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

1. 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

2. 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….

3. 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.

4. ..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.

5. 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.

6. ..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.

7. 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.

8. 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

9. cell gene protein protein gene cell protein cell gene Current Emphasis Hey buddy… Don’t you know you genes, proteins and organelles are in my territory now!! Live Cell Slide animation is complete Please wait till slide animation is complete

10. 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

11. 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……..

12. 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

13. 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

14. 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

15. Many Spectra in Flow Cytometry Roederer, et al

16. Multi-Component Systems Amnis Corp Slide animation is complete http://www.amnis.com

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

18. High through-put flow cytometry - Multiplexing

19. High Through-Put Flow Cytometry Dr. Larry Sklar, Cytometry 44:83-90 (2001)

20. Multispectral microscopy – Not more colors!!! Greyscale image Multispectral image Color image Expansion/rebirth of the Landsat Concept from the 1970s

21. Multispectral microscopy Purdue Spectral Imaging Project

22. Lyot filter (static) Single bandpass 750 700 650 600 Measured center wavelength (nm) 550 500 450 400 400 450 500 550 600 650 700 750 Wavelength “dialed-in” LCTF (randomly tunable) 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

23. Characteristic Spectra Conventional RGB Image Spectrally segmented Image Wavelength (nm) High-resolution cytology segmentation NOTE: this slide has animation – you should wait till it finishes Slide animation is complete 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

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

25. Multispectral Imaging – Zeiss Meta Ability to select a range of wavelengths As desired by the user

26. Visualization of morphology ofcells 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

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

28. 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

29. Combinatorial based classification using multivariate analysis Robinson et al - Cytometry 12:82-90, 1991

30. 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 Modern optical microscopy NOTE: this slide has animation – you should wait till it finishes Slide animation is complete Core technologies

31. 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

32. 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

33. 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

34. Additional Material for Discussion • The following materials were incorporated to highlight a number of educational facilities. Appendix

35. Some Key Web Linkswww.cyto.purdue.eduhttp://www.cyto.purdue.edu/HCS • Issues that can be addressed: • Discussion Group - Communication • Educational – knowledge development and training • Issues of data file standards • Issues of calibration standards • 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

36. 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

37. www.cyto.purdue.edu/HCSEMAIL me suggested questionsjpr@flowcyt.cyto.purdue.edu Survey of HCS Users

38. Site for Lectures and PresentationsJust under a ton of educational materials! http://www.cyto.purdue.edu/flowcyt/educate/pptslide.htm

39. 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

40. Image Software reviews • Image Analysis

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