AN INTRODUCTION TO HIGH CONTENT SCREENING

Imaging Technology, Assay Development, and Data Analysis in Biology and Drug Discovery

Editors

STEVEN A. HANEY
DOUGLAS BOWMAN
ARIJIT CHAKRAVARTY

Associate Editors

ANTHONY DAVIES
CAROLINE SHAMU

Copyright © 2015 by John Wiley & Sons, Inc. All rights reserved.

Published by John Wiley & Sons, Inc., Hoboken, New Jersey.
Published simultaneously in Canada.

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Library of Congress Cataloging-in-Publication Data:

An introduction to high content screening : imaging technology, assay development, and data analysis in biology and drug discovery / editors, Steven A. Haney and Doug Bowman ; associate editors, Arijit Chakravarty, Anthony Davies, and Caroline Shamu.
       1 online resource.
    Includes bibliographical references and index.
    Description based on print version record and CIP data provided by publisher; resource not viewed.
    ISBN 978-1-118-85941-4 (ePub) – ISBN 978-1-118-85947-6 (Adobe PDF) – ISBN 978-0-470-62456-2 (cloth)
    I. Haney, Steven A., editor. II. Bowman, Doug, active 2014, editor. III. Chakravarty, Arijit, editor. IV. Davies, Anthony, active 2014, editor. V. Shamu, Caroline, editor.
    [DNLM: 1. Drug Design. 2. Systems Biology–methods. 3. Data Interpretation, Statistical. 4. Drug Evaluation, Preclinical–methods. 5. Image Processing, Computer-Assisted–methods. 6. Models, Biological. QV 26.5]
    RM301.25
    615.1′9–dc23

2014027680

CONTENTS

PREFACE

CONTRIBUTORS

1 Introduction

1.1 The Beginning of High Content Screening

1.2 Six Skill Sets Essential for Running HCS Experiments

1.3 Integrating Skill Sets Into A Team

1.4 A Few Words on Experimental Design

1.5 Conclusions

Key Points

Further Reading

References

SECTION 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

2.5 Localization of Fluorophores Within Cells

2.6 Multiplexing Fluorescent Reagents

2.7 Specialized Imaging Applications Derived From Complex Properties of Fluorescence

2.8 Conclusions

Key Points

Further Reading

References

3 Microscopy Fundamentals

3.1 Introducing HCS Hardware

3.2 Deconstructing Light Microscopy

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

4.5 Conclusions

Key Points

Further Reading

References

SECTION 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

5.3 Basic Hardware Decisions

5.4 Data Generation, Analysis, and Retention

5.5 Installation

5.6 Managing the System

5.7 Setting Up Workflows for Researchers

5.8 Conclusions

Key Points

Further Reading

6 Informatics Considerations

6.1 Informatics Infrastructure for High Content Screening

6.2 Using Databases to Store HCS Data

6.3 Mechanics of an Informatics Solution

6.4 Developing Image Analysis Pipelines: Data Management Considerations

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

7.3 A Simple Protocol to Fix and Stain Cells

7.4 Image Capture and Examining Images

7.5 Conclusions

Key Points

Further Reading

Reference

SECTION 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

8.5 Metrics to Results: Putting it all Together

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

9.4 Calculation of Assay Statistics

9.5 Data Analysis: Hit Selection

9.6 IC 50 Determinations

9.7 Conclusions

Key Points

Further Reading

References

10 Analyzing Cell-Level Data

10.1 Introduction

10.2 Understanding General Statistical Terms and Concepts

10.3 Examining Data

10.4 Developing a Data Analysis Plan

10.5 Cell-Level Data Analysis: Comparing Distributions Through Inferential Statistics

10.6 Analyzing Normal (or Transformed) Data

10.7 Analyzing Non-Normal Data

10.8 When to Call For Help

10.9 Conclusions

Key Points

Further Reading

References

SECTION IV ADVANCED WORK

11 Designing Robust Assays

11.1 Introduction

11.2 Common Technical Issues in High Content Assays

11.3 Designing Assays to Minimize Trouble

11.4 Looking for Trouble: Building in Quality Control

11.5 Conclusions

Key Points

Further Reading

References

12 Automation and Screening

12.1 Introduction

12.2 Some Preliminary Considerations

12.3 Laboratory Options

12.4 The Automated Hcs Laboratory

12.5 Conclusions

Key Points

Further Reading

13 High Content Analysis for Tissue Samples

13.1 Introduction

13.2 Design Choices in Setting Up a High Content Assay in Tissue

13.3 System Configuration: Aspects Unique to Tissue-Based HCS

13.4 Data Analysis

13.5 Conclusions

Key Points

Further Reading

References

SECTION V HIGH CONTENT ANALYTICS

14 Factoring and Clustering High Content Data

14.1 Introduction

14.2 Common Unsupervised Learning Methods

14.3 Preparing for an Unsupervised Learning Study

14.4 Conclusions

Key Points

Further Reading

References

15 Supervised Machine Learning

15.1 Introduction

15.2 Foundational Concepts

15.3 Choosing a Machine Learning Algorithm

15.4 When Do You Need Machine Learning, and How Do You Use IT?

15.5 Conclusions

Key Points

Further Reading

Appendix A Websites and Additional Information on Instruments, Reagents, and Instruction

Appendix B A Few Words About One Letter: Using R To Quickly Analyze Hcs Data

B.1 Introduction

B.2 Setting Up R

B.3 Analyzing Data in R

B.4 Where to Go Next

Further Reading

Appendix C Hypothesis Testing for High Content Data: A Refresher

C.1 Introduction

C.2 Defining Simple Hypothesis Testing

C.3 Simple Statistical Tests to Compare Two Groups

C.4 Statistical Tests on Groups of Samples

C.5 Introduction to Regression Models

C.6 Conclusions

Key Concepts

Further Reading

GLOSSARY

TUTORIAL

T.1 Introduction

T.2 The Assays

T.3 The Imager and Image Analysis Software for Image Acquisition

T.4 Lesson 1: Getting Started in Image Acquisition

T.5 Lesson 2: Image Analysis

T.6 Lesson 3: Data Analysis

T.7 Lesson 4: Quality Control in a Cell-Based Assay

INDEX

END USER LICENSE AGREEMENT

List of Tables

Chapter 2

Table 2.1

Table 2.2

Table 2.3

Chapter 4

Table 4.1

Chapter 7

Table 7.1

Chapter 8

Table 8.1

Table 8.2

Chapter 9

Table 9.1

Chapter 10

Table 10.1

Chapter 11

Table 11.1

Table 11.2

Chapter 12

Table 12.1

Appendix B

Table B.1

Appendix C

Table C.1

Tutorial

Table T.1

Table T.2

Table T.3

List of Illustrations

Chapter 1

Figure 1.1 An early automated microscope used in biomedical research. (a) An example of an automated fluorescence microscope. Letters inside the figure are from the original source. The system is outfitted with controlled stage and filter movements (S and F), a push-button console for manual movements (B), a television camera and monitor (T and m) and a video terminal for digitizing video images (v). (b) A video image of a promyelocyte and (c) image analysis of (b), showing, an outline of the nucleus and cell borders, which can be used in automated cell type recognition. Reproduced with permission from [2]. Copyright 1974 John Wiley & Sons.

Figure 1.2 The basic skill sets essential for establishing and running HCS experiments. Skills noted in the figure are discussed in detail in the text.

Chapter 2

Figure 2.1 Anatomy of a fluorescent dye. A diagrammatic representation of an example of a fluorescent probe showing the fluorophore, and the interactions between the localizer (with specific binding region (key)) and the target region on the cell (lock).

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