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

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….

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.

..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.

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.

..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.

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.

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

Current Emphasis

Live Cell

Hey buddy… Don’t you know you genes, proteins and organelles are in my territory now!!

Please wait till slide animation is complete Slide animation is complete

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

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……..

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 10 8 /hour) Very high speed for imaging systems Permanent and accurate alignment Intelligent interfaces and operating systems Direct links with diagnostic expert systems

This is

HIGH

Content

• Huge number of Variable & Parameters • Very High Speed Rapid Identification or Diagnostics • Huge data sets • Opportunity for Rapid classification Much of this can become Real-Time decision making

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

Many Spectra in Flow Cytometry

Roederer, et al

Multi-Component Systems

Amnis Corp http://www.amnis.com

Slide animation is complete

Future integration

Cell Sorter

ProteomeLab™ PF 2D Protein Fractionation System from Beckman-Coulter Eprogen-Beckman-Coulter automated protein separation system http://www.beckman.com/products/instrument/protein/proteomelab_pf2d_dcr.asp

High through-put flow cytometry - Multiplexing

High Through-Put Flow Cytometry

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

Multispectral microscopy – Not more colors!!!

Greyscale image Color image Expansion/rebirth of the Landsat Concept from the 1970s Multispectral image

Multispectral microscopy AOTF Intensified camera CCD camera Camera controller AOTF controller Microscope controller Microscope Monitor PC computer Intensified camera AOTF Purdue Spectral Imaging Project

Enabling Technology: Liquid tunable filters

Lyot filter (static) Single bandpass 750 700 650 600 550 500 450 400 400 450 500 550 600 650 700 Wavelength “dialed-in” High precision and accuracy 750 LCTF (randomly tunable) Slide from Dr. Richard Levenson, CRi, Inc.,35B Cabot Rd.,Woburn, MA 01801, www.cri-inc.com

High-resolution cytology segmentation

NOTE: this slide has animation – you should wait till it finishes Characteristic Spectra Conventional RGB Image Spectrally segmented Image Slide animation is complete Wavelength (nm) 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

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

Multispectral Imaging – Zeiss Meta

Ability to select a range of wavelengths As desired by the user

Visualization of morphology of cells embedded within a collagen matrix NOTE: this slide has animation – you should wait till it finishes Slide animation is complete Publications: http://www.cyto.purdue.edu/flowcyt/research/pub1.htm

Small Intestinal submucosa – BSL-based visualization Publications: http://www.cyto.purdue.edu/flowcyt/research/pub1.htm

Visualization of collagen matrices — laser scanning confocal microscopy using backscattered light NOTE: this slide has animation – you should wait till it finishes Slide animation is complete

Combinatorial based classification using multivariate analysis

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

Modern optical microscopy NOTE: this slide has animation – you should wait till it finishes  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 Nu ance-Micro Core technologies Slide animation is complete

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

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

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

Additional Material for Discussion

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

Appendix

Some Key Web Links www.cyto.purdue.edu

http://www.cyto.purdue.edu/HCS Issues that can be addressed:

1. Discussion Group - Communication 2. Educational – knowledge development and training 3. Issues of data file standards 4. Issues of calibration standards 5. Image processing issues – algorithms and processes

Opportunity?

Discovery Park, Purdue University Center for Applied Cytomics Expanded educational role Can we partner with HCS users and developers Can we train in the basic issues

     

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

www.cyto.purdue.edu/HCS EMAIL me suggested questions [email protected]

Survey of HCS Users

Site for Lectures and Presentations Just under a ton of educational materials!

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

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

Image Software reviews  Image Analysis

Software Tutorials    Dr. Gerald Gregori set of tutorials More software reviews coming Independent evaluation