1 MDS Analytical Technologies
Complete HCS Solution
Turning the Corner on Speed and Flexibility
Gloria Huang
Applications Scientist, MDS Analytical Technologies
2
What is high-content screening?
• Cell-based assays for drug discovery and other research
• Automated fluorescent imaging
• High-throughput microscopic image analysis
0.105
Plate reader Well-by-well data
High Content Screening Cell-by-cell data
What is High Content Screening?
From one well:
⇒one data point
From one well:
• Each cell measured individually
• Multiple measurements per cell
⇒Thousands of data points
examples of HCS assays
Drug discovery
• Transfluor
®for GPCR screening
• Nuclear Translocation
• Cell Counting Cancer research
• Angiogenesis
• Cell Cycle
• Monopolar Spindle Formation
• Live / Dead / Apoptosis CNS
• Neurite Outgrowth
3 TNF- induced NF-kB nuclear translocation.
Andrea L. Kasinski, Yuhong Du, Haian Fu, et. al, Mol Pharmacol 74:654–661, 2008
Axonal Outgrowth
Fenghua Hu and Stephen M. Strittmatter, Yale University School of Medicine J. Neurosci. 28(5):1262–1269, 2008
The N-Terminal Domain of Nogo-A Inhibits Axonal Outgrowth
Inhibition or not?? % of Inhibition ??
Axonal Outgrowth
original image segmented image
• Outgrowth length
• Branches
• Straightness of processes
• Cell body area
Neuron precursor cell differentiation
Exploring the regulation of human neural precursor cell differentiation using arrays of signaling microenvironments
Yoav Soen, Patrick O Brown, et. al , Stanford University Molecular Systems Biology , 2006
Glial-like cells
Neuron-like cells
5 Neuron precursor cell differentiation
Neuron precursor cell differentiation
Global analysis of
differentiation
AR Conformational Change and Subcellular Localization
original image segmented image
Jeremy O. Jones, W. Frank An, and Marc I. Diamond UCSF, Broad Institute of Harvard and MIT, 2009
• Androgen Receptor (AR) o nuclear hormone receptor (NR)
superfamily
o ligand regulated transcription factor
• Ligand binding induces an intramolecular conformational change that brings the N- and C- termini into close proximity
• After ligand activation, AR accumulates in the nucleus, where it binds DNA as a homodimer at specific androgen response elements (AREs) to regulate gene expression.
AR Conformational Change and Subcellular Localization
original image
Jeremy O. Jones, W. Frank An, and Marc I. Diamond UCSF, Broad Institute of Harvard and MIT, 2009
Untreated Cells
Positive Control
↓
AR conformational change↑
Nuclear localization↑
AR conformational change↓
Nuclear localization7 Lipid-droplet formation and utilization
Yi Guo, and Robert V. Farese Jr University of California, San Francisco Nature 453:657, 2008
Lipid-droplet formation and utilization
Nature 453:657, 2008
Functional genomic screen
RNAi-mediated genomic screen
http://mpibcms.biochem.mpg.de/en/rg/lipidrophe/index.html
Mitotic Spindle Assembly
• Genes Required for Mitotic Spindle Assembly in Drosophila S2 Cells
• Department of Cellular and Molecular Pharmacology, University of California, San Francisco
• SCIENCE 316:417–21, 2007
An image- based, genome-wide RNAi screening of metaphase spindle morphology.
Antiangiogenesis in Transgenic Zebrafish
T. Cameron Tran, Blossom Sneed, Cancer Res 67(23), Dec. 2007
9 Genomics to phenomics in the yeast
FEBS Letters 583 (2009) 1656–1661
Franco J. Vizeacoumar, Yolanda Chong, Charles Boone *, Brenda J. Andrews * University of Toronto, Canada
High-Content Analysis
• Cell-by-cell data – more information from each sample
• Sub-cellular analysis – enables more assays
• Multiple assays from a single plate Advantages
• Automatic image acquisition and analysis
• Data storage intensive
• Data management
• Generally requires more user expertise
Challenges
A Complete Solution for High Content Screening
Samples Image
Analysis HCA
Complete for Solution for HCS:
Fully self-contained imaging platform that integrates:• Image acquisition
• Image analysis
• Data analysis
Images
D emo 46 - MeanCu r ve Fitt in g - Hill, EC5 0
Co n ce n tr at ion [ µM] ( B2 AR E 12 5 0- K Iso p ro te r en o l DRC)
1 e -5 1e - 4 0. 00 1 0. 01 0 .1 1
Cell: Vesicle Count (Transfluor)
10 15 20 25
Maximize Resource:
• Ease of Use
• Flexibility
• No third-party resource
MDS Complete Solution for HCS
11 MDS-AT HCS Customers
Acquisition through HCA
Image
Analysis HCA Images
Thumbnail Image array
D emo 46 - Mean Cu r ve Fitt in g - Hill, EC5 0
Co n ce n tr at ion [ µM] ( B2 AR E 12 5 0- K Iso p ro te r en o l DRC)
1 e -5 1e - 4 0. 00 1 0. 01 0 .1 1
Cell: Vesicle Count (Transfluor)
10 15 20 25
Acquisition
MDCStore
Database
ImageXpress
MICRO™Widefield
• CCD-based imager
• Small footprint
• Modular and cost-effective
• Environmental control
• Fluidics
• High-speed laser auto-focus
Microplate Well 2 Well 1
Laser Camera Laser Camera
ImageXpress
MICRO™Widefield
• 300 watt Xenon lamp
• Up to 4 objectives
• Up to 5 filter cubes
• Brightfield/Phase contrast
• Robotic plate handling
13 ImageXpress - image acquisition wizard
Acquisition Wizard
1. Experiment Information 2. Magnification
3. Plate
4. Wavelengths
5. Acquire
Adaptive Acquisition™ for fast multi-site imaging
• Option to set a minimum number of cells per well
• System only collects as many sites as necessary
• Can reduce acquisition time 5-10 fold Minimum needed: 3
Sites captured: 12
Minimum needed: 3 Sites captured: 3
Z-stack as best focus
• MetaXpress captures Z-stack of images
• Each raw image may be only partially in focus
Images courtesy of Min Wu, BD Biosciences
15
Raw z-series data
Z-stack as best focus
MetaXpress calculates best focus image from stack
Images courtesy of Min Wu, BD Biosciences
Best focus algorithm
Journaling in MetaXpress enables more assays
• Design custom protocols
oCapture entire well at 2x
o
Analyze to detect object of interest
o
Automatically center stage around object and zoom in to 40x
oCollect kinetic data at single site
Image Acquisition
• Easy of Use
o Image acquisition wizard o One button access
• Flexibility
o Magnification and wavelength choice o Sample format
o Journaling
MDCStore
Database
Acquisition through HCA
Image
Analysis HCA Images
Thumbnail Image array
D emo 46 - Mean Cu r ve Fitt in g - Hill, EC5 0
Co n ce n tr at ion [ µM] ( B2 AR E 12 5 0- K Iso p ro te r en o l DRC)
1 e -5 1e - 4 0. 00 1 0. 01 0 .1 1
Cell: Vesicle Count (Transfluor)
10 15 20 25
Acquisition
17 MetaXpress – built on the MetaMorph ® backbone
• MetaMorph – a powerful imaging toolbox
o Developed over the last two decades
o Evolved in response to real-world imaging research o Full toolbox of image processing algorithms o Journaling for easy-to-build analysis macros
Number of v erified new references per year citing MetaMorph (source: HireWire Press from Stanford University)
2 2 4 13 25 30 67 92 100 136
186 220 269 310
436 547
718 841
1025 1148
0 200 400 600 800 1000 1200 1400
1986 1987
1988 1989
1990 1991
1992 1993
1994 1995
1996 1997
1998 1999
2000 2001
2002 2003
2004 2005 Year
Number of known published papers
MetaXpress Image Analysis
1. Count Nuclei 2. Cell Scoring 3. Mitotic Index 4. Live/Dead 5. Cell Health 6. Translocation*
7. Translocation Enhanced*
8. Granularity 9. Transfluor*
10.Angiogenesis Tube Formation
11.Neurite Outgrowth Cell Cycle
12.Cell Cycle 13.Monopole Detection 14.Multi Wavelength Cell
Scoring
15.Multi Wavelength Translocation*
16.Cell Proliferation HT*
17.Transfluor HT*
18.Nuclear Translocation HT*
19.Micronuclei
The Solution for High-Throughput Imaging
• Remove analysis bottleneck: faster than image acquisition
• License the power required
• Modified Analysis Settings? Quickly reanalyze entire screens
Image analysis in minutes with parallel processing
19 Image Analysis
• Easy of Use
o Application Modules o Simple configurations
• Flexibility
o Work with multiple assays/kits o Journaling
o Import images from other imaging instruments
MDCStore
Database
Acquisition through HCA
Image
Analysis HCA Images
Thumbnail Image array
D emo 46 - Mean Cu r ve Fitt in g - Hill, EC5 0
Co n ce n tr at ion [ µM] ( B2 AR E 12 5 0- K Iso p ro te r en o l DRC)
1 e -5 1e - 4 0. 00 1 0. 01 0 .1 1
Cell: Vesicle Count (Transfluor)
10 15 20 25
