Handbook of Single-Cell Technologies

Preface Contents About the Editors Contributors Part I: Physical Methods for Single Cell Therapy and Analysis 1 Light-Induced Cellular Delivery and Analysis Introduction Light-Induced Intracellular Delivery from Bulk to Single-Cell Photoporation Direct Laser-Cell Interaction Indirect Laser-Cell Interaction Pulsed Laser Systems for Optoporation Nanosecond Laser Femtosecond Laser Modifications of the Method Applications of Single-Cell Surgery Conclusion References 2 Mechanoporation: Toward Single Cell Approaches Introduction Types of Mechanoporation Particle Bombardment Microinjection Nanoneedle AFM Tip Microinjection Using Microfluidic Platforms Parallel Delivery Oscillating Nanoneedle Array Pressure Driven Devices Hydrodynamic Effect Sonoporation Shear Flow Devices A Cone and Plate Shearing Device Microchannel-Based Devices Mechanical Confinement High-Throughput Parallel Mechanical Confinement Device Microfluidic Devices Constriction Channel-Based Delivery Cell Squeeze Platform DNA Transfection Via Mechanical and Electrical Cell Membrane Disruption Advantages and Limitations of Mechanoporation Future Prospects Summary References 3 Single-Cell Electroporation Introduction Brief History Operation Mechanism Challenges in Macroscale Electroporation Landscape Transformation Electroporation Theories Intracellular Molecular Transport Cell Membrane Resealing Dynamics Microscale Electroporation Patch-Clamp Based Techniques Patch-Free Techniques Feedback-Driven Approaches Conclusion and Future Outlook References 4 Microinjection for Single-Cell Analysis and Therapy Introduction Development of Microinjection History of Microinjection Types of Cargos Used for Microinjection Types of Host Cells Used in Microinjection Instruments for Microinjection Microinjection for Single-Cell Analysis Injection for Analysis of Changes in Cells Transgenic Animal Production Modern Advancements for Increasing Efficiency Microinjection for Single-Cell Therapy Intracytoplasmic Sperm Injection for Treatment of Infertility Gene Therapy by Injection Microneedles for Therapeutic Uses Advantages Disadvantages Future Perspective and Conclusion References Part II: Fluidic System and Integration 5 Single-Cell Manipulation Introduction Flow Modeling of Hydrodynamic Cell Manipulation Fluidic Resistance Versus Electric Resistance Conservation of Mass and Energy Versus Kirchhoff´s Current Law and Voltage Law Division of Pressure and Flow Rate Versus Division of Voltage and Current Examples of Microfluidic Manipulation Single-Cell Trapping Co-culture of Single Cells Deforming, Sorting, and Separating Single Cells Integration with Other Methods Optical Integration Dielectrophoretic Integration Magnetic Integration Acoustic Integration Conclusion References 6 Single Cell Manipulation Using Macro-scale Actuator Introduction Transfer Function of Macro-to-Micro Manipulation (Mizoue et al. 2017) The Driving Mechanism Mechanical Model System Identification Through Experiments On-Chip Transmitter for Enhancing Manipulation Speed (Monzawa et al. 2015) Advantages of Using an Actuation Transmitter Mechanical Model and Theoretical Basis Experimental Validation on Manipulation with an Actuation Transmitter Recent Works and Applications Summary References 7 Inertial Microfluidics for Single-Cell Manipulation and Analysis Introduction Underlying Physics Inertial Migration Dean Flow Viscoelasticity Guidelines for Designing Spiral Inertial Microfluidics Dimensionless Numbers Design Guidelines Cross-Sectional Dimensions Channel Length (Loop Number) Other Structural Parameters Other Functional Units Operational Parameters Improved Understandings on Spiral Inertial Microfluidics Application Guidelines for Spiral Inertial Microfluidics Focusing/Ordering Separation Concentration/Microfiltration Conclusion and Future Perspective References 8 Digital Microfluidics for Single Cell Manipulation and Analysis Introduction DMF Systems DMF Technology Electrowetting-on-Dielectric Electrodewetting Liquid Dielectrophoresis Dielectrophoresis Optoelectrowetting Optoelectronic Tweezers Magnetic Force Fabrication of DMF DMF Manipulation and Analysis of a Single Cell Adherent Cell Suspension Cell Long-Term Culture Related Applications of DMF Cell Sorting and Concentrating 3D Cell Culture Diagnosis and Clinical Application Conclusions and Future Outlook References 9 Single-Cell Separation Introduction Conventional Cell Separation Techniques Centrifugation Fluorescence-Activated Cell Sorting Magnetic-Activated Cell Sorting Laser Capture Microdissection Manual Cell Picking Microfluidic Single-Cell Separation Techniques Microfluidic Passive Separation Techniques Filter-Based Separation Deterministic Lateral Displacement Hydrodynamic Separation Non-inertial Hydrodynamic Separation Inertial Hydrodynamic Separation Microfluidic Active Separation Techniques Dielectrophoresis (DEP) Magnetic Separation Acoustophoresis Affinity-Based Separation Comparison Between Different Microfluidic Separation Techniques Conclusion References 10 Technologies for Automated Single Cell Isolation Introduction Cell Samples Size and Morphology of Cells Sample Preparation: Single-Cell Suspensions Basic Considerations, Definitions, and Classifications for Single-Cell Isolation Automated Single-Cell Isolation Technologies Limiting Dilution Suitability for Microbial Cells Fluorescence-Activated Cell Sorting Suitability for Microbial Cells Single-Cell Dispensing (SCD) Suitability for Microbial Cells Microfluidic Single-Cell Isolation Hydrodynamic Trapping Vertical Trapping in Nanowells Suitability for Microbial Cells Droplet Microfluidics Single-Cell Manipulation (on Microfluidic Chips) by Directed External Forces Optical Tweezers (OT) Dielectrophoresis (DEP) Suitability for Microbial Cells Automated Micromanipulators Discussion and Conclusion References 11 Dual-Well Microfluidic Technique for Single Cell Isolation and Long-Term Clonal Culture Introduction Applications of Monoclonal Cell Culture Overview of Single Cell Isolation Methods for Clonal Culture The Design Concept of the Dual-Well Technique Fabrication of Dual-Well Device Fabrication of Master Molds by Photolithography Technology Molding PDMS Device with the Master Molds Dual-Well Device Preparation for Single Cell Isolation Cell Suspension Preparation for Single Cell Isolation with Dual-Well Device Single Cell Isolation and Clonal Culture with Dual-Well Device Culture Medium Replacement of the Dual-Well Device Discussion The Effect of Capture Well Depth on Single Cell Efficiency The Effect of Washing Flow Rate on Single Cell Efficiency The Effect of Cell Type on Single Cell Capture Efficiency The Effect of Device Flipping on Cell Transfer Efficiency Single Cell-Derived Clonal Colonies and Stem Cell Differentiation in the Microwells of the Dual-Well Device Conclusion References 12 Single-Cell Cultivation Utilizing Microfluidic Systems Introduction Purpose of Single-Cell Cultivation Morphology Proliferation and Differentiation Migration Genomics, Transcriptomics, Proteomics, and Metabolomics Epigenomics Single-Cell Cultivation in Microfluidic Devices Cell Coculture Neural Coculture Coculture of Tumor Cells to Neighboring Cells Coculture of Neurons and Cancer Cells Molecule-Induced Cellular Behaviors Molecular Cue-Guided Neuron Molecule and Immune Cell Interaction Molecule and Microbe Interaction Regeneration Axotomy Stem Cell-Based Regeneration Further Limitations and Future Prospects Conclusion References 13 Integrated Microwell Array Technologies for Single Cell Analysis Introduction Microfabrication of a Microwell Array Material and Design Considerations Soft Photolithography for Fast Prototyping of Microwell Arrays Hydrophilic-in-Hydrophobic Microwells Polyethylene Glycol Microwells for Reduced Non-specific Adsorption Single Cell Docking Docking Strategies: Manual Administration of Cells Docking Strategies: Microfluidics-Assisted Administration of Cells Continuous Flow Microfluidics Digital Microfluidics Single Cell Manipulation Optical Manipulation Magnetic Manipulation Mechanical Manipulation Electric Manipulation Applications Single Cell Drug Screening Single Cell Omics Detection of Single Cell Secreted Products Other Applications Conclusion References 14 Micro- and Nanopore Technologies for Single-Cell Analysis Coulter Principle A History Behind the Invention of Coulter Principle Single-Cell Counting Mechanism Single-Cell Analysis with Conventional Coulter Counters Solid-State Micro- and Nanopores: Structures and Fabrication Procedures Electron and Ion Beam Milling Dielectric Breakdown Glass Nanopipette Lithographically Defined Cross-Membrane Nanopore Tunable Nanopore Material Coating (ALD, SEM, Molecule Coating) Focused Ion Beam Lithography Electron Beam Lithographed Micro-/Nanochannel Nanoimprint Lithography Carbon Nanotubes Solid-State Micro- and Nanopores: Functions Beyond Particle Sizing Single-Particle Shape Analysis Single-Particle Surface Charge Measurements Intermolecular Interactions Single-Cell Analysis Using Micro- and Nanopores Volume Discrimination of Single Cells Using Advanced Multichannel Sensors Single-Cell Shape Analysis Using Low Aspect Ratio Pores AI-Driven Resistive Pulse Analysis for Discriminating Single Cells Biorecognition Pore Sensors Potential and Challenges for Total Cell Analysis Conclusion References 15 Technologies for Single-Cell Printing and Patterning Introduction Acoustophoresis and Fluorescence-Activated Cell Sorting Laser and Vacuum Microdissection Impedance-Based Single-Cell Printing Optical Platforms for Single-Cell Printing Inkjet-Based Single-Cell Printing Microcontact Printing Droplet-Based Patterning of Single Cells Single Microbial Cell Printing Conclusion References 16 Microfluidic Device with Removable Electrodes for Single Cell Electrical Characterization Introduction Principle of Impedance Measurement inside Microfluidic Channel Microfluidic Device and Impedance Measurement Cell Culture Device Fabrication Device Operation Electrical Measurement Procedure Impedance Measurement inside Microfluidic Impedance of Cell Population Impedance of Single Particle Conclusions References 17 Microfluidic and Nanomaterial Approach for Virology Introduction General Understanding of Host-Viral Interaction How Do Hosts Respond to Intruders and What Is Viral Feedback? Microfluidic Technique in the Field of Virology Flow-Based Channel Microfluidics Droplet-Based Microfluidics Electric Field-Based Digital Microfluidics (DMF) Microfluidic and Nanoparticle-Based Diagnostic Devices Paper-Based Device Gold Nanoparticles Microfluidics for Host-Viral Interaction Study Conclusion Future Direction References Part III: Chemical Methods for Single Cell Technology 18 Liposome-Mediated Material Transfer in Single Cells Introduction Preparation and Physical Characteristics of Liposomes Liposome Fusion as a Model System Transfer of Materials from Liposomes into Cells Single-Cell Modification Techniques Discussion and Conclusion References 19 The Art of Therapeutic Antibody Discovery: Finding Them One Cell at a Time Introduction Technological Arts for Monoclonal Antibody Discoveries Immortalization of Memory B Cells Humanized Mice Single B Cell Cloning Single-Cell Antibody Nanowells Application of Single-Cell Antibody Discovery in the Human Conditions Dengue Antibody Dependent Enhancement (ADE) Neutralizing Antibodies in Dengue Autoimmune Diseases Monoclonal Antibody Treatments in Autoimmune Diseases Cytokine Targeting Biologic Therapies in Autoimmune Diseases Anti-Tumor Necrosis Factor-α (TNF-α) Anti-interleukin (IL)-6 Anti-IL-17 T and B Cell Depleting Biologic Therapies in Autoimmune Disease Challenges in Monoclonal Antibody Therapy Conclusion References 20 Screening of Antigen-Specific Antibody-Secreting Cells Introduction Importance of mAb Discovery and Production Conventional Methods in Screening ASCs Hybridoma Production EBV Immortalization Phage Display Flow Cytometry Single-Cell Screening Methods for ASCs Modified Flow Cytometry Microwell Arrays Droplet Microfluidics Conclusion References 21 Biochemical Analysis of Secreted Molecules by Individual Cells Introduction Secretion Analysis Single-Cell Analysis of Secretion Technologies for Single-Cell Analysis of Secretion ELISpot and Derivatives Cytometry-Based Methods Micro- and Nanowell Assays Microchamber Assays Droplet-Based Microfluidics Label-Free Optical Methods Limitation of the Current Technologies and Future Directions Conclusion References Part IV: Single Cell Omics 22 Single Cell Genomics Introduction Methods of Whole Genome Amplification for Single Cell Genomics Single Cell Genomics Is Revolutionizing Cancer Biology Somatic Mosaicisms in Development and Disease Clinical Applications of Single Cell Genomics Single Cell Genomics in Microbiology Epigenetics Meets Single Cell Technology Conclusion References 23 Single-Cell Proteomics Introduction Technology Platforms and Methodologies for Single-Cell Proteomics Capillary Electrophoresis (CE) Chemical Cytometry Microfluidics Mass Spectrometry (MS) Flow Cytometry Mass Cytometry Challenges, Limitations, and Outlook References 24 Single Cell Pull-Down for Characterization of Protein Complexes Introduction Fluorescence Methods for Characterization of Protein Complex High-Throughput Screening of Protein-Protein Interactions Single Molecule Fluorescence Detection of Protein Complexes Single Cell Pull-Down Surface Chemistry for Single Cell Pull-Down Live Cell Micropatterning High Affinity Capturing of Target Protein Complexes from a Single Cell Dissociation Kinetics of Protein Complex Determination of the Stoichiometry of Protein Complexes Automated Large-Scale Single Molecule Analysis Single Cell Pull-Down for Label Free Detection Conclusions References 25 Single-Cell Transcriptomics Introduction Techniques for Single-Cell Transcriptome Analysis Single-Cell Isolation cDNA Library for sc-qPCR cDNA Library for scRNA-seq Methodology Selection Single-Cell Transcriptome in Stem Cell Biology Cellular Classification Developmental Studies Stem Cells in Adult Tissues Single-Cell Transcriptomics for Immunology Research Studies on Innate Immune System Studies on Adaptive Immune System Studies on Immune System and Cancer Single-Cell Transcriptomics in Cancer Research Intratumor Heterogeneity Led by the Tumor Microenvironment Study of Minor Populations in Cancer Circulating Tumor Cells Current Issues and Future Approaches Conclusions References 26 Single-Cell Transcriptome Sequencing Using Microfluidics Introduction: The Importance of Cellular Heterogeneity and Single-Cell Analysis Cellular Heterogeneity in Cell Biology Advantages of Single-Cell Analysis Using Microfluidics for Single-Cell Analysis Biochemistry of Single-Cell RNA-Seq Generation of Barcoded Beads Pairing a Barcoded Bead with a Cell Using Microfluidics Droplet-Based Microfluidics for Bead-Cell Pairing Using Microwell for Bead-Cell Pairing Bead-Cell Pairing Using Hydrodynamic Cell Capture Basics of Gene Sequencing and Data Analysis Gene Sequencing of Single-Cell Library Read Alignment to Quantify Gene Expression of Each Cell Statistical Analysis of Single-Cell Data Extension from Single-Cell Transcriptomics to Multiomics Challenges and Future Directions Reliability of Single-Cell RNA-Seq Fixation and Storage of Biological Samples Challenges in Single-Cell Multiomics Analysis Conclusion References Part V: Single Cell Analysis in Systems Biology and Biocatalysis 27 Single-Cell Phenotyping of Complex Heterogeneous Tissue Introduction Mass Matters: Shortfalls of Population Averaging Transcriptomics Through Population Averaging Will scRNAseq Be the Ultimate Solution? ``Clothes Make the Man´´: What Single-Cell Phenotyping Has to Offer Over Population Averaging The Coccygeal Bovine IVD as a Research Model Micro-Niche Style Living of Resident IVD Cells Heterogeneity Detection Through Transcript Analysis on a Cell-by-Cell Approach Cell Velocity as an Indicator for Behavioral Heterogeneity of Cell Populations Materials and Methods IVD Tissue Source and Cell Culture Conditions Nucleic Acid Hybridization Chromogenic Transcript Detection Transcript Detection Through Fluorescent Imaging Histology Post-SISH Immunohistochemistry for Protein Detection Cell Velocity Measurements Conclusions References 28 Record the Single Cell Signal Pathway Introduction Signal Transduction in Cells State-of-the-Art Methods Challenges of Rapid Mixing Concept and Theory Microfluidic Circuit Design Chip Fabrication and System Assembly Deflection Switching Time Quantification of Specific Phospho-Proteins Single-Cell Fluorescence Intensity Quantification Conclusion References 29 Single-Cell Microencapsulation for Evolution and Discovery of Biocatalysts Introduction Ultrahigh-Throughput Enzyme Screens: Directed Evolution and Discovery Single-Cell Encapsulation in Microfluidic Droplets Stochastic Encapsulation Deterministic Single-Cell Encapsulation Techniques Microfluidic Workflow for Directed Evolution of Enzymes Construction of Multistep Workflows for Single-Cell Assays Considerations for Setting Up Single Cell Assays Library Construction and DNA Transformation Single-Cell Phenotypic Variations Sensitivity of Fluorescence Detection Operation of a Microdroplet Sorter Formats for Single-Cell Biocatalyst Functional Screens Single-Cell Lysate Assays Single-Cell Internal Expression and Surface Display Single-Cell Secretion Assays Functional Metagenomics and Bioprospecting In Vitro Workflows as Artificial Single Cells Limitations and Future Prospects of Single-Cell Microencapsulation Conclusions References 30 Analytical Techniques for Single-Cell Studies in Microbiology Introduction Cytometry Flow Cytometry Imaging Flow Cytometry Scanning Cytometry Quantitative Optical Microscopy Brightfield Microscopy Fluorescence Microscopy Scanning Probe Microscopy Scanning Atomic Force Microscopy Scanning Electrochemical Microscopy Nanoscale Secondary Ion Mass Spectrometry Rotational-Vibrational Spectroscopy Conclusion References Part VI: Single Cell Technologies in Cancer 31 Single Cell Adhesion in Cancer Progression Introduction Cell Adhesion Molecules Cadherins Integrins Selectin Immunoglobulin Superfamily Cell-To-Extracellular Matrix (ECM) Adhesion Role of Cell Adhesion in Cancer Progression Epithelial-Mesenchymal Transition (EMT) Tumor Invasion Angiogenesis Intravasation Extravasation Techniques to Study Cell Adhesion and Migration Bulk Adhesion Measurements Centrifugation Assays Hydrodynamic Shear Assays Wash Assay Parallel Plate System Rotating Disk System Radial Flow System High-Throughput Cone and Plate (HT-CAP)-Electric Cell-Substrate Impedance Sensing (ECIS) Instrumentation Single Cell Techniques Micropipette Aspiration Step-Pressure Technique Biomembrane Force Probe Micropipette Aspiration (Narrow Sense) Atomic Force Microscopy (AFM) Nanofork and Line-Patterned Substratum Optical Tweezer Instrumentation Measuring Cell-to-Extracellular Matrix (ECM) Adhesion Strength Förster Resonance Energy Transfer (FRET) Cellular Traction Force Quantitative Intravital Microscopy Fluorescent Models to Study Tumor Heterogeneity Optical Imaging Windows References 32 Single-Cell Technologies for Cancer Therapy Introduction Heterogeneity of Single Cancer Cells The Origin of Cancer Cell Characteristics and Variability Vertical or Horizontal Gene Transfer in Single Cancer Cells Single-Cell Epigenetics Cancer Cells and Immune Response Virus Defense and Small RNA in Single Cells Cellular Surface Antigen, Immunity, and Canceration Microbiome and Canceration Communication and Application of Gut Microbiome Single Cell Technologies in Cancer Diagnosis and Treatment Single Cell Sequencing for Cancer Diagnosis and Biomarker Molecular Technology and Cancer Immunotherapy at Single Cell Resolution CRISPR: Treating Cancer and Posing Other Cancer Risk TCR and CAR-T at Single T Cells Resolution Real-Time Images in Single Cells Photodynamic Therapy Techniques for Single Cell Analysis Single Cell Partitioning Methods-Mechanical Partitioning and Microfluidic Device Circulating Tumor Cells Measurement PCR and NGS Single Cell qPCR NGS Sequencing in Cancer Treatment and Future Applications of Single Cell Analysis Cellular ``Barcode´´ Map Protein Crystallization Technology CyTOF and IscA Magnetic Protein TEM/Cryo-EM/SEM/Confocal 3D Images Global Projects, Database, and Bioinformatics Database and Bioinformatics from Single Cells or a Single Cell Global Projects at Single-Cell Resolution Human Genome Project (HGP) and Human Genome Diversity Project (HGDP) The Haplotype Map (HapMap) Human Genome Diversity Project (HGDP) The Human Proteome Project and Comparative Serum Proteomics Project Comparative Serum Proteomics Project Human Longevity Project The Cancer Genome Atlas (TCGA) Human Cell Atlas (HCA) TCR Diversity Database-ImmunoMap and Human Microbiome Project (HMP) High-Tech Omics-Based Patient Evaluation (HOPE) Project Personal Medicine: Gene Sequencing, Gene Drugs, and Precision Therapy at Single-Cell Resolution Anticancer Drug Development and Personal Medicine Cancer Vaccine Drug Testing and Organoids from Single Stem Cells Antibody Drugs and T Cells Immunotherapy Molecular Synthesis and Bio-genetical Engineering Drugs to Kill Drug-Resistant Drug-Drug Interaction Database and Drug Release Bioinfomatics and Analytical Framework for Single-Cell Meta-Analysis Algorithm, Grouping, and Data Visualization of Datasets from Single Cells Principal Component Analysis (PCA) Ingenuity Pathway Analysis Business Market: Single Cells Related Technology and Personalized Medicine Single Cell Specific Biomarkers and Service for Immunotherapy Human Longevity AI/Deep Learning and Service in Personal Medicine IBM Watson Conclusions References 33 Analytical Technology for Single-Cancer-Cell Analysis Introduction Conventional Analytical Technology for Cancer Cell Analysis Two-Dimensional (2D) Cell-Based Assay Three-Dimensional (3D) Cell-Based Assay Analytical Technology for Single-Cancer-Cell Analysis Single-Cell Sequencing Single-Cell Isolation Single-Cell Sequencing Single-Cell Analysis and Data Computation Inherent Traits of Single Cells Mechanical Traits of Single Cells Size Deformability Electrokinetic Properties Conclusion References 34 Transmembrane Receptor Dynamics as Biophysical Markers for Assessing Cancer Cells Applications of SPT/SMT in Biology Principle of Single-Particle Tracking (SPT) and Trajectory Analysis Structure-Property-Function-Disease Paradigm of TReD Assay Quantification of Metastatic Potential of Breast and Prostate Cancer Cells Biomolecular Mechanisms Guiding the Dynamics of Transmembrane Receptors Deep Learning-Based TReD Assay with Better Accuracy Future Direction References Part VII: Flow Cytometry for Single Cell Analysis 35 Single-Cell Impedance Flow Cytometry Introduction Conventional Characterization Approaches Patch Clamping Electrorotation Dielectrophoresis Impedance Flow Cytometry Prototype Demonstration Microfluidic Impedance Flow Cytometry Microfluidic Impedance Flow Cytometry Based on Constriction Channels Conclusion and Outlook References 36 Cytometry of Single Cell in Biology and Medicine Introduction Mechanisms of Cytometry Application of Cytometry in Microbial Study Total Bacterial Cell Count Bacterial Viability Analysis Specific Microbial Identification Multifunction Analysis Conclusions Application of Cytometry in Rare Cell Analysis Phenotype Analysis Genotype Analysis Conclusions Application of Cytometry in Medicine Fluorescent Staining Drug Discovery in Immunology and Receptor Pharmacology Conclusions Conclusions and Perspectives References Part VIII: Spectrum Analysis, Methods, Targets, Imaging, and Applications 37 Single Cell Electrophysiology Introduction Membrane Electrophysiology Cell Membrane Resting Membrane Potential and Action Potential Hodgkin-Huxley Model Neuron-Electrode Interface and Transfer Function from the Point Contact Model Recording Model Intracellular Recording Sharp Microelectrode Patch Electrode Cell-Attached Patch Whole-Cell Recording Patch Inside-Out Patch Outside -Out Patch Loose Patch Voltage Clamp Current Clamp Extracellular Recording Microelectrode Arrays (MEAs) Complementary Metal Oxide Semiconductor (CMOS) Thin-Film Transistor Array Technology Comparison Current Research Noise in Electrophysiological Measurements Thermal Noise Shot Noise Dielectric Noise Excess Noise Other Forms of Noise and General Precautions Summary References 38 Mechanical and Microwave Resonators for Sensing and Sizing Single Cells Introduction Single-Cell Characterization with Microelectromechical Systems (MEMS) MEMS Sensors and Early Efforts Adherent Cell Measurements with MEMS Sensors Working Inside Liquid Suspended Cell Measurements with Suspended Microchannel Resonators Single-Cell Sensing with Microfluidics-Integrated Microwave Sensors (MIMS) Conclusion References 39 Molecular Force Spectroscopy on Cells: Physiological Functions of Cell Adhesion Introduction Biochemical Aspects of SCFS Molecular Recognition Through ECM Application of SCFS in Nanobiotechnology Study of Single-Cell Structure and Migration Single-Molecule Force Spectroscopy (SMFS) Conclusion References 40 Micro-tweezers and Force Microscopy Techniques for Single-Cell Mechanobiological Analysis Introduction Micro-tweezers for Single-Cell Mechanical Stimulation Optical Tweezers Magnetic Tweezers Acoustic Tweezers Micropipette Aspiration Microfluidic Shear Device Force Microscopy Techniques for Single Cell Force Mapping Traction Force Microscopy 3D Traction Force Microscopy Elastic Micropost Arrays Fluorescence Resonance Energy Transfer-Based Sensors Atomic Force Microscopy Summary and Future Outlook References 41 Mass Spectrometry for Single-Cell Analysis Introduction Mass Spectrometry (MS) Techniques Electrospray Ionization Mass Spectrometry (ESI-MS) Technique Secondary Ion Mass Spectrometry (SIMS) Laser Deposition/Ionization Mass Spectrometry (LDI-MS) Inductively Coupled Plasma Mass Spectrometry (ICP-MS) Future Prospects of Mass Spectrometry Conclusions References 42 Acoustic Tweezers for Single-Cell Manipulation Introduction Principles and Theory of Acoustic Tweezers Wave Generation Theory of Acoustic Tweezers Acoustic Tweezer Technologies Standing Wave Tweezers Traveling Wave Tweezers Acoustic Streaming Tweezers Applications of Acoustic Tweezers in Single-Cell Studies Cell Printing/Patterning Cell Separation Standing SAW-Based Cell Separation Standing BAW-Based Cell Separation Traveling SAW-Based Cell Separation Cell Sorting Cell Imaging Cell Signaling Cell Stretching and Poration Conclusion References Index Preface Contents About the Editors Contributors Part I: Physical Methods for Single Cell Therapy and Analysis 1 Light-Induced Cellular Delivery and Analysis Introduction Light-Induced Intracellular Delivery from Bulk to Single-Cell Photoporation Direct Laser-Cell Interaction Indirect Laser-Cell Interaction Pulsed Laser Systems for Optoporation Nanosecond Laser Femtosecond Laser Modifications of the Method Applications of Single-Cell Surgery Conclusion References 2 Mechanoporation: Toward Single Cell Approaches Introduction Types of Mechanoporation Particle Bombardment Microinjection Nanoneedle AFM Tip Microinjection Using Microfluidic Platforms Parallel Delivery Oscillating Nanoneedle Array Pressure Driven Devices Hydrodynamic Effect Sonoporation Shear Flow Devices A Cone and Plate Shearing Device Microchannel-Based Devices Mechanical Confinement High-Throughput Parallel Mechanical Confinement Device Microfluidic Devices Constriction Channel-Based Delivery Cell Squeeze Platform DNA Transfection Via Mechanical and Electrical Cell Membrane Disruption Advantages and Limitations of Mechanoporation Future Prospects Summary References 3 Single-Cell Electroporation Introduction Brief History Operation Mechanism Challenges in Macroscale Electroporation Landscape Transformation Electroporation Theories Intracellular Molecular Transport Cell Membrane Resealing Dynamics Microscale Electroporation Patch-Clamp Based Techniques Patch-Free Techniques Feedback-Driven Approaches Conclusion and Future Outlook References 4 Microinjection for Single-Cell Analysis and Therapy Introduction Development of Microinjection History of Microinjection Types of Cargos Used for Microinjection Types of Host Cells Used in Microinjection Instruments for Microinjection Microinjection for Single-Cell Analysis Injection for Analysis of Changes in Cells Transgenic Animal Production Modern Advancements for Increasing Efficiency Microinjection for Single-Cell Therapy Intracytoplasmic Sperm Injection for Treatment of Infertility Gene Therapy by Injection Microneedles for Therapeutic Uses Advantages Disadvantages Future Perspective and Conclusion References Part II: Fluidic System and Integration 5 Single-Cell Manipulation Introduction Flow Modeling of Hydrodynamic Cell Manipulation Fluidic Resistance Versus Electric Resistance Conservation of Mass and Energy Versus Kirchhoff ́s Current Law and Voltage Law Division of Pressure and Flow Rate Versus Division of Voltage and Current Examples of Microfluidic Manipulation Single-Cell Trapping Co-culture of Single Cells Deforming, Sorting, and Separating Single Cells Integration with Other Methods Optical Integration Dielectrophoretic Integration Magnetic Integration Acoustic Integration Conclusion References 6 Single Cell Manipulation Using Macro-scale Actuator Introduction Transfer Function of Macro-to-Micro Manipulation (Mizoue et al. 2017) The Driving Mechanism Mechanical Model System Identification Through Experiments On-Chip Transmitter for Enhancing Manipulation Speed (Monzawa et al. 2015) Advantages of Using an Actuation Transmitter Mechanical Model and Theoretical Basis Experimental Validation on Manipulation with an Actuation Transmitter Recent Works and Applications Summary References 7 Inertial Microfluidics for Single-Cell Manipulation and Analysis Introduction Underlying Physics Inertial Migration Dean Flow Viscoelasticity Guidelines for Designing Spiral Inertial Microfluidics Dimensionless Numbers Design Guidelines Cross-Sectional Dimensions Channel Length (Loop Number) Other Structural Parameters Other Functional Units Operational Parameters Improved Understandings on Spiral Inertial Microfluidics Application Guidelines for Spiral Inertial Microfluidics Focusing/Ordering Separation Concentration/Microfiltration Conclusion and Future Perspective References 8 Digital Microfluidics for Single Cell Manipulation and Analysis Introduction DMF Systems DMF Technology Electrowetting-on-Dielectric Electrodewetting Liquid Dielectrophoresis Dielectrophoresis Optoelectrowetting Optoelectronic Tweezers Magnetic Force Fabrication of DMF DMF Manipulation and Analysis of a Single Cell Adherent Cell Suspension Cell Long-Term Culture Related Applications of DMF Cell Sorting and Concentrating 3D Cell Culture Diagnosis and Clinical Application Conclusions and Future Outlook References 9 Single-Cell Separation Introduction Conventional Cell Separation Techniques Centrifugation Fluorescence-Activated Cell Sorting Magnetic-Activated Cell Sorting Laser Capture Microdissection Manual Cell Picking Microfluidic Single-Cell Separation Techniques Microfluidic Passive Separation Techniques Filter-Based Separation Deterministic Lateral Displacement Hydrodynamic Separation Non-inertial Hydrodynamic Separation Inertial Hydrodynamic Separation Microfluidic Active Separation Techniques Dielectrophoresis (DEP) Magnetic Separation Acoustophoresis Affinity-Based Separation Comparison Between Different Microfluidic Separation Techniques Conclusion References 10 Technologies for Automated Single Cell Isolation Introduction Cell Samples Size and Morphology of Cells Sample Preparation: Single-Cell Suspensions Basic Considerations, Definitions, and Classifications for Single-Cell Isolation Automated Single-Cell Isolation Technologies Limiting Dilution Suitability for Microbial Cells Fluorescence-Activated Cell Sorting Suitability for Microbial Cells Single-Cell Dispensing (SCD) Suitability for Microbial Cells Microfluidic Single-Cell Isolation Hydrodynamic Trapping Vertical Trapping in Nanowells Suitability for Microbial Cells Droplet Microfluidics Single-Cell Manipulation (on Microfluidic Chips) by Directed External Forces Optical Tweezers (OT) Dielectrophoresis (DEP) Suitability for Microbial Cells Automated Micromanipulators Discussion and Conclusion References 11 Dual-Well Microfluidic Technique for Single Cell Isolation and Long-Term Clonal Culture Introduction Applications of Monoclonal Cell Culture Overview of Single Cell Isolation Methods for Clonal Culture The Design Concept of the Dual-Well Technique Fabrication of Dual-Well Device Fabrication of Master Molds by Photolithography Technology Molding PDMS Device with the Master Molds Dual-Well Device Preparation for Single Cell Isolation Cell Suspension Preparation for Single Cell Isolation with Dual-Well Device Single Cell Isolation and Clonal Culture with Dual-Well Device Culture Medium Replacement of the Dual-Well Device Discussion The Effect of Capture Well Depth on Single Cell Efficiency The Effect of Washing Flow Rate on Single Cell Efficiency The Effect of Cell Type on Single Cell Capture Efficiency The Effect of Device Flipping on Cell Transfer Efficiency Single Cell-Derived Clonal Colonies and Stem Cell Differentiation in the Microwells of the Dual-Well Device Conclusion References 12 Single-Cell Cultivation Utilizing Microfluidic Systems Introduction Purpose of Single-Cell Cultivation Morphology Proliferation and Differentiation Migration Genomics, Transcriptomics, Proteomics, and Metabolomics Epigenomics Single-Cell Cultivation in Microfluidic Devices Cell Coculture Neural Coculture Coculture of Tumor Cells to Neighboring Cells Coculture of Neurons and Cancer Cells Molecule-Induced Cellular Behaviors Molecular Cue-Guided Neuron Molecule and Immune Cell Interaction Molecule and Microbe Interaction Regeneration Axotomy Stem Cell-Based Regeneration Further Limitations and Future Prospects Conclusion References 13 Integrated Microwell Array Technologies for Single Cell Analysis Introduction Microfabrication of a Microwell Array Material and Design Considerations Soft Photolithography for Fast Prototyping of Microwell Arrays Hydrophilic-in-Hydrophobic Microwells Polyethylene Glycol Microwells for Reduced Non-specific Adsorption Single Cell Docking Docking Strategies: Manual Administration of Cells Docking Strategies: Microfluidics-Assisted Administration of Cells Continuous Flow Microfluidics Digital Microfluidics Single Cell Manipulation Optical Manipulation Magnetic Manipulation Mechanical Manipulation Electric Manipulation Applications Single Cell Drug Screening Single Cell Omics Detection of Single Cell Secreted Products Other Applications Conclusion References 14 Micro- and Nanopore Technologies for Single-Cell Analysis Coulter Principle A History Behind the Invention of Coulter Principle Single-Cell Counting Mechanism Single-Cell Analysis with Conventional Coulter Counters Solid-State Micro- and Nanopores: Structures and Fabrication Procedures Electron and Ion Beam Milling Dielectric Breakdown Glass Nanopipette Lithographically Defined Cross-Membrane Nanopore Tunable Nanopore Material Coating (ALD, SEM, Molecule Coating) Focused Ion Beam Lithography Electron Beam Lithographed Micro-/Nanochannel Nanoimprint Lithography Carbon Nanotubes Solid-State Micro- and Nanopores: Functions Beyond Particle Sizing Single-Particle Shape Analysis Single-Particle Surface Charge Measurements Intermolecular Interactions Single-Cell Analysis Using Micro- and Nanopores Volume Discrimination of Single Cells Using Advanced Multichannel Sensors Single-Cell Shape Analysis Using Low Aspect Ratio Pores AI-Driven Resistive Pulse Analysis for Discriminating Single Cells Biorecognition Pore Sensors Potential and Challenges for Total Cell Analysis Conclusion References 15 Technologies for Single-Cell Printing and Patterning Introduction Acoustophoresis and Fluorescence-Activated Cell Sorting Laser and Vacuum Microdissection Impedance-Based Single-Cell Printing Optical Platforms for Single-Cell Printing Inkjet-Based Single-Cell Printing Microcontact Printing Droplet-Based Patterning of Single Cells Single Microbial Cell Printing Conclusion References 16 Microfluidic Device with Removable Electrodes for Single Cell Electrical Characterization Introduction Principle of Impedance Measurement inside Microfluidic Channel Microfluidic Device and Impedance Measurement Cell Culture Device Fabrication Device Operation Electrical Measurement Procedure Impedance Measurement inside Microfluidic Impedance of Cell Population Impedance of Single Particle Conclusions References 17 Microfluidic and Nanomaterial Approach for Virology Introduction General Understanding of Host-Viral Interaction How Do Hosts Respond to Intruders and What Is Viral Feedback? Microfluidic Technique in the Field of Virology Flow-Based Channel Microfluidics Droplet-Based Microfluidics Electric Field-Based Digital Microfluidics (DMF) Microfluidic and Nanoparticle-Based Diagnostic Devices Paper-Based Device Gold Nanoparticles Microfluidics for Host-Viral Interaction Study Conclusion Future Direction References Part III: Chemical Methods for Single Cell Technology 18 Liposome-Mediated Material Transfer in Single Cells Introduction Preparation and Physical Characteristics of Liposomes Liposome Fusion as a Model System Transfer of Materials from Liposomes into Cells Single-Cell Modification Techniques Discussion and Conclusion References 19 The Art of Therapeutic Antibody Discovery: Finding Them One Cell at a Time Introduction Technological Arts for Monoclonal Antibody Discoveries Immortalization of Memory B Cells Humanized Mice Single B Cell Cloning Single-Cell Antibody Nanowells Application of Single-Cell Antibody Discovery in the Human Conditions Dengue Antibody Dependent Enhancement (ADE) Neutralizing Antibodies in Dengue Autoimmune Diseases Monoclonal Antibody Treatments in Autoimmune Diseases Cytokine Targeting Biologic Therapies in Autoimmune Diseases Anti-Tumor Necrosis Factor-α (TNF-α) Anti-interleukin (IL)-6 Anti-IL-17 T and B Cell Depleting Biologic Therapies in Autoimmune Disease Challenges in Monoclonal Antibody Therapy Conclusion References 20 Screening of Antigen-Specific Antibody-Secreting Cells Introduction Importance of mAb Discovery and Production Conventional Methods in Screening ASCs Hybridoma Production EBV Immortalization Phage Display Flow Cytometry Single-Cell Screening Methods for ASCs Modified Flow Cytometry Microwell Arrays Droplet Microfluidics Conclusion References 21 Biochemical Analysis of Secreted Molecules by Individual Cells Introduction Secretion Analysis Single-Cell Analysis of Secretion Technologies for Single-Cell Analysis of Secretion ELISpot and Derivatives Cytometry-Based Methods Micro- and Nanowell Assays Microchamber Assays Droplet-Based Microfluidics Label-Free Optical Methods Limitation of the Current Technologies and Future Directions Conclusion References Part IV: Single Cell Omics 22 Single Cell Genomics Introduction Methods of Whole Genome Amplification for Single Cell Genomics Single Cell Genomics Is Revolutionizing Cancer Biology Somatic Mosaicisms in Development and Disease Clinical Applications of Single Cell Genomics Single Cell Genomics in Microbiology Epigenetics Meets Single Cell Technology Conclusion References 23 Single-Cell Proteomics Introduction Technology Platforms and Methodologies for Single-Cell Proteomics Capillary Electrophoresis (CE) Chemical Cytometry Microfluidics Mass Spectrometry (MS) Flow Cytometry Mass Cytometry Challenges, Limitations, and Outlook References 24 Single Cell Pull-Down for Characterization of Protein Complexes Introduction Fluorescence Methods for Characterization of Protein Complex High-Throughput Screening of Protein-Protein Interactions Single Molecule Fluorescence Detection of Protein Complexes Single Cell Pull-Down Surface Chemistry for Single Cell Pull-Down Live Cell Micropatterning High Affinity Capturing of Target Protein Complexes from a Single Cell Dissociation Kinetics of Protein Complex Determination of the Stoichiometry of Protein Complexes Automated Large-Scale Single Molecule Analysis Single Cell Pull-Down for Label Free Detection Conclusions References 25 Single-Cell Transcriptomics Introduction Techniques for Single-Cell Transcriptome Analysis Single-Cell Isolation cDNA Library for sc-qPCR cDNA Library for scRNA-seq Methodology Selection Single-Cell Transcriptome in Stem Cell Biology Cellular Classification Developmental Studies Stem Cells in Adult Tissues Single-Cell Transcriptomics for Immunology Research Studies on Innate Immune System Studies on Adaptive Immune System Studies on Immune System and Cancer Single-Cell Transcriptomics in Cancer Research Intratumor Heterogeneity Led by the Tumor Microenvironment Study of Minor Populations in Cancer Circulating Tumor Cells Current Issues and Future Approaches Conclusions References 26 Single-Cell Transcriptome Sequencing Using Microfluidics Introduction: The Importance of Cellular Heterogeneity and Single-Cell Analysis Cellular Heterogeneity in Cell Biology Advantages of Single-Cell Analysis Using Microfluidics for Single-Cell Analysis Biochemistry of Single-Cell RNA-Seq Generation of Barcoded Beads Pairing a Barcoded Bead with a Cell Using Microfluidics Droplet-Based Microfluidics for Bead-Cell Pairing Using Microwell for Bead-Cell Pairing Bead-Cell Pairing Using Hydrodynamic Cell Capture Basics of Gene Sequencing and Data Analysis Gene Sequencing of Single-Cell Library Read Alignment to Quantify Gene Expression of Each Cell Statistical Analysis of Single-Cell Data Extension from Single-Cell Transcriptomics to Multiomics Challenges and Future Directions Reliability of Single-Cell RNA-Seq Fixation and Storage of Biological Samples Challenges in Single-Cell Multiomics Analysis Conclusion References Part V: Single Cell Analysis in Systems Biology and Biocatalysis 27 Single-Cell Phenotyping of Complex Heterogeneous Tissue Introduction Mass Matters: Shortfalls of Population Averaging Transcriptomics Through Population Averaging Will scRNAseq Be the Ultimate Solution? ``Clothes Make the Man ́ ́: What Single-Cell Phenotyping Has to Offer Over Population Averaging The Coccygeal Bovine IVD as a Research Model Micro-Niche Style Living of Resident IVD Cells Heterogeneity Detection Through Transcript Analysis on a Cell-by-Cell Approach Cell Velocity as an Indicator for Behavioral Heterogeneity of Cell Populations Materials and Methods IVD Tissue Source and Cell Culture Conditions Nucleic Acid Hybridization Chromogenic Transcript Detection Transcript Detection Through Fluorescent Imaging Histology Post-SISH Immunohistochemistry for Protein Detection Cell Velocity Measurements Conclusions References 28 Record the Single Cell Signal Pathway Introduction Signal Transduction in Cells State-of-the-Art Methods Challenges of Rapid Mixing Concept and Theory Microfluidic Circuit Design Chip Fabrication and System Assembly Deflection Switching Time Quantification of Specific Phospho-Proteins Single-Cell Fluorescence Intensity Quantification Conclusion References 29 Single-Cell Microencapsulation for Evolution and Discovery of Biocatalysts Introduction Ultrahigh-Throughput Enzyme Screens: Directed Evolution and Discovery Single-Cell Encapsulation in Microfluidic Droplets Stochastic Encapsulation Deterministic Single-Cell Encapsulation Techniques Microfluidic Workflow for Directed Evolution of Enzymes Construction of Multistep Workflows for Single-Cell Assays Considerations for Setting Up Single Cell Assays Library Construction and DNA Transformation Single-Cell Phenotypic Variations Sensitivity of Fluorescence Detection Operation of a Microdroplet Sorter Formats for Single-Cell Biocatalyst Functional Screens Single-Cell Lysate Assays Single-Cell Internal Expression and Surface Display Single-Cell Secretion Assays Functional Metagenomics and Bioprospecting In Vitro Workflows as Artificial Single Cells Limitations and Future Prospects of Single-Cell Microencapsulation Conclusions References 30 Analytical Techniques for Single-Cell Studies in Microbiology Introduction Cytometry Flow Cytometry Imaging Flow Cytometry Scanning Cytometry Quantitative Optical Microscopy Brightfield Microscopy Fluorescence Microscopy Scanning Probe Microscopy Scanning Atomic Force Microscopy Scanning Electrochemical Microscopy Nanoscale Secondary Ion Mass Spectrometry Rotational-Vibrational Spectroscopy Conclusion References Part VI: Single Cell Technologies in Cancer 31 Single Cell Adhesion in Cancer Progression Introduction Cell Adhesion Molecules Cadherins Integrins Selectin Immunoglobulin Superfamily Cell-To-Extracellular Matrix (ECM) Adhesion Role of Cell Adhesion in Cancer Progression Epithelial-Mesenchymal Transition (EMT) Tumor Invasion Angiogenesis Intravasation Extravasation Techniques to Study Cell Adhesion and Migration Bulk Adhesion Measurements Centrifugation Assays Hydrodynamic Shear Assays Wash Assay Parallel Plate System Rotating Disk System Radial Flow System High-Throughput Cone and Plate (HT-CAP)-Electric Cell-Substrate Impedance Sensing (ECIS) Instrumentation Single Cell Techniques Micropipette Aspiration Step-Pressure Technique Biomembrane Force Probe Micropipette Aspiration (Narrow Sense) Atomic Force Microscopy (AFM) Nanofork and Line-Patterned Substratum Optical Tweezer Instrumentation Measuring Cell-to-Extracellular Matrix (ECM) Adhesion Strength Förster Resonance Energy Transfer (FRET) Cellular Traction Force Quantitative Intravital Microscopy Fluorescent Models to Study Tumor Heterogeneity Optical Imaging Windows References 32 Single-Cell Technologies for Cancer Therapy Introduction Heterogeneity of Single Cancer Cells The Origin of Cancer Cell Characteristics and Variability Vertical or Horizontal Gene Transfer in Single Cancer Cells Single-Cell Epigenetics Cancer Cells and Immune Response Virus Defense and Small RNA in Single Cells Cellular Surface Antigen, Immunity, and Canceration Microbiome and Canceration Communication and Application of Gut Microbiome Single Cell Technologies in Cancer Diagnosis and Treatment Single Cell Sequencing for Cancer Diagnosis and Biomarker Molecular Technology and Cancer Immunotherapy at Single Cell Resolution CRISPR: Treating Cancer and Posing Other Cancer Risk TCR and CAR-T at Single T Cells Resolution Real-Time Images in Single Cells Photodynamic Therapy Techniques for Single Cell Analysis Single Cell Partitioning Methods-Mechanical Partitioning and Microfluidic Device Circulating Tumor Cells Measurement PCR and NGS Single Cell qPCR NGS Sequencing in Cancer Treatment and Future Applications of Single Cell Analysis Cellular ``Barcode ́ ́ Map Protein Crystallization Technology CyTOF and IscA Magnetic Protein TEM/Cryo-EM/SEM/Confocal 3D Images Global Projects, Database, and Bioinformatics Database and Bioinformatics from Single Cells or a Single Cell Global Projects at Single-Cell Resolution Human Genome Project (HGP) and Human Genome Diversity Project (HGDP) The Haplotype Map (HapMap) Human Genome Diversity Project (HGDP) The Human Proteome Project and Comparative Serum Proteomics Project Comparative Serum Proteomics Project Human Longevity Project The Cancer Genome Atlas (TCGA) Human Cell Atlas (HCA) TCR Diversity Database-ImmunoMap and Human Microbiome Project (HMP) High-Tech Omics-Based Patient Evaluation (HOPE) Project Personal Medicine: Gene Sequencing, Gene Drugs, and Precision Therapy at Single-Cell Resolution Anticancer Drug Development and Personal Medicine Cancer Vaccine Drug Testing and Organoids from Single Stem Cells Antibody Drugs and T Cells Immunotherapy Molecular Synthesis and Bio-genetical Engineering Drugs to Kill Drug-Resistant Drug-Drug Interaction Database and Drug Release Bioinfomatics and Analytical Framework for Single-Cell Meta-Analysis Algorithm, Grouping, and Data Visualization of Datasets from Single Cells Principal Component Analysis (PCA) Ingenuity Pathway Analysis Business Market: Single Cells Related Technology and Personalized Medicine Single Cell Specific Biomarkers and Service for Immunotherapy Human Longevity AI/Deep Learning and Service in Personal Medicine IBM Watson Conclusions References 33 Analytical Technology for Single-Cancer-Cell Analysis Introduction Conventional Analytical Technology for Cancer Cell Analysis Two-Dimensional (2D) Cell-Based Assay Three-Dimensional (3D) Cell-Based Assay Analytical Technology for Single-Cancer-Cell Analysis Single-Cell Sequencing Single-Cell Isolation Single-Cell Sequencing Single-Cell Analysis and Data Computation Inherent Traits of Single Cells Mechanical Traits of Single Cells Size Deformability Electrokinetic Properties Conclusion References 34 Transmembrane Receptor Dynamics as Biophysical Markers for Assessing Cancer Cells Applications of SPT/SMT in Biology Principle of Single-Particle Tracking (SPT) and Trajectory Analysis Structure-Property-Function-Disease Paradigm of TReD Assay Quantification of Metastatic Potential of Breast and Prostate Cancer Cells Biomolecular Mechanisms Guiding the Dynamics of Transmembrane Receptors Deep Learning-Based TReD Assay with Better Accuracy Future Direction References Part VII: Flow Cytometry for Single Cell Analysis 35 Single-Cell Impedance Flow Cytometry Introduction Conventional Characterization Approaches Patch Clamping Electrorotation Dielectrophoresis Impedance Flow Cytometry Prototype Demonstration Microfluidic Impedance Flow Cytometry Microfluidic Impedance Flow Cytometry Based on Constriction Channels Conclusion and Outlook References 36 Cytometry of Single Cell in Biology and Medicine Introduction Mechanisms of Cytometry Application of Cytometry in Microbial Study Total Bacterial Cell Count Bacterial Viability Analysis Specific Microbial Identification Multifunction Analysis Conclusions Application of Cytometry in Rare Cell Analysis Phenotype Analysis Genotype Analysis Conclusions Application of Cytometry in Medicine Fluorescent Staining Drug Discovery in Immunology and Receptor Pharmacology Conclusions Conclusions and Perspectives References Part VIII: Spectrum Analysis, Methods, Targets, Imaging, and Applications 37 Single Cell Electrophysiology Introduction Membrane Electrophysiology Cell Membrane Resting Membrane Potential and Action Potential Hodgkin-Huxley Model Neuron-Electrode Interface and Transfer Function from the Point Contact Model Recording Model Intracellular Recording Sharp Microelectrode Patch Electrode Cell-Attached Patch Whole-Cell Recording Patch Inside-Out Patch Outside -Out Patch Loose Patch Voltage Clamp Current Clamp Extracellular Recording Microelectrode Arrays (MEAs) Complementary Metal Oxide Semiconductor (CMOS) Thin-Film Transistor Array Technology Comparison Current Research Noise in Electrophysiological Measurements Thermal Noise Shot Noise Dielectric Noise Excess Noise Other Forms of Noise and General Precautions Summary References 38 Mechanical and Microwave Resonators for Sensing and Sizing Single Cells Introduction Single-Cell Characterization with Microelectromechical Systems (MEMS) MEMS Sensors and Early Efforts Adherent Cell Measurements with MEMS Sensors Working Inside Liquid Suspended Cell Measurements with Suspended Microchannel Resonators Single-Cell Sensing with Microfluidics-Integrated Microwave Sensors (MIMS) Conclusion References 39 Molecular Force Spectroscopy on Cells: Physiological Functions of Cell Adhesion Introduction Biochemical Aspects of SCFS Molecular Recognition Through ECM Application of SCFS in Nanobiotechnology Study of Single-Cell Structure and Migration Single-Molecule Force Spectroscopy (SMFS) Conclusion References 40 Micro-tweezers and Force Microscopy Techniques for Single-Cell Mechanobiological Analysis Introduction Micro-tweezers for Single-Cell Mechanical Stimulation Optical Tweezers Magnetic Tweezers Acoustic Tweezers Micropipette Aspiration Microfluidic Shear Device Force Microscopy Techniques for Single Cell Force Mapping Traction Force Microscopy 3D Traction Force Microscopy Elastic Micropost Arrays Fluorescence Resonance Energy Transfer-Based Sensors Atomic Force Microscopy Summary and Future Outlook References 41 Mass Spectrometry for Single-Cell Analysis Introduction Mass Spectrometry (MS) Techniques Electrospray Ionization Mass Spectrometry (ESI-MS) Technique Secondary Ion Mass Spectrometry (SIMS) Laser Deposition/Ionization Mass Spectrometry (LDI-MS) Inductively Coupled Plasma Mass Spectrometry (ICP-MS) Future Prospects of Mass Spectrometry Conclusions References 42 Acoustic Tweezers for Single-Cell Manipulation Introduction Principles and Theory of Acoustic Tweezers Wave Generation Theory of Acoustic Tweezers Acoustic Tweezer Technologies Standing Wave Tweezers Traveling Wave Tweezers Acoustic Streaming Tweezers Applications of Acoustic Tweezers in Single-Cell Studies Cell Printing/Patterning Cell Separation Standing SAW-Based Cell Separation Standing BAW-Based Cell Separation Traveling SAW-Based Cell Separation Cell Sorting Cell Imaging Cell Signaling Cell Stretching and Poration Conclusion References Index This book provides a brief overview of single-cell analysis using recent advanced technologies. The different sections cover different aspect of single cell analysis and applications with their advantages, limitations, and future challenges. The book has covered how different physical energies such as optical, electrical, and mechanical energy have been applied for single cell therapy and analysis. The recent advanced micro/nanofluidic devices have been employed for single-cell counting, manipulation, cultivation, separation, isolation, lysis, printing and patterning and host-viral interaction at single-cell level. Various chemical approaches for single-cell analysis have been discussed, such as liposome mediated materials transfer at single-cell and their analysis, discovery of antibody via single-cell, high-throughput screening of antigen-specific antibody-secreting cells, and biomolecular secretion analysis of individual cells. Moreover, different single-cell omics such as genomics, proteomics and transcriptomics have been discussed using microfluidic technologies as well as conventional approaches. The role of single cell analysis in system biology and biocatalysis have been discussed in detail. The book describes single-cell phenotyping of heterogeneous tissue, stimulation, and instant reaction quenching technology for biochemical kinetic analysis, large scale single-cell assay for the identification of biocatalysts and analytical techniques for single-cell studies in microbiology. The role of single-cell analysis in cancer, such as single-cell adhesion and cancer progression, single-cell technologies for cancer therapy, analytical technology for single cancer cell analysis, and biophysical markers for cancer cell analysis have been discussed. The flow cytometry based high throughput single-cell analysis have been well emphasized. Finally this book has covered single-cell electrophysiology, single-cell sensing and size measurement using mechanical and microwave resonators, molecular force spectroscopy for cell adhesion measurement, micro-tweezers and force microscopy techniques for single-cell mechanobiological analysis, mass spectrometry and acoustic tweezers for single-cell manipulation and analysis. This book is intended for academic and industrial researchers, undergraduate and graduate students in the fields of biomedical engineering, bio-micro/nanoengineering, and bio-micro/nano fabrication for single-cell analysis. It can be used for courses on bio-MEMS/bio-NEMS, biomicrofluidics, bio-micro/nanofabrications, micro/nanofluidics, biophysics, single cell analysis, bionanotechnology, drug delivery systems and biomedical microdevices. Collective contributions from respected experts, have brought diverse aspects of single-cell technologies in a single hand book. This will benefit researchers and practitioners in the biotechnology industry for different diseases analysis, therapeutics, diagnostics, drug discovery, drug screening etc. In addition to hard copies, the book will be available online and will often be updated by the authors