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O-2
HMIHost�MicrobeInteractomics
High throughput high�content imaging and
analysis of live and fixed cells
Jurgen Karczewski / June 13th 2013 Cat�Agrofood Workshop
Host�Microbe Interactomics Group Wageningen University E�mail: [email protected]
HMIHost�MicrobeInteractomics
Introduction
� Postdoc at Host Microbe Interactomics (HMI)
� Background Biology, Toxicology, Immunology
� Microscopy, Confocal Laser Scanning Microscopy
� Signaling at the Interface between Host�Microbe
� Development of Tools and Assays
� Development and Management of the High Content Platform at HMI
� Cat�Agrofood Investment: BD Pathway 885
HMIHost�MicrobeInteractomics
Introduction II
� High Content Imaging
� The BD Pathway 885 microscope
� Segmentation
� Host Microbe Interactions
� Infection/Invasion Assay
� Transcriptional Reporter Assays
� Wounding Assay
HMIHost�MicrobeInteractomics
How Do We Turn These…
HMIHost�MicrobeInteractomics
Into these?
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1.00E-10 1.00E-09 1.00E-08 1.00E-07 1.00E-06 1.00E-05
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EC50: 2.02E-07
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HMIHost�MicrobeInteractomics
Power of Cellular Analysis
Every pixel is like a photomultiplier tube providing position (where) and intensity (how much) information.
Red Green BlueMerged Image
Regions of Interest (ROI)
Channels
HMIHost�MicrobeInteractomics
High Content Cellular Analysis
Data AnalysisImage AnalysisImage AcquisitionBiology
HMIHost�MicrobeInteractomics
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BDTM Pathway 855 � high content high
throughput microscopy
� Live�cell & End�point assays� Environmental chamber � Coverslips�384 wells plates� Liquid handling (robotics)� Confocal or widefield illumination � Laser or image autofocus� Up to 16 colors� multiplexing� High Content Analysis software
� Image processing� Advanced Segmentation� Analysis� reporting� Database Output
� Data mining
HMIHost�MicrobeInteractomics
Features to enhance automation of imaging and analysis
� Environmental control: longer and more complex experiments
� Laser�based autofocus: better plate throughput and less bleaching
� Confocal: sharper images, better segmentation
� Liquid handling: multiple assays after each other
� Montage Mode: multiple images to increase sample size
� Open modular system, additional robotics
HMIHost�MicrobeInteractomics
BD Pathway – Optical Pathway
HMIHost�MicrobeInteractomics
High Content Analysis: need for segmentation
HMIHost�MicrobeInteractomics
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Reporter Cell�lines: Lentiviral Transduction
Kremers G et al. J Cell Sci 2011;124:157�160
Multiplexing
LentiviralFluorescent protein
HMIHost�MicrobeInteractomics
BFP, RFP, mKate2 histon H2B fusion proteins
mTagBFP
mTagRFP mKate2
HMIHost�MicrobeInteractomics
High Content Spectral Strategy
GFP
BFP
RFP
mKate2
YFP
CFP
Histon, RNAi marker
Histon, RNAi marker
Fret pair with CFP
GreenFire Reporter
FRET pair with YFP
Histon?
?
??
?
HMIHost�MicrobeInteractomics
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High Content Analysis Addresses Four Basic Assay Categories
Fluorescence intensity change
• Calcium flux• Phosphorylation• Protein degradation• Image cytometry
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Fluorescence distribution• Cytoplasm to nucleus
(eg NFκκκκB)• Cytoplasm to plasma
membrane (eg PKCαααα)• Plasma membrane to
organelle (egTransfluor GPCR)
• Receptor internalization
• Protein co-localization
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Morphology
• Neurite outgrowth• Angiogenesis• Cell differentiation• Apoptosis
3
Cell Movement
• Chemotaxis/migration• Wound healing• Metastasis/invasion• Long-term tracking
4
HMIHost�MicrobeInteractomics
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Host Microbe Interactions
Marco et al. Curr. Opin. Biotechnol. 2006
HMIHost�MicrobeInteractomics
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Automated infection assays – with high content
data extraction
E.g. Average no of bacteria per cell, percentage of infected
cells, average intensity of bacterial spots per cell, etc..
Applications in identifying host receptors, bacterial genes
required for infection, screening anti�infectives, inhibitors
HMIHost�MicrobeInteractomics
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High Content Screening: Transcriptional reporter assays
� NFkB:inflammatory pathways.� AP�1� Interferon (IRF)� STAT1,2,3� CREB� NFAT� C/EBP� Wnt, Notch, c�Jun� PPARy� RA� Foxo� Ahr
HMIHost�MicrobeInteractomics
pGreenfire Reporter: Fluorescence and Luminescence
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HMIHost�MicrobeInteractomics
HTSScratcher� wounding assays
HMIHost�MicrobeInteractomics
HTS Scratcher
� Controlled spring counterforce ensures the downward movement is smooth and controlled.
� Adjustable scratch length � 1�12 mm
� Versatility � can be used for 96 and 384�well plates
� Wounding assay can be combined with other reporters
Beijnum, JR, VUMC
HMIHost�MicrobeInteractomics
Summary� BD Pathway
� Very suitable for automated live cell imaging and extraction of high content information from cells and tissue (multiplexing).
� Screening of compound libraries, gene silencing arrays, mutant (transposon) libraries, meta�genomic libraries
� High Content Imaging Data can be integrated with genomics, proteomics, metabolomics datasets
