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 .............................................. 1
1.2 Experimental Approach ................................... 2
1.3 Single Particle Tracking within Living Cells ............ 6
1.4 Pre-Messenger RNA Splicing .............................. 7
1.5 Intranuclear Splicing Factor Tracking ................... 8
1.6 Intranuclear U1 snRNP Splicing Factor Binding .......... 10
1.7 Events in Speckles ..................................... 10
1.8 Intranuclear U1 snRNP Mobility ......................... 11
1.9 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 ........................................... 19
2.2 AFM on Paracrystalline Cell Surface Layers of C.
glutamicum: Protein Sequence Information and
Morphology ............................................. 20
2.3 Imaging of Living C. glutamicum Cells with Molecular
Resolution: Genes, Transcriptional Regulation and
Morphology ............................................. 23
2.4 Single Molecule Force Spectroscopy on Specific
Protein-DNA Complexes: Transcriptional Regulation
in S. meliloti ......................................... 25
2.5 Effector-Induced Protein-DNA Binding on the Single
Molecule Level: Quorum Sensing in S. meliloti .......... 29
2.6 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 ........................................... 39
3.2 Tomography with the Electron Microscope - a Practical
Perspective ............................................ 42
3.2.1 Sample Preparation .............................. 42
3.2.2 Instrumental and Technical Requirements ......... 48
3.2.3 Alignment, Reconstruction and Visualization ..... 54
3.3 Molecular Interpretation of Cellular Tomograms ......... 58
3.4 Outlook: The Future is Bright .......................... 61
References .................................................. 65
Part II Single Cell Analysis: Technologies
4 Single Cell Proteomics ...................................... 69
Norman J. Dovichi, Shen Нu, David Michels, Danqian Mao,
and Amy Dambrowitz
4.1 Introduction ........................................... 69
4.2 The Challenge .......................................... 70
4.3 Single Cell Proteomics: Mass Spectrometry .............. 71
4.4 Single Cell Separations ................................ 72
4.5 Ultrasensitive Protein Analysis: Capillary
Electrophoresis with Laser-Induced Fluorescence
Detection .............................................. 74
4.6 Capillary Sieving Electrophoresis of Proteins from
a Single Cancer Cell ................................... 75
4.7 Cell Cycle-dependent Single Cell Capillary Sieving
Electrophoresis ........................................ 77
4.8 Tentative Identification of Proteins in Single Cell
Electropherograms ...................................... 78
4.9 Capillary Micellar and Submicellar Separation of
Proteins from a Single Cell ............................ 79
4.10 Two-Dimensional Capillary Electrophoresis of
Proteins in a Single Cell .............................. 80
4.11 Single Copy Detection of Specific Proteins in Single
Cells .................................................. 83
4.12 Conclusion ............................................. 85
References .................................................. 87
5 Protein Analysis of Single Cells in Microfluidic Format ..... 91
Alexandra Ros and Dominik Creif
5.1 Introduction ........................................... 91
5.2 Microfluidic Single Cell Analysis Concept .............. 93
5.2.1 Single Cell Selection and Trapping .............. 93
5.2.2 Single Cell Lysis ............................... 95
5.3 Single Cell Electrophoretic Separation and Detection
of Proteins ............................................ 96
5.3.1 Label-Based Fluorescence Detection .............. 98
5.3.2 Label-Free Fluorescence Detection ............... 99
5.3.2.1 UV-LIF in Quartz Microfluidic
Devices ................................ 99
5.3.2.2 UV-LIF in PDMS Microfluidic Devices .... 99
5.3.2.3 Single Cell UV-LIF Electrophoretic
Analysis .............................. 102
5.4 Future Directions in Single Cell Analysis ............. 103
References ................................................. 104
6 Single Cell Mass Spectrometry .............................. 109
Elena V. Romanova, Stanislav S. Rubakhin, Eric
B. Monroe, and Jonathan V. Sweedler
6.1 Introduction .......................................... 109
6.2 Considerations for Single Cell Chemical
Microanalysis using Mass Spectrometry ................. 110
6.3 Mass Spectrometry as a Discovery Tool for Chemical
Analysis of Cells ..................................... 111
6.4 Single Cell Mass Spectrometric Applications ........... 115
6.5 Subcellular Profiling ................................. 119
6.6 Imaging Single Cells with MS .......................... 121
6.7 Signaling Molecule Release from Single Cells .......... 124
6.8 Future Developments ................................... 126
References ................................................. 126
7 Single Cell Analysis for Quantitative Systems Biology ...... 135
Luke P. Lee and Dino Di Carlo
7.1 Introduction .......................................... 135
7.2 Misleading Bulk Experiments ........................... 138
7.3 Common Techniques for High-Throughput and High-
Content Single Cell Analysis .......................... 140
7.4 Improved Functionality for High-Throughput Single
Cell Analysis ......................................... 141
7.4.1 Microfluidic Techniques ........................ 141
7.4.2 Array-Based Techniques ......................... 144
7.4.3 High-Content Separation-Based Techniques ....... 147
7.5 Example Studies Enabled by Microfluidic Cell Arrays ... 148
7.5.1 Pore-Forming Dynamics in Single Cells .......... 148
7.5.1.1 Microfluidic Single Cell Arrays with
Fluorescence Imaging .................. 148
7.5.1.2 Toxin-Induced Permeability ............ 148
7.5.1.3 Stochastic Model of Pore Formation .... 149
7.5.1.4 Amount and Size of Pores for Best
Fit Models ............................ 151
7.5.1.5 Concerning the Pore Formation
Mechanism of SLO ...................... 151
7.5.1.6 Conclusions on Pore-Forming Dynamics
in Single Cells ....................... 152
7.5.2 Single Cell Culture and Analysis ............... 153
7.5.2.1 Single Cell Trapping Arrays ........... 154
7.5.2.2 Arrayed Single Cell Culture ........... 154
7.5.2.3 Conclusions on Arrayed Single Cell
Culture ............................... 156
7.6 Conclusions and Future Directions ..................... 157
References ................................................. 158
8 Optical Stretcher for Single Cells ......................... 161
Karla Muller, Anatol Fritsch, Tobias Kiessling, Marc
Crosseruschkamp, and Josef A. Kas
8.1 Introduction .......................................... 161
8.2 Theory, Methods and Experimental Setup ................ 163
8.2.1 Fundamentals of Optical Stretching ............. 164
8.2.1.1 Ray Optics ............................ 165
8.2.1.2 Resulting Forces ...................... 167
8.2.2 Microfluidics - Laminar Flow ................... 169
8.3 Applications .......................................... 170
8.3.1 Cancer Diagnostics ............................. 171
8.3.2 Minimally Invasive Analysis .................... 172
8.3.3 Stem Cell Characterization ..................... 173
8.4 Outlook ............................................... 173
References ................................................. 174
Part III Single Cell Analysis: Applications
9 Single Cell Immunology ..................................... 175
Ulrich Walter and Jan Buer
9.1 Introduction .......................................... 175
9.2 Single Cell Gene Expression Profiling ................. 175
9.2.1 Single Cell (Multiplex) RT-PCR ................. 175
9.2.2 Quantitative Single Cell Multiplex RT-PCR ...... 179
9.3 Fluorescence-Activated Cell Sorting ................... 180
9.4 Live Cell Fluorescence Microscopy ..................... 183
9.4.1 Confocal Laser Scanning Microscopy ............. 183
9.4.2 Total Internal Reflection Fluorescence
Microscopy ..................................... 185
9.4.3 Forster Resonance Energy Transfer Imaging ...... 186
9.4.4 Two-Photon Laser Scanning Microscopy ........... 186
9.5 Other Techniques for Single Cell Analysis ............. 187
9.5.1 Enzyme-Linked Immunospot Assay ................. 187
9.5.2 In Situ Hybridization .......................... 188
9.5.3 Electron Microscopy ............................ 188
9.6 Conclusions and Outlook ............................... 189
References ................................................. 189
10 Molecular Characterization of Rare Single Tumor Cells ...... 197
James F. Leary
10.1 Introduction ......................................... 197
10.1.1 Importance of Rare Cells ...................... 197
10.1.2 Detection of Rare Tumor Cells ................. 198
10.2 Finding Rare Event Tumor Cells in Multidimensional
Data .................................................. 199
10.2.1 Rare Event Sampling Statistics ................ 200
10.2.2 High-Speed Sorting of Rare Cells .............. 203
10.2.3 Sorting Speeds must be Fast Enough to be
Practical ..................................... 203
10.2.4 Limits in Sorting Speeds and Purities ......... 204
10.3 Classification of Rare Tumor Cells ................... 205
10.3.1 Using Classifiers to Sort Rare Tumor Cells .... 208
10.4 Molecular Characterization of Sorted Tumor Cell
Cells ................................................. 209
10.4.1 Model Cell Systems ............................ 209
10.4.2 Design of PCR Primers to Detect the PTEN
Gene Region ................................... 209
10.4.3 Processing BT-549 Human Breast Cancer Cells ... 210
10.5 Detection of Mutated Sequences in Tumor Suppressor
Genes ................................................. 211
10.5.1 Detection of Mutations in Breast Cancer
Tumor Suppressor Genes by High-Throughput
Flow Cytometry, Single Cell Sorting and
Single Cell Sequencing ......................... 211
10.5.2 Single Cell Sorting for Mutational Analysis
by PCR ......................................... 214
10.5.3 ТА Cloning ..................................... 215
10.5.4 Single Cell Analysis of Gene Expression
Profiles ....................................... 216
10.6 Conclusions and Discussion ............................ 219
References ................................................. 219
11 Single Cell Heterogeneity .................................. 223
Edgar A. Arriaga
11.1 Introduction .......................................... 223
11.2 Measuring Heterogeneity using Single Cell
Techniques ............................................ 224
11.2.1 Optical Well Arrays ............................ 225
11.2.2 Capillary Electrophoresis Analysis of
Organelles Released from Single Cells .......... 225
11.3 Describing Cellular Heterogeneities and
Subpopulations ........................................ 227
11.4 Origins of Cellular Heterogeneity ..................... 228
11.5 Identifying Extrinsic and Intrinsic Noise Sources ..... 230
11.5.1 Validation of the Flow Cytometry
Measurements ................................... 230
11.5.2 Noise Dissection ............................... 232
11.5.3 Identification of Deviant Gene Products ........ 232
11.5.4 Correlation of Gene Products with Potential
Sources of Noise ............................... 232
11.6 Concluding Remarks .................................... 233
References ................................................. 234
12 Genome and Transcriptome Analysis of Single Tumor Cells .... 235
Bemhard Polzer, Claudia H. Hartmann, and Christoph
A. Klein
12.1 Introduction .......................................... 235
12.2 Detection and Malignant Origin of Disseminated
Cancer Cells .......................................... 235
12.3 Methods for Amplifying Genomic DNA of Single Cells .... 237
12.4 Studying the Genome of Single Disseminated Cancer
Cells ................................................. 239
12.5 The Need for Higher Resolution: Array CGH of Single
Cells ................................................. 240
12.6 Studying the Gene Expression of Single Disseminated
Cancer Cells .......................................... 241
12.7 Combined Genome and Transcriptome Analysis of
Single Disseminated Cancer Cells ...................... 246
References ................................................. 246
Index ......................................................... 251
|
