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Course

 

Monday, January 10

Pre-Conference Advanced High-Content Analysis Course*

Course Instructors:

  • Anthony M. Davies, Ph.D., Director, High-Content Research Facility, Clinical Medicine, Trinity College
  • Paul A. Johnston, Ph.D., Research Associate Professor, Department of Pharmacology & Chemical Biology, University of Pittsburgh Drug Discovery Institute
  • Karol Kozak, Ph.D., Head, Data Handling Unit & High-Content Screening, ETH Zurich

8:30-9:00 am Registration for the Advanced Workshop and Morning Coffee

 

Advanced Workshop Part I: Instrumentation and Automation

9:00-9:45 An Introduction to High-Content Screening and Analysis Technologies

Anthony M. Davies, Ph.D., Director, High-Content Research Facility, Clinical Medicine, Trinity College, Ireland

Descriptions and Capabilities of HCA Platforms

Associated with the rapid and sustained expansion of the HCA market has been a proportional rise in the number of commercially available cellular imaging and analysis platforms. In the face of this ever increasing number of choices and configurations it has now become all the more important to not only gain a clear understanding of the capabilities of these technologies, but also the direction in which their development is heading. In this section of the workshop we will cover:

  • HCA platforms: An examination of the functionality and unique selling points of the numerous HCA systems currently on the market
  • Making the RIGHT choice for your lab: An assessment of the most widely used methods employed when choosing the HCA instrument
  • Training your personnel for maximum productivity
  • Effective strategies for managing equipment and users

9:45-10:30 A Guide to Setting up an HCA Lab

Anthony M. Davies, Ph.D., Director, High-Content Research Facility, Clinical Medicine, Trinity College, Ireland

Imaging, Robotics and People

It is clear that a well-developed strategy for the setting up and running of an HCA lab will ensure the fastest route to attaining the productivity that this technology promises. To guarantee a swift and trouble-free deployment of this technology there are a number of key steps that need to be considered, including:

  • Procurement planning and justification for purchase of equipment
  • ­How to stay friends with your equipment vendor
  • Robotics, automation and software: What’s available and what will you need?

10:30-11:00 Networking Coffee Break

 

Advanced Workshop Part II: Assay Development

11:00 am-12:30 pm HCA/HCS Assay Development

Paul A. Johnston, Ph.D., Research Associate Professor, Department of Pharmacology & Chemical Biology, Drug Discovery Institute, University of Pittsburgh School of Medicine


The development of high-content analysis/screening assays (HCA/HCS) involves the optimization of sample preparation methods, image acquisition procedures, and image analysis algorithms.

HCS sample preparation is a complex, multi-component process that includes selection and optimization of cell line, microtiter plate, fixative, permeabilization buffer, blocking buffer, wash buffer, primary and secondary antibodies, and fluorescent probes. The choices made for automating cell plating, compound treatment, and sample preparation will also have a significant impact on the biology and the consistency of
HCS assays.

Image acquisition for HCS assays requires input on the objective, the number of channels to be acquired, the excitation and emission filters, focal offsets required relative to the autofocus point, exposure times, and number of image fields that need to be captured. The choice of magnification (objective) profoundly affects HCS assay performance and throughput by impacting the resolution, field of view, detection sensitivity, and the output from the image analysis algorithm. The selection of fluorophores, filter performance and mode of image acquisition all impact the sensitivity, signal to background, signal-to-noise, and throughput of the HCS system.

Image analysis can be achieved at several levels: pixels, objects, semantic concepts, and at the pattern and knowledge level. Digital images are composed of pixels, or squares of uniform grey values captured by a CCD camera or PMT that are assigned to objects established through segmentation. The user defines the objects and features to be extracted automatically from every image prior to the analysis procedure. The selection and optimization of the final image analysis parameters typically involves the iterative use of a training set of images, most commonly the assay controls for the top and bottom of the HCS assay signal window.

To illustrate the process and selections that are required during the HCS assay development process, several case histories will be presented.

12:30-1:30 Lunch on Your Own

 

Advanced Workshop Part III: Data Analysis and Management

1:30-3:00 Practical Handling and Data Analysis of High-Content Screening Data

Karol Kozak, Ph.D., Head, Data Handling Unit & High Content Screening, ETH Zurich

This part of the course focuses on the analysis of data derived from HCS. High-Content Screening (HCS) using automated microscopy is an upcoming methodology for the investigation of cellular processes and their alteration by multiple chemical or genetic perturbations. The analysis of the large amount of data generated in HCS experiments represents a significant challenge and is currently a bottleneck in many screening projects. This workshop session reviews the different ways to analyze and manage large sets of HCS data, including the questions that can be asked and the challenges in interpreting the measurements. The main data mining approaches used in HCS, such as image descriptors, computations and classification algorithms, will be outlined.

Objectives:

  • What do we do with all those data?
  • Specify the steps of HCS data analysis
  • Very simple data management and data structures
  • List the issues associated with HCS data analysis
  • List various data standards
  • Open source versus commercial software applications
  • Role of workflow systems in HCS
  • Laboratory Information Management Systems (LIMS)
  • Quality control and normalization
  • Explain what filtering is and how it should be properly used
  • List different pattern discovery techniques such as clustering, classification
  • Explain how biological interpretation is linked to pattern discovery
  • List the different modules of an HCS informatics system

3:00-3:30 Networking Refreshment Break

 

Advanced Workshop Part IV: Discussion

3:30-5:00 Open discussion with all participants

5:00 Close of Day

*Separate Registration Required


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