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An Introduction to High Content Screening : Imaging Technology, Assay Development, and Data Analysis in Biology and Drug Discovery.

By: Contributor(s): Publisher: Somerset : John Wiley & Sons, Incorporated, 2015Copyright date: ©2015Edition: 1st edDescription: 1 online resource (350 pages)Content type:
  • text
Media type:
  • computer
Carrier type:
  • online resource
ISBN:
  • 9781118859476
Subject(s): Genre/Form: Additional physical formats: Print version:: An Introduction to High Content Screening : Imaging Technology, Assay Development, and Data Analysis in Biology and Drug DiscoveryDDC classification:
  • 615.1/9
LOC classification:
  • RM301.25 -- .I587 2015eb
Online resources:
Contents:
Intro -- An Introduction to High Content Screening -- Contents -- Preface -- Contributors -- 1 Introduction -- 1.1 The Beginning of High Content Screening -- 1.2 Six Skill Sets Essential for Running HCS Experiments -- 1.2.1 Biology -- 1.2.2 Microscopy -- 1.2.3 HCS Instrumentation (Platform Manager) -- 1.2.4 Image Analysis -- 1.2.5 Statistical Analysis -- 1.2.6 Information Technology Support -- 1.3 Integrating Skill Sets Into A Team -- 1.4 A Few Words on Experimental Design -- 1.5 Conclusions -- Key Points -- Further Reading -- References -- Part I First Principles -- 2 Fluorescence and Cell Labeling -- 2.1 Introduction -- 2.2 Anatomy of Fluorescent Probes, Labels, and Dyes -- 2.3 Stokes Shift and Biological Fluorophores -- 2.4 Fluorophore Properties -- 2.4.1 The Extinction Coefficient (Efficiency of Absorption) -- 2.4.2 Quantum Yield -- 2.4.3 Fluorescence Lifetime -- 2.4.4 Loss of Signal (Fading or Signal Degradation) -- 2.5 Localization of Fluorophores Within Cells -- 2.5.1 Nuclear Stains -- 2.5.2 Fluorescent Proteins -- 2.5.3 Localization Agents -- 2.5.4 Issues that Affect Fluorescent Reagent Choice -- 2.6 Multiplexing Fluorescent Reagents -- 2.7 Specialized Imaging Applications Derived From Complex Properties of Fluorescence -- 2.7.1 Förster Resonance Energy Transfer -- 2.7.2 Fluorescence Lifetime Imaging/Förster Resonance Energy Transfer -- 2.8 Conclusions -- Key Points -- Further Reading -- References -- 3 Microscopy Fundamentals -- 3.1 Introducing HCS Hardware -- 3.1.1 The HCS Imager and the Microscope -- 3.1.2 Common uses of HCS that Require Specific Hardware Adaptations -- 3.2 Deconstructing Light Microscopy -- 3.2.1 The Light Source(s) -- 3.2.2 The Filter Cube -- 3.2.3 The Objective -- 3.2.4 The Camera -- 3.3 Using the Imager to Collect Data -- 3.4 Conclusions -- Key Points -- Further Reading -- References -- 4 Image Processing.
4.1 Overview of Image Processing and Image Analysis in HCS -- 4.2 What is a Digital Image? -- 4.3 "Addressing" Pixel Values in Image Analysis Algorithms -- 4.4 Image Analysis Workflow -- 4.4.1 Step 1: Image Preprocessing -- 4.4.2 Step 2: Image Thresholding and Segmentation -- 4.4.3 Step 3: Calculation of Image Features -- 4.4.4 Step 4. Collation and Summary of Features -- 4.4.5 Step 5: Data Export and Feature Data -- 4.5 Conclusions -- Key Points -- Further Reading -- References -- Part II Getting Started -- 5 A General Guide to Selecting and Setting Up A High Content Imaging Platform -- 5.1 Determining Expectations of the Hcs System -- 5.2 Establishing an HC Platform Acquisition Team -- 5.2.1 The Platform Manager -- 5.2.2 The Department or Research Head -- 5.2.3 Facilities Management/Lab Operations -- 5.2.4 Local and Institutional IT Personnel -- 5.3 Basic Hardware Decisions -- 5.3.1 Consider the Needs of the Users and the Lab Setting -- 5.3.2 Instrumentation Options -- 5.4 Data Generation, Analysis, and Retention -- 5.4.1 Image Acquisition Software -- 5.4.2 Data Storage -- 5.4.3 Image Analysis Software -- 5.4.4 System Configuration -- 5.5 Installation -- 5.5.1 Overview -- 5.5.2 Ownership of Technical Issues -- 5.6 Managing the System -- 5.6.1 System Maintenance -- 5.6.2 New User Training -- 5.6.3 Scheduling Time on the System -- 5.6.4 Billing -- 5.7 Setting Up Workflows for Researchers -- 5.7.1 Introducing Scientists to HCS and the Imager -- 5.7.2 Superusers -- 5.7.3 Initial Experiments and Assay Development -- 5.8 Conclusions -- Key Points -- Further Reading -- 6 Informatics Considerations -- 6.1 Informatics Infrastructure for High Content Screening -- 6.1.1 The Scope of the Data Management Challenge -- 6.1.2 Do-It-Yourself Data Storage Solutions -- 6.1.3 Working with Central IT Departments -- 6.2 Using Databases to Store HCS Data.
6.2.1 Introduction -- 6.2.2 Types of Data -- 6.2.3 Databases -- 6.2.4 Basic Features of an HCS Database -- 6.3 Mechanics of an Informatics Solution -- 6.3.1 Introduction -- 6.3.2 Data Life Cycle Management -- 6.4 Developing Image Analysis Pipelines: Data Management Considerations -- 6.4.1 Using Commercial Image Analysis Software -- 6.4.2 Using Custom Image Analysis Pipelines -- 6.4.3 Data Duplication and Uncontrolled Data Growth -- 6.4.4 Metadata Loss -- 6.4.5 Data Movement, Network Bandwidth Limitations, and the Challenges of Moving Large Datasets -- 6.4.6 Problems with Handling Very Large Numbers of Files -- 6.4.7 Parallel Data Processing -- 6.4.8 Workflow Documentation and Automation -- 6.4.9 Software Development and Maintenance: Managing Software Development Projects -- 6.4.10 Software Sharing, User Training -- 6.4.11 Image Repositories -- 6.5 Compliance With Emerging Data Standards -- 6.6 Conclusions -- Key Points -- Further Reading -- References -- 7 Basic High Content Assay Development -- 7.1 Introduction -- 7.2 Initial Technical Considerations for Developing a High Content Assay -- 7.2.1 Plate Type -- 7.2.2 Choice and Use of Staining Reagents and of Positive and Negative Controls -- 7.2.3 Plate Layout -- 7.2.4 Replicates -- 7.2.5 Cell Plating Density -- 7.3 A Simple Protocol to Fix and Stain Cells -- 7.3.1 Washing Cells -- 7.3.2 Fixing Cells -- 7.3.3 Permeabilization -- 7.3.4 Blocking -- 7.3.5 Postblocking Washes -- 7.3.6 Primary Antibody Application -- 7.3.7 Postprimary Antibody Washes -- 7.3.8 Secondary Antibodies -- 7.3.9 Postsecondary Washes -- 7.4 Image Capture and Examining Images -- 7.4.1 Resolution, Magnification, and Image Exposure -- 7.4.2 Number of Cells to Acquire for the Image Analysis Phase -- 7.4.3 Performance of Positive and Negative Controls -- 7.5 Conclusions -- Key Points -- Further Reading -- Reference.
Part III Analyzing Data -- 8 Designing Metrics for High Content Assays -- 8.1 Introduction: Features, Metrics, Results -- 8.2 Looking at Features -- 8.3 Metrics and Results: The Metric is The Message -- 8.4 Types of High Content Assays and Their Metrics -- 8.4.1 Intensity -- 8.4.2 Area Above Threshold -- 8.4.3 Spot Counting, Including Nuclei or Cell Counting -- 8.4.4 Translocation -- 8.4.5 Morphology -- 8.5 Metrics to Results: Putting it all Together -- 8.5.1 Basic Assay Measurements -- 8.5.2 Use of Multiple Independent Measurements to Assess a Perturbation -- 8.5.3 Integrating Multiple Features to Measure a Phenotypic Response -- 8.6 Conclusions -- Key Points -- Further Reading -- References -- 9 Analyzing Well-Level Data -- 9.1 Introduction -- 9.2 Reviewing Data -- 9.3 Plate and Control Normalizations of Data -- 9.3.1 Ratio or Percent of Control -- 9.3.2 z-Score or Robust z-score -- 9.3.3 B-score -- 9.3.4 Mixed Effects Models -- 9.4 Calculation of Assay Statistics -- 9.4.1 Signal to Background Ratio (SB) -- 9.4.2 Signal to Noise Ratio (SN) -- 9.4.3 Z' Statistic -- 9.4.4 V Factor -- 9.4.5 Strictly Standardized Mean Difference -- 9.5 Data Analysis: Hit Selection -- 9.5.1 Rank Order -- 9.5.2 Mean +/− k SD -- 9.5.3 Median +/− k MAD -- 9.5.4 SSMD -- 9.5.5 t-Test -- 9.6 IC 50 Determinations -- 9.6.1 Overview -- 9.6.2 Challenges of Fitting Data to a Standard Dose-Response Curve and Potential Biological Insights from Imaging Data -- 9.7 Conclusions -- Key Points -- Further Reading -- References -- 10 Analyzing Cell-Level Data -- 10.1 Introduction -- 10.2 Understanding General Statistical Terms and Concepts -- 10.2.1 Normal and Non-normal Distributions -- 10.2.2 Normalizing or Transforming Data -- 10.2.3 Robust Statistics -- 10.2.4 Parametric and Nonparametric Statistics -- 10.3 Examining Data -- 10.3.1 Descriptive Statistics -- 10.3.2 Data Visualization.
10.3.3 Transformation of Data -- 10.4 Developing a Data Analysis Plan -- 10.4.1 Review the Summary Statistics -- 10.4.2 Determine the Distribution of the Data -- 10.4.3 Consider Transforming Non-normal Data -- 10.5 Cell-Level Data Analysis: Comparing Distributions Through Inferential Statistics -- 10.6 Analyzing Normal (or Transformed) Data -- 10.6.1 The t-Test -- 10.6.2 ANOVA Tests -- 10.7 Analyzing Non-Normal Data -- 10.7.1 Wilcoxons Rank Sum -- 10.7.2 Kruskal-Wallis ANOVA -- 10.7.3 The Kolmogrov-Smirnoff (KS) Statistic -- 10.7.4 Bootstrapping -- 10.8 When to Call For Help -- 10.9 Conclusions -- Key Points -- Further Reading -- References -- Part IV Advanced Work -- 11 Designing Robust Assays -- 11.1 Introduction -- 11.2 Common Technical Issues in High Content Assays -- 11.2.1 At the Bench -- 11.2.2 During Image Analysis -- 11.3 Designing Assays to Minimize Trouble -- 11.3.1 Choosing the Right Antibodies -- 11.3.2 Optimizing Your Antibodies -- 11.3.3 Preparation of Samples and Effects on Fluorescence -- 11.3.4 Planning Ahead with Image Analysis -- 11.4 Looking for Trouble: Building in Quality Control -- 11.4.1 Using Controls for QC -- 11.4.2 Uniformity Plates -- 11.4.3 Monitoring Assay Statistics -- 11.4.4 Monitoring Meta-data -- 11.4.5 Visually Inspect Images via Montages or Random Sampling -- 11.4.6 Lock down Standard Operating Procedures (SOPs) -- 11.5 Conclusions -- Key Points -- Further Reading -- References -- 12 Automation and Screening -- 12.1 Introduction -- 12.2 Some Preliminary Considerations -- 12.2.1 Assay or Screen? -- 12.2.2 To Automate or Not? -- 12.3 Laboratory Options -- 12.3.1 Workstation versus Fully Automated Systems -- 12.3.2 Liquid Handler/Reagent Dispenser/Plate Washer Requirements -- 12.3.3 Barcode Reading Requirements -- 12.3.4 Vendor Selection Issues -- 12.3.5 Highly Customized versus More General Systems and Software.
12.3.6 Managing Expectations About Automation.
Summary: Using a collaborative and interdisciplinary author base with experience in the pharmaceutical industry and academia, this book is a practical resource for high content (HC) techniques.  Instructs readers on the fundamentals of high content screening (HCS) techniques  Focuses on practical and widely-used techniques like image processing and multiparametric assays  Breaks down HCS into individual modules for training and connects them at the end  Includes a tutorial chapter that works through sample HCS assays, glossary, and detailed appendices.
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Intro -- An Introduction to High Content Screening -- Contents -- Preface -- Contributors -- 1 Introduction -- 1.1 The Beginning of High Content Screening -- 1.2 Six Skill Sets Essential for Running HCS Experiments -- 1.2.1 Biology -- 1.2.2 Microscopy -- 1.2.3 HCS Instrumentation (Platform Manager) -- 1.2.4 Image Analysis -- 1.2.5 Statistical Analysis -- 1.2.6 Information Technology Support -- 1.3 Integrating Skill Sets Into A Team -- 1.4 A Few Words on Experimental Design -- 1.5 Conclusions -- Key Points -- Further Reading -- References -- Part I First Principles -- 2 Fluorescence and Cell Labeling -- 2.1 Introduction -- 2.2 Anatomy of Fluorescent Probes, Labels, and Dyes -- 2.3 Stokes Shift and Biological Fluorophores -- 2.4 Fluorophore Properties -- 2.4.1 The Extinction Coefficient (Efficiency of Absorption) -- 2.4.2 Quantum Yield -- 2.4.3 Fluorescence Lifetime -- 2.4.4 Loss of Signal (Fading or Signal Degradation) -- 2.5 Localization of Fluorophores Within Cells -- 2.5.1 Nuclear Stains -- 2.5.2 Fluorescent Proteins -- 2.5.3 Localization Agents -- 2.5.4 Issues that Affect Fluorescent Reagent Choice -- 2.6 Multiplexing Fluorescent Reagents -- 2.7 Specialized Imaging Applications Derived From Complex Properties of Fluorescence -- 2.7.1 Förster Resonance Energy Transfer -- 2.7.2 Fluorescence Lifetime Imaging/Förster Resonance Energy Transfer -- 2.8 Conclusions -- Key Points -- Further Reading -- References -- 3 Microscopy Fundamentals -- 3.1 Introducing HCS Hardware -- 3.1.1 The HCS Imager and the Microscope -- 3.1.2 Common uses of HCS that Require Specific Hardware Adaptations -- 3.2 Deconstructing Light Microscopy -- 3.2.1 The Light Source(s) -- 3.2.2 The Filter Cube -- 3.2.3 The Objective -- 3.2.4 The Camera -- 3.3 Using the Imager to Collect Data -- 3.4 Conclusions -- Key Points -- Further Reading -- References -- 4 Image Processing.

4.1 Overview of Image Processing and Image Analysis in HCS -- 4.2 What is a Digital Image? -- 4.3 "Addressing" Pixel Values in Image Analysis Algorithms -- 4.4 Image Analysis Workflow -- 4.4.1 Step 1: Image Preprocessing -- 4.4.2 Step 2: Image Thresholding and Segmentation -- 4.4.3 Step 3: Calculation of Image Features -- 4.4.4 Step 4. Collation and Summary of Features -- 4.4.5 Step 5: Data Export and Feature Data -- 4.5 Conclusions -- Key Points -- Further Reading -- References -- Part II Getting Started -- 5 A General Guide to Selecting and Setting Up A High Content Imaging Platform -- 5.1 Determining Expectations of the Hcs System -- 5.2 Establishing an HC Platform Acquisition Team -- 5.2.1 The Platform Manager -- 5.2.2 The Department or Research Head -- 5.2.3 Facilities Management/Lab Operations -- 5.2.4 Local and Institutional IT Personnel -- 5.3 Basic Hardware Decisions -- 5.3.1 Consider the Needs of the Users and the Lab Setting -- 5.3.2 Instrumentation Options -- 5.4 Data Generation, Analysis, and Retention -- 5.4.1 Image Acquisition Software -- 5.4.2 Data Storage -- 5.4.3 Image Analysis Software -- 5.4.4 System Configuration -- 5.5 Installation -- 5.5.1 Overview -- 5.5.2 Ownership of Technical Issues -- 5.6 Managing the System -- 5.6.1 System Maintenance -- 5.6.2 New User Training -- 5.6.3 Scheduling Time on the System -- 5.6.4 Billing -- 5.7 Setting Up Workflows for Researchers -- 5.7.1 Introducing Scientists to HCS and the Imager -- 5.7.2 Superusers -- 5.7.3 Initial Experiments and Assay Development -- 5.8 Conclusions -- Key Points -- Further Reading -- 6 Informatics Considerations -- 6.1 Informatics Infrastructure for High Content Screening -- 6.1.1 The Scope of the Data Management Challenge -- 6.1.2 Do-It-Yourself Data Storage Solutions -- 6.1.3 Working with Central IT Departments -- 6.2 Using Databases to Store HCS Data.

6.2.1 Introduction -- 6.2.2 Types of Data -- 6.2.3 Databases -- 6.2.4 Basic Features of an HCS Database -- 6.3 Mechanics of an Informatics Solution -- 6.3.1 Introduction -- 6.3.2 Data Life Cycle Management -- 6.4 Developing Image Analysis Pipelines: Data Management Considerations -- 6.4.1 Using Commercial Image Analysis Software -- 6.4.2 Using Custom Image Analysis Pipelines -- 6.4.3 Data Duplication and Uncontrolled Data Growth -- 6.4.4 Metadata Loss -- 6.4.5 Data Movement, Network Bandwidth Limitations, and the Challenges of Moving Large Datasets -- 6.4.6 Problems with Handling Very Large Numbers of Files -- 6.4.7 Parallel Data Processing -- 6.4.8 Workflow Documentation and Automation -- 6.4.9 Software Development and Maintenance: Managing Software Development Projects -- 6.4.10 Software Sharing, User Training -- 6.4.11 Image Repositories -- 6.5 Compliance With Emerging Data Standards -- 6.6 Conclusions -- Key Points -- Further Reading -- References -- 7 Basic High Content Assay Development -- 7.1 Introduction -- 7.2 Initial Technical Considerations for Developing a High Content Assay -- 7.2.1 Plate Type -- 7.2.2 Choice and Use of Staining Reagents and of Positive and Negative Controls -- 7.2.3 Plate Layout -- 7.2.4 Replicates -- 7.2.5 Cell Plating Density -- 7.3 A Simple Protocol to Fix and Stain Cells -- 7.3.1 Washing Cells -- 7.3.2 Fixing Cells -- 7.3.3 Permeabilization -- 7.3.4 Blocking -- 7.3.5 Postblocking Washes -- 7.3.6 Primary Antibody Application -- 7.3.7 Postprimary Antibody Washes -- 7.3.8 Secondary Antibodies -- 7.3.9 Postsecondary Washes -- 7.4 Image Capture and Examining Images -- 7.4.1 Resolution, Magnification, and Image Exposure -- 7.4.2 Number of Cells to Acquire for the Image Analysis Phase -- 7.4.3 Performance of Positive and Negative Controls -- 7.5 Conclusions -- Key Points -- Further Reading -- Reference.

Part III Analyzing Data -- 8 Designing Metrics for High Content Assays -- 8.1 Introduction: Features, Metrics, Results -- 8.2 Looking at Features -- 8.3 Metrics and Results: The Metric is The Message -- 8.4 Types of High Content Assays and Their Metrics -- 8.4.1 Intensity -- 8.4.2 Area Above Threshold -- 8.4.3 Spot Counting, Including Nuclei or Cell Counting -- 8.4.4 Translocation -- 8.4.5 Morphology -- 8.5 Metrics to Results: Putting it all Together -- 8.5.1 Basic Assay Measurements -- 8.5.2 Use of Multiple Independent Measurements to Assess a Perturbation -- 8.5.3 Integrating Multiple Features to Measure a Phenotypic Response -- 8.6 Conclusions -- Key Points -- Further Reading -- References -- 9 Analyzing Well-Level Data -- 9.1 Introduction -- 9.2 Reviewing Data -- 9.3 Plate and Control Normalizations of Data -- 9.3.1 Ratio or Percent of Control -- 9.3.2 z-Score or Robust z-score -- 9.3.3 B-score -- 9.3.4 Mixed Effects Models -- 9.4 Calculation of Assay Statistics -- 9.4.1 Signal to Background Ratio (SB) -- 9.4.2 Signal to Noise Ratio (SN) -- 9.4.3 Z' Statistic -- 9.4.4 V Factor -- 9.4.5 Strictly Standardized Mean Difference -- 9.5 Data Analysis: Hit Selection -- 9.5.1 Rank Order -- 9.5.2 Mean +/− k SD -- 9.5.3 Median +/− k MAD -- 9.5.4 SSMD -- 9.5.5 t-Test -- 9.6 IC 50 Determinations -- 9.6.1 Overview -- 9.6.2 Challenges of Fitting Data to a Standard Dose-Response Curve and Potential Biological Insights from Imaging Data -- 9.7 Conclusions -- Key Points -- Further Reading -- References -- 10 Analyzing Cell-Level Data -- 10.1 Introduction -- 10.2 Understanding General Statistical Terms and Concepts -- 10.2.1 Normal and Non-normal Distributions -- 10.2.2 Normalizing or Transforming Data -- 10.2.3 Robust Statistics -- 10.2.4 Parametric and Nonparametric Statistics -- 10.3 Examining Data -- 10.3.1 Descriptive Statistics -- 10.3.2 Data Visualization.

10.3.3 Transformation of Data -- 10.4 Developing a Data Analysis Plan -- 10.4.1 Review the Summary Statistics -- 10.4.2 Determine the Distribution of the Data -- 10.4.3 Consider Transforming Non-normal Data -- 10.5 Cell-Level Data Analysis: Comparing Distributions Through Inferential Statistics -- 10.6 Analyzing Normal (or Transformed) Data -- 10.6.1 The t-Test -- 10.6.2 ANOVA Tests -- 10.7 Analyzing Non-Normal Data -- 10.7.1 Wilcoxons Rank Sum -- 10.7.2 Kruskal-Wallis ANOVA -- 10.7.3 The Kolmogrov-Smirnoff (KS) Statistic -- 10.7.4 Bootstrapping -- 10.8 When to Call For Help -- 10.9 Conclusions -- Key Points -- Further Reading -- References -- Part IV Advanced Work -- 11 Designing Robust Assays -- 11.1 Introduction -- 11.2 Common Technical Issues in High Content Assays -- 11.2.1 At the Bench -- 11.2.2 During Image Analysis -- 11.3 Designing Assays to Minimize Trouble -- 11.3.1 Choosing the Right Antibodies -- 11.3.2 Optimizing Your Antibodies -- 11.3.3 Preparation of Samples and Effects on Fluorescence -- 11.3.4 Planning Ahead with Image Analysis -- 11.4 Looking for Trouble: Building in Quality Control -- 11.4.1 Using Controls for QC -- 11.4.2 Uniformity Plates -- 11.4.3 Monitoring Assay Statistics -- 11.4.4 Monitoring Meta-data -- 11.4.5 Visually Inspect Images via Montages or Random Sampling -- 11.4.6 Lock down Standard Operating Procedures (SOPs) -- 11.5 Conclusions -- Key Points -- Further Reading -- References -- 12 Automation and Screening -- 12.1 Introduction -- 12.2 Some Preliminary Considerations -- 12.2.1 Assay or Screen? -- 12.2.2 To Automate or Not? -- 12.3 Laboratory Options -- 12.3.1 Workstation versus Fully Automated Systems -- 12.3.2 Liquid Handler/Reagent Dispenser/Plate Washer Requirements -- 12.3.3 Barcode Reading Requirements -- 12.3.4 Vendor Selection Issues -- 12.3.5 Highly Customized versus More General Systems and Software.

12.3.6 Managing Expectations About Automation.

Using a collaborative and interdisciplinary author base with experience in the pharmaceutical industry and academia, this book is a practical resource for high content (HC) techniques.  Instructs readers on the fundamentals of high content screening (HCS) techniques  Focuses on practical and widely-used techniques like image processing and multiparametric assays  Breaks down HCS into individual modules for training and connects them at the end  Includes a tutorial chapter that works through sample HCS assays, glossary, and detailed appendices.

Description based on publisher supplied metadata and other sources.

Electronic reproduction. Ann Arbor, Michigan : ProQuest Ebook Central, 2019. Available via World Wide Web. Access may be limited to ProQuest Ebook Central affiliated libraries.

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