HighContent Screening: Emerging Hardware and Software Technologies

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High-Content ScreeningEmerging Hardware and
Software Technologies

• Seungtaek Lee
• Automated Biotechnology
• Merck Co

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Outline

• HCS Introduction
• 4 Components of HCS/HCA
• 1. Cell Preparation and Labeling
• 2. Image Acquisition
• 3. Image Analysis
• 4. Data Management and Informatics
• Current Bottlenecks

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HTS Strategy

• Why Followup with HCS/Imaging?
• More representative biological models.
• Direct and dynamic measurement of biological
  changes that occur when cells are treated with
  compounds or siRNA
• Obtain rich and diverse set of measurements using
  multi-parametric analysis and multiplexing (High
  Content)
• Multiple probes, toxicity, fluorescent compounds,
  liquid handling error.
• False positive identification
• Individual cellular analysis
• Visual verification

• Keys to HCS
• Understanding the biology (Assays)
• Proper Tools/Instruments (Imager)
• Image and data analysis (Software)
• Informatics (Image/Data management)

Imaging!
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Step 1 Cellular Assay and Imaging Preparation

• Determine cell type, assay technology (marker),
  and phenotype
• Kinetics
• Agonist/Antagonist dose response
• DMSO tolerance
• Plate type
• Optimize fixation method

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Immunofluorescence/Neuronal Applications
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DNA Damage and Mitotic Arrest
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GFP Applications
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Fixing and Staining Protocols
GFP
Wash and Image
Seed cells (24 hrs)
Add cmpd (10 min)
Add DRAQ5/ Formaldehyde (10 min)
Add agonist (1hr)
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Step 2 Image Acquisition

• Widefield vs. Confocal
• Excitation source and filters
• Autofocus
• Environmental control
• Field of view
• Live/on-the-fly image analysis

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IN Cell Analyzer 3000

• Line scanning, confocal system
• Simultaneous, three color imaging
• Large field of view and rapid scan rate
• Up to 500 cells per image
• Scan rate
• 96 wells 2.5 - 5 min
• 384 wells 8 - 18 min
• Throughput gt35,000 wells per day
• Live or fixed cell assays
• Real-time data analysis
• Integrated software with multiple analysis
  modules

GE
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HCS Imaging Systems
Opera Evotec
iCyte - CompuCyte
IN Cell 1000 - GE
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Widefield
Confocal
Tubulin
Phospho-MAP
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Step 3 Image Analysis

• Canned modules vs. scripting (macro)
• Receptor trafficking
• Object intensity/co-localization
• Translocation
• Cell cycle
• Morphology
• Neurite length
• Toxicity
• Micro Nuclei
• MULTIPARAMETRIC analysis (in one assay)

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Neurite Length Measurements
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Comparison of Algorithms
New Variable Spot Size Algorithm (Vesicles
identified are shown by the white bitmap overlay)
Original Granularity Algorithm (vesicles
identified are shown by the grain boxes within
the cells bounding box)
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Confirmation Screen Data from the

Granularity and Variable Spot Size Algorithms
Granularity Algorithm
Average fluorescent intensity of vesicles per
cell were measured using both algorithms.
Images were captured with 2x2 binning
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Step 4 Image Management and Informatics

• Image storage and archive
• Image and data retrieval, reanalysis, and
  visualization
• Report generation

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Image Volume Management
Image
1MB/Image
(2 color, 0.375x0.375 mm image)
Well
2MB/Well
(2 images/well)
1GB/Plate
Plate
(384 well plate)
100 GB/Screen
Screen
(100 plates/screen)
Includes assay development
Year
1 TB (10 screens)
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HCS Campaign Workflow
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Bottlenecks

• Cellular Assay cell preparation
• Relatively low throughput
• Washes, fixes, staining
• 96 or 384-well format
• Image Management and Data Visualization
• Terabytes of images/data
• Archival, retrieval, visualization, reports

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GNF/Opera Automated Imaging System (Cellular
Assay and Imaging)

• Staubli robot
• 384-well plate washer/dispensers
• Ventilation
• Centrifuge
• Plate storage (static hotel)
• 486 plate incubation/refrigeration
• Small volume (nl) transfer
• Automated Error reporting
• Evotecs Opera

3D movie courtesy of GNF (Genomics Institute of
the Novartis Research Foundation)
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GNF/Opera Automated Imaging System (Cellular
Assay and Imaging)

• Cell plating
• Compound transfer
• Transfection
• Incubation (4C/37C)
• Live-cell imaging
• Fixation (PFA)
• Washes with minimal cell loss
• Confocal Imaging in 384 and 1536
• Automated data handling

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Informatics
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HTS Strategy
Screen Entire Library Confirmation Second
ary Assays
Imaging!
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Acknowledgements

• Merck Co, Inc.
• Bonnie Howell
• David Ross
• Marc Ferrer
• Jeremy McGarvey
• Erica Stec
• Anthony Kreamer
• Ken Wilke
• Jonathan Schneeweis
• Xinnian Li
• Jim Inglese
• Francesca Santini
• Berta Strulovici

• GNF
• Avi Spier
• Norak
• Carson Loomis
• Robert Oakley
• Christine Hudson
• Cellomics
• Mark Collins
• Tim Harris
• Bill Karsh