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 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
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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….
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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.
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..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.
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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.
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..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.
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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.
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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
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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!!
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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
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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……..
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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
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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
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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
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Many Spectra in Flow Cytometry
Roederer, et al
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Multi-Component Systems Amnis Corp
http://www.amnis.com
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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
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High through-put flow cytometry - Multiplexing
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High Through-Put Flow Cytometry
Dr. Larry Sklar, Cytometry 44:83-90 (2001)
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Multispectral microscopy – Not more colors!!!
Color imageMultispectralimage
Greyscaleimage
Expansion/rebirth of the Landsat Concept from the 1970s
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Multispectral microscopy
Camera controller
AOTF controller
Microscope controller
PC computer
Monitor
Intensified camera
CCD camera
AOTF
Microscope
Intensifiedcamera
AOTF
Purdue Spectral Imaging Project
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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
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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
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Nuance-Micro
Slide from Dr. Richard Levenson, CRi, Inc.,35B Cabot Rd.,Woburn, MA 01801, www.cri-inc.com
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Multispectral Imaging – Zeiss Meta
Ability to select a range of wavelengthsAs desired by the user
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Visualization of morphology of cells embedded within a collagen matrix
Publications: http://www.cyto.purdue.edu/flowcyt/research/pub1.htm
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Small Intestinal submucosa – BSL-based visualization
Publications: http://www.cyto.purdue.edu/flowcyt/research/pub1.htm
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Visualization of collagen matrices — laser scanning confocal microscopy using backscattered light
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Combinatorial based classification using multivariate analysis
Robinson et al - Cytometry 12:82-90, 1991
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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
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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
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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
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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
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Additional Material for Discussion
The following materials were incorporated to highlight a number of educational facilities.
Appendix
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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
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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
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Site for Lectures and PresentationsJust under a ton of educational materials!
http://www.cyto.purdue.edu/flowcyt/educate/pptslide.htm
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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
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Image Software reviews
Image Analysis
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Software Tutorials
Dr. Gerald Gregori set of tutorials
More software reviews coming
Independent evaluation