High Content Imaging and Flow Cytometry with Phaedra: can we integrate with Knime?”

Frans Cornelissen

Informatics CoE, Janssen Pharmaceutical Companies of

Johnson & Johnson

What’s in a name…

Plate-based

High-

Content

Analysis,

Evaluation

&

Dynamic

Reliability

Assurance

LIMS-integrated screening tool.

Can start from images, or from images + numerical data.

End point is compound result, single-well or dose-response.

Strong focus on user interaction, quality control functions and annotation.

GOALS of PHAEDRA

• Seamlessly integrated data access.

– Images and numerical data.

– Cell level, well level, and plate level data with full drill-down capacity.

– Full experimental context, including e.g. compound structures.

– Dose-Response Curve Fitting

– Upload to Data Warehouse

• Data hub between different systems.

– All types of HCS imagers.

– Experimental setups from different LIMS environments.

– Capable of accepting data from other [imaging-analysis] equipment.

• Bring data analysis to a higher level.

– Highly interactive, customizable environment.

– Statistical tools for univariate & multivariate data analysis.

– Full QC & Annotation options.

– Search & Report facilities.

Architecture & Design

4

Java language

Oracle database

OSGI dynamic module system

• Permits cross-platform deployment.

• Developer familiarity.

• Multiple implementations available.

• Highly efficient image compression.

• Fast extract Images on different ROI/Scales/Quality.

• Rapid incorporation of new tools.

• Dynamic management of modules and updates.

Eclipse/RCP app framework.

• Highly configurable user environment.

• Add functionality from BioClipse, OpenChrom, …

JPEG 2000 images

• Database links with LIMS systems.

OpenGL, ICS, R, MATLAB, Javascript, PDF, JFreeChart, …

(thanks to these standards/Technologies) most of PHAEDRA was developed in ~ 12 months

Experiment Definition

Plate Definition & Preparation : TiboLims, Laplace, …

Plate Definition

Plate Scan

Plate Scan: Mias-2, Opera, Incell,

& Analysis: Dci, Matlab, A

Capella,…

Metadata

Plate Preparation

Images

Image Analysis

Cell + Well Data &

Overlays

ABCD FEDS

PHAEDRA

Import Archival Validation

& Approval

Search, Report,

Print

Heatmaps Charts

Visualization XX50 calcs

Images, Plate, Cell

&Well Annotation

Link

Data Warehouse

Search & Vizualization

PDF files

Rules, Features, Preferences, ..

Create & Edit

Protocols

HCA : Systems & Process Interconnections

Yellow boxes: can be done in or initated from PHAEDRA

Annotations

Features-Kinetics

Image Types

Well Entities

Wells

Plates

Experiments (runs, batches, plate stack)

Protocols (e.g. diff cell lines)

Protocol Classes Neo

Neo Cell Line ABC

Batchname 1

Plate 36570152 Plate 36584432

Well 1 Well 2

Compound, Concentration Well Results

Mean Area, length, perimeter, intensity

Well Annotations

Images

Raw Image Channels

Overlay Result Images

Cell Results

Area, length, perimeter, intensity

Cell Annotations

Well 3

Batchname 2

Neo Cell Line DEF

Batchname 3

PHAEDRA DB : Main Entity Definitions

Drill down

PHAEDRA UI: Extensive, Flexible GUI based on RCP + OSGI, an

Industry-Standard Open Source Framework

Consists of…

All Views can Talk to each Other

Assemble views and create own perspectives

One perspective, 3 views

Perspectives can co-exist

User can have multiple, cooperating perspectives open at

the same time

Eclipse/RCP Framework

8

Dockable GUI

45+ Views

Customizable perspectives

Univariate Tools

Multivariate Tools

PHAEDRA: general GUI Look and Feel

Perspective open/switch

User & data source indicator/selector:

DVL, PPR, PRD

Subsystem Traffic Light

Health Indicator

RAM usage indicator & Garbage Collector

Multiple Views

Heatmaps

charts

Images

Image Detail

EXPLORER

Dynamic Drill-Down from Compound to Cell

10

DR Curve Plate Heat Map

Well Spider

Scatter Plot of Cells

Every Open View reacts immediately to all actions/selections

Quality Control & Annotation

11

• Strong focus on supporting interactive QC of the HCS data set.

• Users can add annotations to:

• Wells

• Cells

• Regions on images

• Invalid wells can be excluded:

• By selecting wells manually.

• By defining filter criteria, (RULES) e.g. on number of cells detected.

• By selecting wells on well scatter plots.

• Exclusion of data points triggers [automatic] recalculation.

• Validation (User), Approval(Manager)

12

PHAEDRA: Validation: Well Selection Rules Queries: e.g. Indicate all well images where #branches > 30

Efficient : • Can apply any rule to select wells, then invalidate all selected • Rules can be stored / reused

Advanced Data Analysis Tools Built-In

• MATLAB integrated to support advanced Image Analysis

3D Colony Formation 3D Neurites & Spines

Analysis of low-resolution, whole-well brightfield Z-stack.

High-resolution confocal Z-stack.

Advanced Data Analysis Tools Built-In

• R engine for statistics and dose-response curve analysis – Supports rejection of outliers or selection of an alternative model.

– Multiple measured features are fitted in parallel.

• Javascript tool – Data transformations to generate ‘Calculated Features’.

PHAEDRA: Protocol Class Configuration

Each aspect of each

Protocol can be fully and individually

configured

Users/ Roles / Access Rights

Each aspect of each

FEATURE can be fully and individually

configured

PHAEDRA: Protocol – FEATURE Configuration

For calculated (derived) features

For dose-response curves

17

PHAEDRA: Protocols: IMAGE Configuration

Extensive, Comprehensive:

Each aspect of each

Image Channel can be fully and individually

configured

Other config panels

PHAEDRA: Analysis & Import: in & outputs

1 : drag input folder

2 : Select output Protocol (defines the analysis)

3 : go to the analysis settings

PHAEDRA: Analysis & Import: settings

check Settings (optionally)

Experimental cluster support

Settings are saved with the data

PHAEDRA: Analysis & Import: preparation/test

Test a few wells or do

Full Analysis of all plates in

this experiment

Preparation: Input Folders are “validated”

PHAEDRA: Analysis & Import: execution & import

analysis: all wells are analyzed ; runs MULTITHREADED in background while continue other work

There is also an import-only perspective, to integrate 3th party analysis results & images

PHAEDRA: Image Visualization

Complete : Linked Hierarchical Image views from

PLATE to WELL to CELL

PHAEDRA: JPEG 2000 Images, JPX format

JP2 compression: • best compression for natural imagery •Less artefacts, very flexible&performant file format

JPX format: • multiple layers stored in 1 file • each layer is optimally compressed

Enables fast random access and on the fly Generation of arbitrary sized thumbnails

Each protocol has its own import +compression settings

PHAEDRA: Show/Add Well & Cell Annotations

Cell rejection & Well recalculation

PHAEDRA: Data Visualization

Statistics, Histograms, Scatter, Trends, Spiders, … , for Experiments, Plate, Well & Cell data

Protocol Experiment Plate

PHAEDRA: Data Visualization

Multiple plate Info Tabs for each Plate

Hide/show , Maximize Views

Cell Histograms & Statistics

• All views from all perspectives can be combined at will • Views can be placed and scaled as needed • Users can save any combination as new personal perspectives

Fitting

Reject outliers & refit

Batch Compound Validation/Approval

Invalidated & Disapproved compounds will NOT be uploaded to the Data Warehouse

Original state = unchecked

Display: 1200 compounds/7200 curves

Invalidated state

Validated state

Column Grouping

Freezing Columns

Images & data are linked : up to individual cell/organell

Bi-directional hightlighting

31

PHAEDRA: Search & Report (1)

User can Combine any number of search Rules, Save searches

for later use, Drill down in Collections, etc.

PHAEDRA: Search & Report (2)

Everything that can be shown on screen can be put in

a PDF Report

EXAMPLES: 3D Tumor Colony analysis

Max. Intensity Projection Images

Colony Size

Colony Location

Max. Intensity Projection Image

Quality Control: Uniformity of colony location

EXAMPLES : 3D Tumor Colony analysis

Colony Size Position & Size

EXAMPLES: 3D Neurite & Spine Analysis

Max. intensity Projection

Thumbnails

Effect of Pilocarpine

EXAMPLES: 3D Neurite & Spine Analysis

ICS: ~ 60 K / image

Multiple Real-time Interactive 3D Views

Multi-channel fluorescence:

Show arbitrary

image & overlay combinations,

set transparency, Stretch individual

channels

Epithelial -Mesenchymal Transition Assay (oncology)

Same file, different channels

3D

3D-Dicom 3D : slices LSM ICS JPG TIFF PNG NIFTI

Analyze GIF

BioRAD …

Phaedra as a multi-format, multi-dimensional image viewer

39

Summary - PHAEDRA: 50 Linked Views

12 Linked Chart Types

PHAEDRA: >150 Context Actions (only highest level shown)

Intuitive, Extensive All actions

available as context menu

items

PHAEDRA: >100 Preferences

Extensive: About Everything that can be shown on screen can be configured - customised

Flexible: Centrally Stored per User ; User can log in on any PC and reuse his preferences

FEDS: JP2 Plate Images,Overlays & Data in PDF

Interactive !

JP2 in PDF: Best of Several Worlds: • Highest Compression • Standard Archival of Image + Data • Interactivity

cell/well data as attachments

PHAEDRA: Development

PHAEDRA: Future • Extend Image Analysis Toolkit for new protocols • Finalize Cell-level Rejection & Recalculation • Kinetics (see below) & Flow Cytometry (next slide)

End

Ari de Groot, Frederick Michielssen, Pieter van Nuffel, Michael Geerts

Flow Cytometry in PHAEDRA: R & Knime

FCM == workflow ! Compensation Transformation Gating strategies Counting

Need to show data & scatterplots at different stages in the gating, HilLite subpopulations in all plots

=> KNIME!

produce high-quality graphics:

R + Reporting

FCM in PHAEDRA: R & Knime

Planning: • develop nodes to :

• read/write • FCS files (FCS 3.1 spec) • gate files (Gating-ML spec) • ACS files (archiving in FCM, ZIP +TOC.xml contains all data files of 1 plate)

• automatically/ interactively define rectangular/polygon gates on scatterplots Lots of Questions: • can we integrate the Knime application with Phaedra ? • can we execute a WF iteratively on all wells of a plate (headless – no UI) • can we execute all R calls within a single (static, persistent) instance (in order to reduce data transport back & forth)