Preface ...................................................... XIII
List of Contributors ........................................... XV
1 Origin, Current Status, and Future Perspectives of
Chemical Cytometry ........................................... 1
Norman J. Dovichi
1.1 The Cell and Cytometry .................................. 1
1.2 Flow and Image Cytometry ................................ 1
1.3 Chemical Cytometry ...................................... 2
1.3.1 Prehistory of Chemical Cytometry -
Microchemistry, Microspectroscopy, and
Microseparations ................................. 3
1.3.2 Early History of Chemical Cytometry - Before
Ultrasensitive Detection ......................... 4
1.3.3 Prehistory of Chemical Cytometry - The Origins
of Capillary-based Separations ................... 5
1.3.4 Prehistory of Chemical Cytometry - The Origins
of Ultrasensitive Detection ...................... 6
1.3.5 Prehistory of Chemical Cytometry -
The Polymerase Chain Reaction and Fluorescence-
based DNA Sequencing ............................. 6
1.3.6 Chemical Cytometry: Protein Analysis -
The First Experiments ............................ 7
1.3.7 Chemical Cytometry: Protein Analysis - Cell
Lysis ............................................ 7
1.3.8 Chemical Cytometry: Protein Analysis - Native
Fluorescence ..................................... 8
1.3.9 Chemical Cytometry: Protein Analysis -
On-column Labeling ............................... 9
1.3.10 Chemical Cytometry: Protein Analysis - Two-
dimensional Capillary Electrophoresis ........... 10
1.4 Chemical Cytometry of DNA and mRNA ..................... 10
1.5 Metabolic Cytometry .................................... 10
1.5.1 Directed Metabolic Cytometry .................... 11
1.5.2 Shotgun Metabolic Cytometry ..................... 11
1.6 Future Perspectives on Instrumentation for Chemical
Cytometry .............................................. 12
1.6.1 Chemical Cytometry of Primary Cells Is the
Wave of the Future .............................. 12
1.6.2 Challenges - Tissue Dissociation Can Introduce
Changes in a Cell's Composition ................. 12
1.6.3 Challenges - Cells Need to Be Fixed to Prevent
Changes Associated with Sample Handling ......... 13
1.6.4 Challenges - The Connection Between the Real
World and the World of Chemical Cytometry ....... 13
Acknowledgments ............................................. 14
References .................................................. 14
2 Metabolic Cytometry - The Study of Clycosphingolipid
Metabolism in Single Primary Cells of the Dorsal Root
Ganglia ..................................................... 21
Colin D. Whitmore, Jillian Prendergast, David Essaka,
Ole Hindgaul, Monica P. Palcic, Ronald L. Schnaar, and
Norman J. Dovichi
2.1 Introduction ........................................... 21
2.1.1 Glycosphingolipids Play an Important Role in
Neuronal Membranes .............................. 21
2.1.2 The Metabolism of Glycosphingolipids Is Quite
Complex ......................................... 21
2.1.3 Expression of Glycosphingolipids Is Quite
Heterogeneous in Primary Tissues ................ 22
2.1.4 Metabolic Cytometry Has Been Used to
Characterize Glycosphingolipid Metabolism in
Model Cell Systems .............................. 23
2.2 Material and Methods ................................... 24
2.2.1 Reagents ........................................ 24
2.2.2 Cells and Cell Culture .......................... 24
2.2.3 Uptake of Fluorescent Gmi ....................... 25
2.2.4 Homogenate and Single Cell Preparation .......... 25
2.2.5 Capillary Electrophoresis ....................... 25
2.3 Results and Discussion ................................. 26
2.3.1 Dorsal Root Ganglia Homogenate .................. 26
2.3.2 DRG Single Cells ................................ 26
2.4 Conclusions ............................................ 29
Acknowledgments ............................................. 29
References .................................................. 29
3 Cell Signaling Studied at the Single-cell Level ............. 31
Angela Proctor, Shan Yang, Sumith Kottegoda, Michael
Brenner, Ryan M. Phillips, Christopher E. Sims, and
Nancy L. Allbritton
3.1 Introduction ........................................... 31
3.2 Analytes Examined and Reporters Used ................... 32
3.2.1 Reporter Properties ............................. 32
3.2.2 Probing System Function ......................... 33
3.2.3 Peptides as Reporters ........................... 33
3.2.4 Lipids as Reporters ............................. 35
3.2.5 Secondary Reporters ............................. 35
3.3 Cell Preparation and Reporter Loading .................. 35
3.3.1 Disruptive Loading Methods ...................... 36
3.3.2 Nondisruptive Loading Methods ................... 36
3.4 Cell Lysis and Sampling Techniques ..................... 37
3.4.1 Chemical Lysis .................................. 37
3.4.2 Hypotonic Lysis ................................. 39
3.4.3 Laser Lysis ..................................... 39
3.4.4 Electrical Lysis ................................ 40
3.4.5 Sampling Techniques ............................. 41
3.4.6 Whole Cell Sampling ............................. 41
3.4.7 Subcellular Sampling ............................ 42
3.5 Electrophoresis Separation Conditions .................. 43
3.5.1 Buffer Exchange ................................. 43
3.5.2 No Buffer Exchange .............................. 44
3.5.3 Microchannel Electrophoretic Separations ........ 45
3.6 Detection .............................................. 46
3.6.1 Instrumentation ................................. 46
3.6.1.1 Excitation ............................. 46
3.6.1.2 Fiber-optic Cable-based Excitation
Pathway ................................ 48
3.6.1.3 Emission ............................... 48
3.6.1.4 Data Collection and Analysis ........... 48
3.6.2 Limit of Detection (LOD) ........................ 49
3.6.3 Detection in a Microfluidic Device .............. 49
3.7 Automation and Throughput .............................. 50
3.7.1 Increasing Throughput ........................... 50
3.7.2 Capillary-based Devices ......................... 51
3.7.3 Microfluidic-based Devices ...................... 52
References .................................................. 52
4 Ultrasensitive Detection of Low-copy-number Molecules from
Single Cells ................................................ 55
Kangning Ren, Hongkai Wu
4.1 Introduction ........................................... 55
4.2 Microchip Designs for Single-cell Analysis and/or
Cell Manipulation ...................................... 55
4.3 Ultrasensitive Detection Methods for Single-cell
Analysis ............................................... 57
4.3.1 Fluorescence Detection Method ................... 57
4.3.2 Fluorescence Labeling ........................... 58
4.3.3 Optical Configuration ........................... 60
4.3.4 Molecule-counting Algorithm ..................... 63
4.4 Single-cell Analysis with Single-molecule Sensitivity
on Integrated Microfluidic Chip ........................ 65
4.4.1 Microfluidic Chip Fabrication ................... 66
4.4.2 Analysis of β2AR in SF9 Cells ................... 67
4.4.3 Analysis of Synechococcus ....................... 68
4.4.3.1 Electrophoretic Separation of
Synechococcus Lysate ................... 68
4.4.3.2 Synechococcus Analysis Procedure ....... 69
4.5 Conclusions ............................................ 71
References ............................................. 71
5 Capillary Electrophoresis of Nucleic Acids at the Single-
cell Level .................................................. 75
Ni Li and Wenwan Zhong
5.1 Introduction ........................................... 75
5.2 On-line Cell Analysis .................................. 76
5.2.1 Cell Injection and Lysis ........................ 76
5.2.2 In-column DNA or RNA Amplification with
Integrated Devices .............................. 80
5.2.2.1 Stream-lined Instrumental Setup ........ 80
5.2.2.2 Optimization for Reactions ............. 84
5.2.2.3 Analysis of Amplified Products ......... 87
5.3 Direct Gene and Gene Expression Analysis Without
Amplification
5.4 Potential Alternative Techniques for Single-cell
Gene and Gene Expression Analysis ....................... 88
5.5 Conclusions ............................................. 90
References ................................................... 90
6 Microfluidic Technology for Single-cell Analysis ............ 93
Yan Chen and Jiang F. Zhong
6.1 Introduction ........................................... 93
6.1.1 Limitation of Current Technology ................ 93
6.1.2 Microfluidic Devices ............................ 94
6.2 Biological Significance of Single-cell Analysis ........ 94
6.2.1 Investigate Gene Regulation in Consecutive
Developmental Stages ............................
6.2.2 Identifying Cancer Stem Cells (CSCs) Molecular
Signature ....................................... 95
6.3 Microfluidic Devices in Our Laboratories ............... 96
6.3.1 Microfluidic Single-cell mRNA Extraction
Device .......................................... 97
6.3.2 Functional Components of Single-cell Analysis
Devices ......................................... 97
6.3.3 Manipulation of Single Cells .................... 99
6.4 Materials, Methods, and Protocols ..................... 100
6.4.1 Materials ...................................... 100
6.4.2 Methods ........................................ 101
6.4.3 Device Operation Protocols ..................... 102
6.4.3.1 Microfluidic Chip Control ............. 102
6.4.3.2 Column Construction and Cell Lysis .... 103
6.4.3.3 Capturing mRNA, Synthesizing First
Strand cDNA, and Recovery of cDNA ..... 103
6.4.3.4 Analysis of Single-cell cDNA .......... 103
6.5 Conclusions ........................................... 104
References ............................................ 105
7 On-chip Electroporation and Electrofusion for Single-cell
Engineering ................................................ 107
Ana Valero and Albert van den Berg
7.1 Introduction .......................................... 107
7.2 Single-cell Electroporation in Microfluidic Devices ... 108
7.2.1 Microdevices for Analyzing Cellular
Properties or Intracellular Content ............ 110
7.2.2 Electroporation Microdevices for Cell
Inactivation ................................... 112
7.2.3 Electroporation Microdevices for Gene
Transfection ................................... 113
7.3 Single-cell Electrofusion in Microfluidic Devices ..... 117
7.4 Conclusions ........................................... 120
References ................................................. 121
8 Electroporative Flow Cytometry for Single-cell Analysis .... 125
Chang Lu, Jun Wang, Ning Bao, and Hsiang-Yu Wang
8.1 Introduction .......................................... 225
8.2 Flow-through Electroporation under Constant Voltage ... 126
8.3 Electroporative Flow Cytometry for Detecting Protein
Translocation ......................................... 128
8.4 Electroporative Flow Cytometry for Measuring
Single-cell Biomechanics .............................. 134
8.5 Electroporative Flow Cytometry for Selectively
Releasing and Analyzing Specific Intracellular
Molecules ............................................. 137
8.6 Conclusions ........................................... 139
Acknowledgments ............................................ 139
References ................................................. 139
9 Ultrasensitive Analysis of Individual Cells via Droplet
Microfluidics .............................................. 143
Robert M. Lorenz and Daniel T. Chiu
9.1 Introduction .......................................... 143
9.2 Droplet Properties .................................... 143
9.3 Droplet Generation .................................... 144
9.4 Cell Encapsulation .................................... 246
9.5 Droplet Manipulation .................................. 147
9.6 Droplet Concentration Control ......................... 149
9.7 Temperature Control of Droplets ....................... 150
9.8 Detection in Droplets ................................. 252
9.9 Conclusions ........................................... 255
10 Probing Exocytosis at Single Cells Using
Electrochemistry ........................................... 259
Yan Dong, Michael L. Heien, Michael E. Kurczy, and Andrew
G. Ewing
10.1 Introduction .......................................... 159
10.2 Measurement Requirements .............................. 160
10.3 Electrode Fabrication ................................. 160
10.4 Measurements at Single Cells .......................... 161
10.5 Fusion Pore Dynamics .................................. 163
10.5.1 Studying Fusion Pore in Living Cells ........... 163
10.5.2 Studying Fusion Pore in Artificial Cells ....... 165
10.5.3 Flickering Fusion Pore ......................... 167
10.6 Conclusions ........................................... 170
Acknowledgments ............................................ 171
References ................................................. 171
11 Electrochemical Determination of Enzyme Activity in
Single Cells ............................................... 175
Wenrui Jin
11.1 Introduction .......................................... 175
11.2 Electrochemical Detection Coupled with Capillary
Electrophoresis ....................................... 175
11.2.1 Determination of Activity of Glucose-6-
phosphate Dehydrogenase (G6PDH) in Single
Human Erythrocytes ............................. 177
11.2.2 Separation and Determination of Activity of
Alkaline Phosphatase (ALP) Isoenzymes in
Single BALB/c Fibrolast Cells of Mouse Bone
Marrow ......................................... 178
11.2.3 Separation and Determination of Activity of
Lactate Dehydrogenase (LDH) Isoenzymes in
Single Rat Glioma Cells ........................ 179
11.3 Voltammetry ........................................... 280
11.4 Scanning Electrochemical Microscopy (SECM) ............ 183
11.5 High-throughput ECD ................................... 188
11.6 Perspective ........................................... 190
Acknowledgment ............................................. 190
References ................................................. 191
12 Single-cell Mass Spectrometry .............................. 197
Ann Knolhoff, Stanislaw Rubakhin, and Jonathan
V. Sweedler
12.1 Introduction .......................................... 297
12.2 Mass Spectrometry ..................................... 198
12.2.1 Matrix-assisted Laser Desorption/Ionization .... 199
12.2.1.1 Sample Preparation for Single-cell
MALDI ................................. 200
12.2.1.2 Recent Applications of Single-cell
MALDI ................................. 201
12.2.2 Secondary Ion Mass Spectrometry ................ 205
12.2.2.1 Sample Preparation for Single-cell
SIMS .................................. 206
12.2.2.2 Recent Applications of Single-cell
SIMS .................................. 207
12.2.3 Electrospray Ionization ........................ 210
12.2.3.1 Recent Applications of Single-cell
ESI ................................... 210
12.2.4 Other MS Approaches ............................ 212
12.3 Overall Outlook for Single-cell MS .................... 213
Acknowledgments ............................................ 213
References ................................................. 213
13 Optical Sensing Arrays for Single-cell Analysis ............ 219
Ragnhild D. Whitaker and David R. Walt
13.1 Introduction to Fiber-optic Single-cell Arrays ........ 219
13.2 Advantages of Fiber-optic Single-cell Arrays .......... 220
13.3 Fiber-optic Arrays .................................... 222
13.4 Single-cell Arrays for Bacteria ....................... 223
13.4.1 Array Fabrication ............................. 223
13.4.2 Labeling and Detection of Cellular Responses
in Bacteria Arrays ............................ 224
13.5 Single-cell Arrays for Yeast .......................... 226
13.5.1 Array Fabrication .............................. 226
13.5.2 Labeling and Detection of Cellular Responses
in Yeast Arrays ................................ 228
13.6 Single-cell Arrays for Mammalian Cells ................ 229
13.7 Image and Data Analysis for Single-cell Arrays ... 234
13.8 Summary .......................................... 234
References ................................................. 236
Index ......................................................... 239
|
