Contents. Foreword V Preface XIII List of Contributors XVII. Single Cell Analysis : Imaging

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1 VII Foreword V Preface XIII List of Contributors XVII Part I Single Cell Analysis : Imaging 1 Single Molecule Fluorescence Monitoring in Eukaryotic Cells: Intranuclear Dynamics of Splicing Factors 1 Ulrich Kubitscheck 1.1 Motivation Experimental Approach Single Particle Tracking within Living Cells Pre-Messenger RNA Splicing Intranuclear Splicing Factor Tracking Intranuclear U1 snrnp Splicing Factor Binding Events in Speckles Intranuclear U1 snrnp Mobility Perspectives of Single Molecule Microscopy 13 References 15 2 Gene Classification and Quantitative Analysis of Gene Regulation in Bacteria using Single Cell Atomic Force Microscopy and Single Molecule Force Spectroscopy 19 Robert Ros and Nicole Hansmeier 2.1 Introduction AFM on Paracrystalline Cell Surface Layers of C. glutamicum: Protein Sequence Information and Morphology Imaging of Living C. glutamicum Cells with Molecular Resolution: Genes, Transcriptional Regulation and Morphology Single Molecule Force Spectroscopy on Specific Protein DNA Complexes: Transcriptional Regulation in S. meliloti 25 Single Cell Analysis: Technologies and Applications. Edited by Dario Anselmetti Copyright Ó 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN:

2 VIII 2.5 Effector-Induced Protein DNA Binding on the Single Molecule Level: Quorum Sensing in S. meliloti Conclusion 32 References 33 3 Cellular Cryo-Electron Tomography (CET): Towards a Voyage to the Inner Space of Cells 39 Juergen M. Plitzko 3.1 Introduction Tomography with the Electron Microscope a Practical Perspective Sample Preparation Instrumental and Technical Requirements Alignment, Reconstruction and Visualization Molecular Interpretation of Cellular Tomograms Outlook: The Future is Bright 61 References 65 Part II Single Cell Analysis: Technologies 4 Single Cell Proteomics 69 Norman J. Dovichi, Shen Hu, David Michels, Danqian Mao, and Amy Dambrowitz 4.1 Introduction The Challenge Single Cell Proteomics: Mass Spectrometry Single Cell Separations Ultrasensitive Protein Analysis: Capillary Electrophoresis with Laser-Induced Fluorescence Detection Capillary Sieving Electrophoresis of Proteins from a Single Cancer Cell Cell Cycle-dependent Single Cell Capillary Sieving Electrophoresis Tentative Identification of Proteins in Single Cell Electropherograms Capillary Micellar and Submicellar Separation of Proteins from a Single Cell Two-Dimensional Capillary Electrophoresis of Proteins in a Single Cell Single Copy Detection of Specific Proteins in Single Cells Conclusion 85 References 87 5 Protein Analysis of Single Cells in Microfluidic Format 91 Alexandra Ros and Dominik Greif 5.1 Introduction 91

3 IX 5.2 Microfluidic Single Cell Analysis Concept Single Cell Selection and Trapping Single Cell Lysis Single Cell Electrophoretic Separation and Detection of Proteins Label-Based Fluorescence Detection Label-Free Fluorescence Detection UV-LIF in Quartz Microfluidic Devices UV-LIF in PDMS Microfluidic Devices Single Cell UV-LIF Electrophoretic Analysis Future Directions in Single Cell Analysis 103 References Single Cell Mass Spectrometry 109 Elena V. Romanova, Stanislav S. Rubakhin, Eric B. Monroe, and Jonathan V. Sweedler 6.1 Introduction Considerations for Single Cell Chemical Microanalysis using Mass Spectrometry Mass Spectrometry as a Discovery Tool for Chemical Analysis of Cells Single Cell Mass Spectrometric Applications Subcellular Profiling Imaging Single Cells with MS Signaling Molecule Release from Single Cells Future Developments 126 References Single Cell Analysis for Quantitative Systems Biology 135 Luke P. Lee and Dino Di Carlo 7.1 Introduction Misleading Bulk Experiments Common Techniques for High-Throughput and High-Content Single Cell Analysis Improved Functionality for High-Throughput Single Cell Analysis Microfluidic Techniques Array-Based Techniques High-Content Separation-Based Techniques Example Studies Enabled by Microfluidic Cell Arrays Pore-Forming Dynamics in Single Cells Microfluidic Single Cell Arrays with Fluorescence Imaging Toxin-Induced Permeability Stochastic Model of Pore Formation Amount and Size of Pores for Best Fit Models Concerning the Pore Formation Mechanism of SLO 151

4 X Conclusions on Pore-Forming Dynamics in Single Cells Single Cell Culture and Analysis Single Cell Trapping Arrays Arrayed Single Cell Culture Conclusions on Arrayed Single Cell Culture Conclusions and Future Directions 157 References Optical Stretcher for Single Cells 161 Karla Müller, Anatol Fritsch, Tobias Kiessling, Marc Grosserüschkamp, and Josef A. Käs 8.1 Introduction Theory, Methods and Experimental Setup Fundamentals of Optical Stretching Ray Optics Resulting Forces Microfluidics Laminar Flow Applications Cancer Diagnostics Minimally Invasive Analysis Stem Cell Characterization Outlook 173 References 174 Part III Single Cell Analysis: Applications 9 Single Cell Immunology 175 Ulrich Walter and Jan Buer 9.1 Introduction Single Cell Gene Expression Profiling Single Cell (Multiplex) RT-PCR Quantitative Single Cell Multiplex RT-PCR Fluorescence-Activated Cell Sorting Live Cell Fluorescence Microscopy Confocal Laser Scanning Microscopy Total Internal Reflection Fluorescence Microscopy Förster Resonance Energy Transfer Imaging Two-Photon Laser Scanning Microscopy Other Techniques for Single Cell Analysis Enzyme-Linked Immunospot Assay In Situ Hybridization Electron Microscopy Conclusions and Outlook 189 References 189

5 XI 10 Molecular Characterization of Rare Single Tumor Cells 197 James F. Leary 10.1 Introduction Importance of Rare Cells Detection of Rare Tumor Cells Finding Rare Event Tumor Cells in Multidimensional Data Rare Event Sampling Statistics High-Speed Sorting of Rare Cells Sorting Speeds must be Fast Enough to be Practical Limits in Sorting Speeds and Purities Classification of Rare Tumor Cells Using Classifiers to Sort Rare Tumor Cells Molecular Characterization of Sorted Tumor Cell Cells Model Cell Systems Design of PCR Primers to Detect the PTEN Gene Region Processing BT-549 Human Breast Cancer Cells Detection of Mutated Sequences in Tumor Suppressor Genes Detection of Mutations in Breast Cancer Tumor Suppressor Genes by High-Throughput Flow Cytometry, Single Cell Sorting and Single Cell Sequencing Single Cell Sorting for Mutational Analysis by PCR TA Cloning Single Cell Analysis of Gene Expression Profiles Conclusions and Discussion 219 References Single Cell Heterogeneity 223 Edgar A. Arriaga 11.1 Introduction Measuring Heterogeneity using Single Cell Techniques Optical Well Arrays Capillary Electrophoresis Analysis of Organelles Released from Single Cells Describing Cellular Heterogeneities and Subpopulations Origins of Cellular Heterogeneity Identifying Extrinsic and Intrinsic Noise Sources Validation of the Flow Cytometry Measurements Noise Dissection Identification of Deviant Gene Products Correlation of Gene Products with Potential Sources of Noise Concluding Remarks 233 References 234

6 XII 12 Genome and Transcriptome Analysis of Single Tumor Cells 235 Bernhard Polzer, Claudia H. Hartmann, and Christoph A. Klein 12.1 Introduction Detection and Malignant Origin of Disseminated Cancer Cells Methods for Amplifying Genomic DNA of Single Cells Studying the Genome of Single Disseminated Cancer Cells The Need for Higher Resolution: Array CGH of Single Cells Studying the Gene Expression of Single Disseminated Cancer Cells Combined Genome and Transcriptome Analysis of Single Disseminated Cancer Cells 246 References 246 Index 251