Techniques in Confocal Microscopy

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 public’s awareness of diabetes. Conn’s 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.

Part I. Practical Considerations and Equipment
1. Theoretical Basis of Confocal Microscopy
Robert H. Webb
2. Practical Considerations in Acquiring Biological Signals from Confocal Microscope
Hao He, Rose Chik Ying Ong, Kam Tai Chan, Ho Pui Ho, Siu Kai Kong
3. Equipment for Mass Storage and Processing of Data
Guy Cox
4. Antifading Agents for Confocal Fluorescence Microscopy
Miguel Berrios, Kimberly A. Conlon, and David E. Colflesh
5. Mounting Techniques for Confocal Microscopy
Manabu Kagayama and Yasuyuki Sasano
6. Preparation of Whole Mounts and Thick Sections for Confocal Microscopy
Hisashi Hashimoto, Hiroshi Ishikawa, and Moriaki Kusakabe
7. Use of Confocal Microscopy to Investigate Cell Structure and Function
Ghassan Bkaily, Danielle Jacques
8. Combining Laser Scanning Confocal Microscopy and Electron Microscopy
Xuejun Sun

Part II. Functional Approaches
9. Volume Measurements in Confocal Microscopy
Carlos B. Mantilla, Y.S. Prakash and Gary C. Sieck
10. Quantitation of Phagocytosis by Confocal Microscopy
George F. Babcock and Chad T Robinson MS
11. Receptor-Ligand Internalization
Guido Orlandini, Nicoletta Ronda, Rita Gatti, Gian Carlo Gazzola, and Alberico Borghetti
12. Quantitative Imaging of Metabolism by Two-Photon Excitation Microscopy
David W. Piston and Susan M. Knobel
13. Trafficking of the Androgen Receptor
Virginie Georget, Béatrice Terouanne, Jean-Claude Nicolas, and Charles Sultan
14. Localization of Proteases and Peptide Receptors by Confocal Microscopy
Kanti D. Bhoola, Celia J. Snyman and Carlos D. Figueroa
15. Multiphoton Excitation Microscopy, Confocal Microscopy, and Spectroscopy of Living Cells and Tissues: Functional Metabolic Imaging of Human Skin in Vivo
Barry R. Masters, Peter T. C. So, Ki Hean Kim, Christof Buehler, and Enrico Gratton
16. Video-Rate, Scannng Slit Confocal Microscopy of Living Human Cornea in Vivo: Three-Dimensional Confocal Microscopy of the Eye
Barry R. Masters and Matthias Böhnke
17. In Vivo Imaging of Mammalian Central Nervous System Neurons with the In Vivo Confocal Neuroimaging (ICON) Method
Sylvia Prilloff, Petra Henrich-Noack, Ralf Engelmann, Bernhard A. Sabel
18. Identification of Viral Infection by Confocal Microscopy
David N. Howell and Sara E. Miller
19. Membrane Trafficking
Sabine Kupzig, San San Lee, and George Banting

Part III. Green Fluorescent Protein
20. Monitoring of Protein Secretion with Green Fluorescent Protein
Christoph Kaether and Hans-Hermann Gerdes
21. Comparison of Enhanced Green Fluorescent Protein and Its Destabilized Form as Transcription Reporters
Xiaoning Zhao, Tommy Duong, Chiao-Chian Huang, Steven R. Kain, and Xianqiang Li
22. Measuring Protein Degradation with Green Fluorescent Protein
Stephen R. Cronin and Randolph Y. Hampton
23. Studying Nuclear Receptors with Green Fluorescent Protein Fusions
Gordon L. Hager
24. Signaling, Desensitization, and Trafficking of G Protein-Coupled Receptors Revealed by Green Fluorescent Protein Conjugates
Larry S. Barak, Jie Zhang, Stephen S. G. Ferguson, Stephane A. Laporte, and Marc G. Caron
25. Fluorescent Proteins in Single- and Multicolor Flow Cytometry
Lonnie Lybarger and Robert Chervenak
26. Jellyfish Green Fluorescent Protein: A Tool for Studying Ion Channels and Second-Messenger Signaling in Neurons
L. A. C. Blair, K. K.Bence, and J. Marshall
27. Expression of Green Fluorescent Protein and Inositol 1,4,5-Triphosphate Receptor in Xenopus laevis Oocytes
Atshushi Miyawaki, Julie M. Matheson, Lee G. Sayers, Akira Muto, Takayuki Michikawa, Teiichi Furuichi, and Katsuhiko Mikoshiba
28. Confocal Imaging of Ca2+, pH, Electrical Potential, and Membrane Permeability in Single Living Cells
John J. Lemasters, Donna R. Trollinger, Ting Qian, Wayne E. Cascio, and Hisayuki Ohata

Part IV. Laser Capture Microdissection
29. Laser Capture Microdissection and Its Applications in Genomics and Proteomics
James L. Wittliff
30. Going in Vivo with Laser Microdissection
Anette Mayer, Monika Stich, Dieter Brocksch, Karin Schütze, and Georgia Lahr
31. Flourescence in Situ Hybridization of LCM-Isolated Nuclei from Paraffin Sections
Douglas J. Demetrick, Sabita K. Murthy, and Lisa M. DiFrancesco