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Technology Integration for Analysis of High Content/Throughput Cellular Data: The Cytomics Approach J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering Purdue University This presentation will discuss current ideas for analysis of live cell data incorporating multivariate approaches. It will outline the major problems faced by present generation technologies and provide insight into future advances. Key to the success of future technologies will be an understanding of informatics and high-speed data processing including advanced image analysis. www.cyto.purdue.edu

J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering

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Technology Integration for Analysis of High Content/Throughput Cellular Data: The Cytomics Approach. J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering Purdue University - PowerPoint PPT Presentation

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Page 1: J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering

Technology Integration for Analysis of High Content/Throughput Cellular Data: The Cytomics Approach

J. Paul RobinsonProfessor of Immunopharmacology &Professor of Biomedical EngineeringPurdue University

This presentation will discuss current ideas for analysis of live cell data incorporating multivariate approaches. It will outline the major problems faced by present generation technologies and provide insight into future advances. Key to the success of future technologies will be an understanding of informatics and high-speed data processing including advanced image analysis.

www.cyto.purdue.edu

Page 2: J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering

Goals of this Presentation

Introduce Cytomics Identify current & forthcoming

issues & technologies Call attention to issues that need to

be addressed

Note: Added for the web version of this presentation:

These slides were perfect!! In Powerpoint. However, as amazing as it might seem, the Powerpoint web converter is pretty much the usual Microsoft Disaster Product (MDP).

So, many animations and boxes with text look strange and the text often fails to remain inside the boxes, lines are everwhere….

Page 3: J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering

What is the Cytomics Approach? Discovering the functional relationships between the

cell (Cytome) and the metabolic pathways (Proteomics-proteome) resulting from genetic control mechanisms (Genomics-genome) –

Some relate Cytomics to what is being termed functional genomics.

By definition we are expanding the information being collected in every system because we also want functional data, not just morphological, phenotypic or genotypic.

Page 4: J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering

..the cell is the ultimate functional endpoint…

Cytomics is going to be important because it is the cell that is the ultimate functional endpoint. The cell is the minimal functional unit within our physiology and thus the functional unit that can be manipulated.

Complexity of cell function is only part of why Cytomics will become a major field of study. Every cell is different. By studying each cell's unique function, that cell type can be further modeled for subsequent analysis using statistical techniques.

As the field of tissue engineering explodes, it will not be long before cellular engineering will be a most important component of which an essential element will be a full understanding of Cytomics.

Page 5: J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering

Cytomic-realignment….

Within a short time, no pharmaceutical company will operate without encompassing the essential features of Cytomics

Drugs design will operate at the level of modified cellular functions, cytome-alignment or cytomic-realignment will become the "cellular form" of tissue engineering.

Page 6: J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering

..how does the cell operate… This knowledge will require a better-than-ever

understanding of how the cell operates, how to measure cell function, and how to characterize the live cell in minute detail.

Single-cell analysis techniques will become enhanced and exquisitely sensitive.

New technologies must be developed and new analytical tools will be required to extract these new data.

Of these analytical tools, informatics will continue to play a crucial role in cell biology.

Page 7: J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering

The big link…

Cytomics links technology to functional biology

Cytomics relates measurement & detection to structure & function

Cytomics integrates tools like flow cytometry, image cytometry, etc. with proteomics.

Page 8: J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering

Cytomics….summary so far

Integration of technologies Functional role of system components Relates measurement & detection to structure &

function Brings together traditional cytometry and non

traditional cytometry Informatics now assumes a primary rather than a

secondary role in cytomics

Page 9: J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering

Current Emphasis

gene

protein

cell

cell

protein

gene

protein

cell

gene

Live Cell

Hey buddy…Don’t you know you

genes, proteins and

organelles are in my territory

now!!

Slide animationis completePlease wait till slide animation is complete

Page 10: J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering

Systems Integration

Analytical Cytology Flow cytometry Single cell analysis systems Tissue analysis (after cell separation)

Image Cytometry & Analysis Single cells Tissues and sections Cell culture systems 3D and 4D cell culture environments

Proteomics Proteins from specific cell populations Rapid identification

Page 11: J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering

Imaging Technologies?

• DNA arrays

• “Quantitative” fluorescence assays

• High Throughput assays (96-384-1536 well plates)

• Elispot

• Drug effect assays, Cyto-toxicity

• Toxicology assays

• Apoptosis

• Cell proliferation, Cell ploidy

• Cytoplasm-to-nucleus transportation

• Hormone receptors, Growth factors

• Gene amplification or deletion, Gene fusion

• Chromosome imbalances

• And the list goes on……..

Page 12: J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering

Next Generation Instruments…

40 fluorescent colors (40-50 variables & 100-200 parameters) Lots of other spatial measurements Lifetime Hyperspectral Imaging - Spectral unmixing/deconvolution technologies Multiple probe systems Complex analytical tools – informatics approaches 120,000 events/s for flow systems (4 x 108/hour) Very high speed for imaging systems Permanent and accurate alignment Intelligent interfaces and operating systems Direct links with diagnostic expert systems

Page 13: J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering

This is HIGH Content

• Huge number of Variable & Parameters

• Very High Speed

• Huge data sets• Opportunity for Rapid classification

RapidIdentification

orDiagnostics

Much of this can becomeReal-Time decision making

Page 14: J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering

Laser Scanning Cytometer

First “modern” high content static cell analysis system

Very high content Moderate speed Very high data

storage required Data-base friendly

Concept first published: Kamentsky & Kamentsky, Cytometry 12:381-7, 1991

Page 15: J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering

Many Spectra in Flow Cytometry

Roederer, et al

Page 16: J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering

Multi-Component Systems Amnis Corp

http://www.amnis.com

Slide animationis complete

Page 17: J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering

Future integration

Eprogen-Beckman-Coulter automated protein separation system

http://www.beckman.com/products/instrument/protein/proteomelab_pf2d_dcr.asp

Cell Sorter

ProteomeLab™ PF 2D Protein Fractionation System from Beckman-Coulter

Page 18: J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering

High through-put flow cytometry - Multiplexing

Page 19: J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering

High Through-Put Flow Cytometry

Dr. Larry Sklar, Cytometry 44:83-90 (2001)

Page 20: J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering

Multispectral microscopy – Not more colors!!!

Color imageMultispectralimage

Greyscaleimage

Expansion/rebirth of the Landsat Concept from the 1970s

Page 21: J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering

Multispectral microscopy

Camera controller

AOTF controller

Microscope controller

PC computer

Monitor

Intensified camera

CCD camera

AOTF

Microscope

Intensifiedcamera

AOTF

Purdue Spectral Imaging Project

Page 22: J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering

Lyot filter (static)

Single bandpass

LCTF (randomly tunable)

400

450

500

550

600

650

700

750

400 450 500 550 600 650 700 750Mea

sure

d c

ente

r w

avel

eng

th (

nm

)

Wavelength “dialed-in”

High precision and accuracy

Enabling Technology: Liquid tunable filters

Slide from Dr. Richard Levenson, CRi, Inc.,35B Cabot Rd.,Woburn, MA 01801, www.cri-inc.com

Page 23: J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering

High-resolution cytology segmentation

ConventionalRGB Image

Spectrallysegmented Image

Wavelength (nm)

CharacteristicSpectra

High spectral resolution increases utility of spectrally responsive indicator dyes

Slide from Dr. Richard Levenson, CRi, Inc.,35B Cabot Rd.,Woburn, MA 01801, www.cri-inc.com

NOTE: this slide has animation – you should wait till it finishes

Slide animationis complete

Page 24: J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering

Nuance-Micro

Slide from Dr. Richard Levenson, CRi, Inc.,35B Cabot Rd.,Woburn, MA 01801, www.cri-inc.com

Page 25: J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering

Multispectral Imaging – Zeiss Meta

Ability to select a range of wavelengthsAs desired by the user

Page 26: J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering

Visualization of morphology of cells embedded within a collagen matrix

Publications: http://www.cyto.purdue.edu/flowcyt/research/pub1.htm

NOTE: this slide has animation – you should wait till it finishes

Slide animationis complete

Page 27: J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering

Small Intestinal submucosa – BSL-based visualization

Publications: http://www.cyto.purdue.edu/flowcyt/research/pub1.htm

Page 28: J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering

Visualization of collagen matrices — laser scanning confocal microscopy using backscattered light

NOTE: this slide has animation – you should wait till it finishes

Slide animationis complete

Page 29: J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering

Combinatorial based classification using multivariate analysis

Robinson et al - Cytometry 12:82-90, 1991

Page 30: J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering

Modern optical microscopy

Confocal microscopy UV+VIS Fluorescence Backscattered laser light Environmental Control

Multiphoton microscopy 2-p and 3-p fluorescence SHG Lifetime (B&H)

Multispectral microscopy Wide-field Confocal (Bio-Rad Rainbow, Zeiss

Meta) Purdue “Spectralfluor” CRI’s Nuance-Micro

Core technologies

NOTE: this slide has animation – you should wait till it finishes

Slide animationis complete

Page 31: J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering

So what does the future look like for data processing?

It’s moving fast! We are pushing multiple

technologies simultaneously Data processing is well beyond

human capacity – Informatics Functional studies bring

exponential complexity Real-Time decision making will be

the next requirement

Page 32: J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering

Is there life after HCS Fortunately HCS is not eternal We are going through an evolution of

rapid technology change We are trying to use current HCS

technologies to do everything – that will change

We all do need to be cautious – some companies will develop great technologies and then go broke! What will you do then?

The only “dream machine” is in a dream

Page 33: J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering

Acknowledgements

Bio-Engineering Bartek Rajwa Jennie Sturgis Wamiq Ahmed Muru Venkatapathi Silas Leavesley Jim Jones Padma Varadharaajan

Microbiology/Biofilms Stephanie Sincock Gerald Gregori

Cytomics Jia Lu Kathy Ragheb Cheryl Holdman Gretchen Lawler

Page 34: J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering

Additional Material for Discussion

The following materials were incorporated to highlight a number of educational facilities.

Appendix

Page 35: J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering

Some Key Web Linkswww.cyto.purdue.eduhttp://www.cyto.purdue.edu/HCS

Issues that can be addressed:

1. Discussion Group - Communication2. Educational – knowledge development and training3. Issues of data file standards4. Issues of calibration standards5. Image processing issues – algorithms and processes

Opportunity?Discovery Park, Purdue UniversityCenter for Applied Cytomics

Expanded educational roleCan we partner with HCS users and developersCan we train in the basic issues

Page 36: J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering

ISAC International Society for Analytical

Cytology Forum to address issues Cell-focused – understands “high

content” Has an existing structure International meeting May 23-28, 2004 in

Montpellier, France www.isac-net.org

Page 37: J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering

www.cyto.purdue.edu/HCSEMAIL me suggested [email protected]

Survey of

HCS Users

Page 38: J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering

Site for Lectures and PresentationsJust under a ton of educational materials!

http://www.cyto.purdue.edu/flowcyt/educate/pptslide.htm

Page 39: J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering

Data standards – Conversion to standard

LData Converts any

instruments specific file to FCS for software analysis

Can be used as independent utility or included in code

Page 40: J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering

Image Software reviews

Image Analysis

Page 41: J. Paul Robinson Professor of Immunopharmacology & Professor of Biomedical Engineering

Software Tutorials

Dr. Gerald Gregori set of tutorials

More software reviews coming

Independent evaluation