporting in Cell-Based Assays
23(24)
Roy Edward
1 Introduction
24(1)
2 The Basic Requirement for Counterstaining
25(2)
3 An Optimized Counterstain for GFP-Based Assays
27(1)
4 Cellular Compartment Segmentation
28(1)
5 Labeling Cells in Live and Fixed End-Point Assays
29(2)
6 Protocol for Differential Dual Nuclear/Cytoplasmic Counterstaining in Live Cells
31(1)
7 Protocols for Differential Dual Nuclear/Cytoplasmic Counterstaining in Fixed Cells
32(1)
8 Measuring Cellular DNA Content
33(1)
9 Protocol: DRAQ5™ DNA Content Measurement by Imaging
34(2)
10 Protocol: Cell Enumeration in "In-Cell Westerns" (After Hannoush, 2008)
36(1)
11 Further Augmentation of Compartment Segmentation
37(1)
12 Cell Enumeration and Morphometrics: Early Sentinels of In Vitro Toxicology
37(2)
13 A Far-Red Viability Dye Derivative
39(1)
14 Protocols: Use of DRAQ7™ to Identify Membrane-Compromised Cells
40(2)
15 Summary
42(5)
Acknowledgment
43(1)
References
43(4)
3 Methods to Measure Actin Treadmilling Rate in Dendritic Spines
47(12)
Mikko Koskinen
Enni Bertling
Pirta Hotulainen
1 Introduction
48(3)
2 Methods
51(8)
Acknowledgments
57(1)
References
57(2)
4 Live-Cell Imaging of Clathrin Coats
59(22)
Comert Kural
Tom Kirchhausen
1 Introduction
60(1)
2 Dynamics of Clathrin Assembly
61(1)
3 Limits of Fluorescence Microscopy
62(1)
4 TIRF Microscopy
63(1)
5 How to Optimize Your TIRF System
64(1)
6 Spinning-Disk Confocal Microscopy
65(1)
7 Spherical Aberration Correction Applied to Spinning-Disk Confocal Microscopy
66(1)
8 Getting Around the Diffraction Limit
67(2)
9 Use of 2D Spinning-Disk Confocal Microscope to Study Clathrin-Mediated Endocytosis at the Ventral and Dorsal Surfaces of a Cell
69(1)
10 Use of TIRF Microscopy to Study Clathrin-Mediated Endocytosis
70(1)
11 The Third Dimension
71(1)
12 3D Tracking in Spinning-Disk Imaging
72(2)
13 Use of 3D Tracking to Monitor Clathrin-Mediated Entry of Reovirus Particles at the Apical Surface of Polarized Cells
74(2)
14 Using the Optimum Pixel and Step Sizes in z-Stacks
76(1)
15 Conclusion
76(5)
Acknowledgments
77(1)
References
77(4)
5 Imaging the Mitotic Spindle
81(24)
Paul S. Maddox
Anne-Marie Ladouceur
Rajesh Ranjan
Jonas Dorn
Hery Ratsima
Damien D'Amours
Amy S. Maddox
1 Introduction
82(1)
2 Modern Light Microscopy
83(10)
3 Examples
93(8)
4 Conclusions
101(4)
Acknowledgments
101(1)
References
101(4)
6 Modeling of Spatial Intracellular Signaling Events in Neurons
105(20)
Wendy C. Wenderski
Susana R. Neves
1 Introduction
105(1)
2 Computational Modeling of Intracellular Signaling
106(6)
3 Imaging Signaling Events in Striatal Neuronal Cultures
112(9)
4 Experimental Validation of Model Predictions
121(4)
Acknowledgments
121(1)
References
121(4)
7 Imaging Chromosome Dynamics in Meiosis in Plants
125(20)
Moira J. Sheehan
Wojciech P. Pawlowski
1 Introduction
126(2)
2 Technical Considerations
128(6)
3 Protocols and Methods
134(11)
Acknowledgments
141(1)
References
141(4)
8 Analyzing Lysosomes in Live Cells
145(14)
Paul R. Pryor
1 Introduction
145(1)
2 Visualizing Lysosomes
146(6)
3 Measuring Lysosomal Ions
152(1)
4 Delivery of Phagocytic Material to the Lysosome
153(1)
5 Endocytosis of Membrane Probes to the Lysosome
154(5)
References
156(3)
9 Detection and Quantification of Biomolecular Association in Living Cells using Single-Molecule Microscopy
159(28)
Mario Brameshuber
Gerhard J. Schutz
1 Movements and Interactions Drive Biomolecular Function
160(2)
2 The Concept: "Please Move to the Dark, so That We Can See You"
162(2)
3 Proofing the Principle
164(2)
4 Some Examples
166(10)
5 A User Guide to TOCCSL
176(3)
6 Analysis
179(2)
7 Conclusions
181(6)
Acknowledgments
182(1)
References
182(5)
10 Visualizing Cardiac Ion Channel Trafficking Pathways
187(16)
James W. Smyth
Robin M. Shaw
1 Introduction
188(2)
2 Introducing Recombinant DNA for Ectopic Expression of Fluorescent Ion Channel Fusion Proteins
190(1)
3 Isolating De Novo Ion Channel Forward Trafficking
191(1)
4 Recreating Cardiomyocyte-Like Membrane Subdomains in Model Cell Systems
192(5)
5 Tools for Visualizing Forward Trafficking and Delivery of Ion Channels In Real Time
197(4)
6 Data Analysis
201(1)
7 Concluding Remarks
201(2)
Acknowledgments
202(1)
References
202(1)
11 Live Cell Imaging of the Cytoskeleton
203(16)
Eve G. Stringham
Nancy Marcus-Gueret
Laura Ramsay
Kristopher L. Schmidt
1 Introduction
204(2)
2 Live Imaging of MTs In Tissue Cultured Cells
206(3)
3 Live Imaging of MTs in Whole Organisms
209(2)
4 Live Imaging of Actin Filaments in C. elegans
211(8)
References
214(5)
12 Fluorescence Single-Molecule Imaging of Actin Turnover and Regulatory Mechanisms
219(14)
Naoki Watanabe
1 Introduction
220(2)
2 Introducing a Low Density of Fluorescent Proteins in XTC Cells
222(2)
3 Observation of XTC Cells Spreading on Poly-L-lysine (PLL)-coated Glass Coverslips
224(5)
4 Data Analysis
229(4)
Acknowledgments
232(1)
References
232(1)
13 Analysis of Cell Dispersion and Migration by Video-Microscopy
233(22)
Bonnomet Arnaud
Terryn Christine
Cutrona Jerome
Jonquet Antoine
Birembaut Philippe
Zahm Jean-Marie
1 Introduction
234(1)
2 Video-Microscope
235(1)
3 Model of Cell Dispersion
236(2)
4 Models of Cell Migration in Sheet
238(5)
5 Model of 3D Cell Migration
243(2)
6 Quantification
245(7)
7 Conclusions
252(3)
References
252(3)
Section II Molecules and Ions
255(230)
14 Real Time Qualitative and Quantitative GLUT4 Translocation Assay
257(16)
Maleppillil Vavachan Vijayakumar
Manoj Kumar Bhat
1 Introduction
258(1)
2 GLUT4 Translocation Assay
259(2)
3 Methods and Procedures
261(9)
4 Conclusions and Future Applications
270(3)
Acknowledgements
270(1)
References
270(3)
15 Advanced Imaging of Cellular Signaling Events
273(18)
Marek Cebecauer
Jana Humpolickova
Jeremie Rossy
1 Introduction
274(1)
2 Selection of Tools and Transfection of Cells
275(1)
3 Imaging of Cell Signaling Events
276(5)
4 Data Analysis
281(6)
5 Summary
287(4)
Acknowledgments
288(1)
References
288(3)
16 Dynamic Imaging of Homo-FRET in Live Cells by Fluorescence Anisotropy Microscopy
291(38)
Subhasri Ghosh
Suvrajit Saha
Debanjan Goswami
Sameera Bilgrami
Satyajit Mayor
1 Introduction
292(1)
2 Theoretical Concepts
293(5)
3 Homo-FRET Measurements in an Imaging Modality: Practical Considerations
298(11)
4 Implementation of High-Resolution Steady-State Anisotropy Imaging Modalities
309(10)
5 Detecting Homo-FRET Using Time Resolved Anisotropy (TRA) Modalities
319(6)
6 Prospects and Perspectives
325(4)
Acknowledgments
325(1)
References
325(4)
17 Time-Resolved Luminescence Resonance Energy Transfer Imaging of Protein-Protein Interactions in Living Cells
329(18)
Harsha E. Rajapakse
Lawrence W. Miller
1 Introduction
330(2)
2 Lanthanide Protein Labels
332(3)
3 Transfection and Cellular Delivery of TMP-Lumi4
335(3)
4 Time-Resolved LRET Microscopy
338(9)
Acknowledgment
344(1)
References
344(3)
18 Imaging of Corticosteroid Receptors in Live Cells
347(16)
Mayumi Nishi
1 Introduction
348(1)
2 Time-Lapse Imaging of GR and MR in a Single Cell
349(5)
3 Subcellular and Intranuclear Dynamics of GR and MR Examined by FRAP Analyses
354(1)
4 Interaction of Corticosteroid Receptors in the Nucleus
354(3)
5 Notes
357(6)
Acknowledgment
360(1)
References
360(3)
19 Investigating Second Messenger Signaling In Vivo
363(20)
Rudiger Rudolf
Mathias Hafner
Marco Mongillo
1 Introduction
364(1)
2 Multimodal Imaging in Living Mouse Skeletal Muscle
365(10)
3 Adaptations to Tissues Other Than Skeletal Muscle
375(5)
4 Concluding Remarks
380(3)
Acknowledgments
380(1)
References
380(3)
20 Probes for Intracellular RNA Imaging in Live Cells
383(18)
Philip J. Santangelo
Eric Alonas
Jeenah Jung
Aaron W. Lifland
Chiara Zurla
1 Introduction
384(1)
2 Imaging RNA in Live Cells
385(1)
3 Limitations of Single-Label Probes and Molecular Beacons
386(3)
4 Imaging Native, Nonengineered RNA in Live Cells with Single RNA Sensitivity Using Exogenous Probes
389(5)
5 Time-Lapse Imaging of Native, Nonengineered β-actin mRNA Granule Dynamics
394(1)
6 Imaging RNA-Protein Colocalization Using MTRIPs
395(1)
7 Conclusions
396(5)
References
397(4)
21 Imaging the Glycome in Living Systems
401(20)
Boyangzi Li
Feiyan Mock
Peng Wu
1 Introduction
402(1)
2 Bioorthogonal Chemistry in Glycan Labeling: Merits and Limitations
403(3)
3 Metabolic Labeling of Cell Surface Fucosylated Glycans in Zebrafish Embryos for Fluorescence Imaging
406(6)
4 Labeling Cell Surface Glycans Bearing the LacNAc Disaccharide in Chinese Hamster Ovary(CHO) Cells for Fluorescence Imaging
412(9)
Acknowledgments
416(1)
References
416(5)
22 Intracellular Magnesium Detection by Fluorescent Indicators
421(24)
Valentina Trapani
Monika Schweigel-Rontgen
Achille Cittadini
Federica I. Wolf
1 Introduction
422(2)
2 Fluorescent Indicators for Magnesium
424(4)
3 Magnesium Detection in Live Cells by Spectrofluorimetry
428(6)
4 Magnesium Detection in Live Cells by Microscopy
434(7)
5 Future Perspectives
441(4)
Acknowledgments
442(1)
References
442(3)
23 Illuminating Mobile Zinc with Fluorescence: From Cuvettes to Live Cells and Tissues
445(24)
Zhen Huang
Stephen J. Lippard
1 Introduction
446(2)
2 A Guide to Fluorescent Zinc Probes
448(9)
3 Representative Procedure for Imaging Mobile Zinc in Live Cells
457(5)
4 Imaging Endogenous Neuronal Zinc in Live Cells and Tissues
462(2)
5 Concluding Remarks
464(5)
Acknowledgments
465(1)
References
465(4)
24 Quantitative Fluorescent Live Cell Imaging of Intracellular Ca2+ and H+ Ions in Malaria Parasites
469(16)
Petra Rohrbach
1 Introduction
470(1)
2 Live Cell Imaging of Intact Malaria Parasites
471(3)
3 Measuring Ion Homeostasis in P. falciparum Using Fluorescent Indicators
474(11)
Acknowledgment
482(1)
References
482(3)
Author Index 485(26)
Subject Index 511
P. Michael Conn is the Senior Vice President for Research and Associate Provost, Texas Tech Health Sciences Center. He is The Robert C. Kimbrough, Professor of Internal Medicine and Cell Biology/Biochemistry. He was previously Director of Research Advocacy and Professor of Physiology and Pharmacology, Cell Biology and Development and Obstetrics and Gynecology at Oregon Health and Science University and Senior Scientist of the Oregon National Primate Research Center (ONPRC). He served for twelve years as Special Assistant to the President and Associate Director of the ONPRC. After receiving a B.S. degree and teaching certification from the University of Michigan (1971), a M.S. from North Carolina State University (1973), and a Ph.D. degree from Baylor College of Medicine (1976), Conn did a fellowship at the NIH, then joined the faculty in the Department of Pharmacology, Duke University Medical Center where he was promoted to Associate Professor in 1982. In 1984, he became Professor and Head of Pharmacology at the University of Iowa College of Medicine, a position he held for eleven years. Conn is known for his research in the area of the cellular and molecular basis of action of gonadotropin releasing hormone action in the pituitary and therapeutic approaches that restore misfolded proteins to function. His work has led to drugs that have benefitted humans and animals. Most recently, he has identified a new class of drugs, pharmacoperones, which act by regulating the intracellular trafficking of receptors, enzymes and ion channels. He has authored or co-authored over 350 publications in this area and written or edited over 200 books, including texts in neurosciences, molecular biology and endocrinology. Conn has served as the editor of many professional journals and book series (Endocrinology, Journal of Clinical Endocrinology and Metabolism, Endocrine, Methods, Progress in Molecular Biology and Translational Science and Contemporary Endocrinology). Conn served on the National Board of Medical Examiners, including two years as chairman of the reproduction and endocrinology committee. The work of his laboratory has been recognized with a MERIT award from the NIH, the J.J. Abel Award of the American Society for Pharmacology and Experimental Therapeutics, the Weitzman, Oppenheimer and Ingbar Awards of the Endocrine Society, the National Science Medal of Mexico (the Miguel Aleman Prize) and the Stevenson Award of Canada. He is the recipient of the Oregon State Award for Discovery, the Media Award of the American College of Neuropsychopharmacology and was named a distinguished Alumnus of Baylor College of Medicine in 2012. Conn is a previous member of Council for the American Society for Cell Biology and the Endocrine Society and is a prior President of the Endocrine Society, during which time he founded the Hormone Foundation and worked with political leadership to heighten the publics awareness of diabetes. Conns students and fellows have gone on to become leaders in industry and academia. He is an elected member of the Mexican Institute of Medicine and a fellow of the American Association for the Advancement of Science. He is the co-author of The Animal Research War (2008) and many articles for the public and academic community on the value of animal research and the dangers posed by animal extremism. His op/eds have appeared in The Washington Post, The LA Times, The Wall Street Journal, the Des Moines Register, and elsewhere. Conn consults with organizations that are influenced by animal extremism and with universities and companies facing challenges from these groups.