Imaging and Spectroscopic Analysis of Living Cells

Imaging and Spectroscopic Analysis of Living Cells

Optical and Spectroscopic Techniques

1st Edition - January 24, 2012

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  • Editor: P. Michael Conn
  • eBook ISBN: 9780123918697
  • Hardcover ISBN: 9780123918574

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This volume of Methods in Enzymology is the first of three 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.

Key Features

  • Expert authors who are leaders in the field
  • Extensively referenced and useful figures and tables
  • Provides hints and tricks to facilitate reproduction of methods


Biochemists, biophysicists, molecular biologists, analytical chemists, pharmacologists and physiologists

Table of Contents

  • Series Page


    Volume in series

    Laser-Induced Radiation Microbeam Technology and Simultaneous Real-Time Fluorescence Imaging in Live Cells

    1. Introduction

    2. Experimental Procedure

    3. Conclusion/Forward Look


    A Cell Biologist's Guide to High Resolution Imaging

    1. Introduction

    2. Physical Limitations on the Resolution of Conventional Microscopy

    3. Preparations for High Resolution Fluorescence Imaging

    4. High Resolution Image Data Acquisition and Processing

    5. Processing

    6. Analysis of High Resolution Image Data

    7. Conclusions


    Applications of Fluorescence Lifetime Spectroscopy and Imaging to Lipid Domains In Vivo

    1. The Challenge of Studying Lipid Domains In Vivo Defied by Fluorescence Lifetimes

    2. Importance of Studies In Vitro to Design and Rationalize Studies In Vivo

    3. Labeling Cell Membranes In Vivo: Lipophilic Probes Versus GFP-Tagged Membrane Proteins

    4. Cuvette Lifetimes and FLIM: In Vivo Applications


    Detecting and Tracking Nonfluorescent Nanoparticle Probes in Live Cells

    1. Introduction

    2. Techniques and Tools

    3. Biological Applications

    4. Cytotoxicity of Nanoparticle Probes

    5. Conclusions and Future Perspective


    Fluorescence Lifetime Microscopy of Tumor Cell Invasion, Drug Delivery, and Cytotoxicity

    1. Introduction

    2. Preparation of Mouse Mammary Tumor Cell Spheroids in a Collagen Matrix

    3. Simultaneous Acquisition of Fluorescence Lifetime and Intensity to Monitor DOX Uptake

    4. Monitoring Drug Uptake Kinetics by FLIM

    5. Conclusions and Outlook


    Measuring Membrane Protein Dynamics in Neurons Using Fluorescence Recovery after Photobleach

    1. Introduction

    2. Experimental Parameters and Preparation

    3. Equipment

    4. Establishing FRAP Conditions

    5. A Basic FRAP Protocol

    6. Analysis of FRAP


    Fluorescent Speckle Microscopy in Cultured Cells

    1. Introduction

    2. Technical Challenges of FSM

    3. Detailed Methodology for FSM in Cultured Cells

    4. Future Challenges


    Green-to-Red Photoconvertible mEosFP-Aided Live Imaging in Plants

    1. Introduction

    2. Expression of mEosFP Fusion Proteins in Plants

    3. Visualization of mEosFP Probes in Plants

    4. Uses of mEosFP Probes in Plants

    5. Post Acquisition Image Processing and Data Creation


    Methods for Cell and Particle Tracking

    1. Introduction

    2. Tracking Approaches

    3. Tracking Tools

    4. Tracking Measures

    5. Tips and Tricks


    Correlative Light-Electron Microscopy

    1. Introduction

    2. Observation of Living Cells and Fixation

    3. Immunolabeling with NANOGOLD

    4. Enhancement of Sample Contrast, Sample Locating, and Embedding

    5. Identification of the Cell of Interest on EPON Blocks

    6. Sample Orientation and EM Sectioning from the Very First Section

    7. Picking up Serial Sections with the Empty Slot Grid

    8. EM Analysis


    Optical Techniques for Imaging Membrane Domains in Live Cells (Live-Cell Palm of Protein Clustering)

    1. Introduction

    2. Sample Preparation and Data Acquisition

    3. Data Analysis


    Single Live Cell Topography and Activity Imaging with the Shear-Force-Based Constant-Distance Scanning Electrochemical Microscope

    1. Introduction

    2. Shear-Force-Based Constant-Distance Scanning Electrochemical Microscopy for Live Cell Studies: Apparatus, Probes, and Operation

    3. Selected Applications of SF-CD-SECM Live Cell Studies


    Visualization of TGN-Endosome Trafficking in Mammalian and Drosophila Cells

    1. Introduction

    2. Molecular Tools

    3. Live-Cell Imaging in Mammalian Cells

    4. Live-Cell Imaging in Drosophila Cells

    5. Conclusion Remarks

    Live Cell Imaging with Chemical Specificity Using Dual Frequency CARS Microscopy

    1. Introduction

    2. “Noninvasive” Live Cell Imaging

    3. Experimental Setup

    4. Maximizing Collection Efficiency for Live Cell Imaging


    Imaging Intracellular Protein Dynamics by Spinning Disk Confocal Microscopy

    1. Introduction

    2. Instrument Design

    3. Combination with Other Imaging Techniques

    4. Specimen Preparation


    Visualizing Dynamic Activities of Signaling Enzymes Using Genetically Encodable Fret-Based Biosensors

    1. Introduction

    2. Generalizable Modular Designs

    3. FRET-Based Biosensors for Monitoring Signaling Enzymes

    4. Example: A-kinase Activity Reporter (AKAR)

    5. Summary and Perspectives


    Live-Cell Imaging of Aquaporin-4 Supramolecular Assembly and Diffusion

    1. Aquaporin-4 (AQP4) and Orthogonal Arrays of Particles

    2. Approaches to Image AQP4 and OAPs

    3. AQP4 Diffusion and OAPs Studied by Quantum Dot Single Particle Tracking

    4. OAP Dynamics and Structure Studied with GFP-AQP4 Chimeras

    5. Single-Molecule Analysis Shows AQP4 Heterotetramers

    6. Photobleaching Reveals Post-Golgi Assembly of OAPs

    7. Super-Resolution Imaging of AQP4 OAPs

    Coiled-Coil Tag–Probe Labeling Methods for Live-Cell Imaging of Membrane Receptors

    1. Introduction

    2. Various Principles Used for Tag–Probe Labeling

    3. Coiled-Coil Tag–Probe Labeling

    4. Applications


    Monitoring Protein Interactions in Living Cells with Fluorescence Lifetime Imaging Microscopy

    1. Introduction

    2. FD FLIM Measurements

    3. Measuring Protein–Protein Interactions in Living Cells Using FLIM–FRET

    4. The Strengths and Limitations of FLIM


    Open Source Tools for Fluorescent Imaging

    1. Why Open Source Software?

    2. Open Source Software for Microscopy Imaging and Analysis

    3. The Future


Product details

  • No. of pages: 560
  • Language: English
  • Copyright: © Academic Press 2012
  • Published: January 24, 2012
  • Imprint: Academic Press
  • eBook ISBN: 9780123918697
  • Hardcover ISBN: 9780123918574

About the Serial Volume Editor

P. Michael Conn

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.

Affiliations and Expertise

Texas Tech University Health Sciences Center, Lubbock, USA

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