Embed Size (px)
Citation preview
CYTOMICS IN THE 21st CENTURY
José-Enrique O’Connor Laboratorio de Citómica
Universidad de Valencia-Centro de Investigación Príncipe Felipe Valencia, Spain
The Evolution of Cytometry: From Cytology to Cytomics
Cytology Cytometry Cytomics
New Frontiers in Cytometry: Cytomics
• Cytomics or the cytometry of complex systems is a novel analytical strategy oriented to the determination of molecular phenotype in single cells based on the study of multiple dynamic and structural features of heterogeneous cellular systems (known as the cytomes)
• The cytomes are composed of several individual and coordinated cell types that form the units of organs, tissues, systems and organisms.
Cytomic analysis determines the molecular and functional phenotype in each
individual cell, resulting from its genotype and the exposure to external influences
The objectives of Cytomics
Cytomics benefits of sensitive instrumentation, of non-destructive (fluorescent and non-fluorescent) markers, and of multiparametric methods, on the integrating context of the individual cell, to reveal and quantitate the molecular complexity and the dynamics of tissues and organisms.
The tools of Cytomics
Novel Cytomic Tools
HTS-Flow Cytometry
Confocal Microscopy
Laser Scanning Cytometry
HCA by Automated Bioimaging
Multispectral Image-in-flow Cytometry
Acoustic Flow Cytometry
Mass-Spectrometry Cytometry
Hydrodynamic Flow Cytometry
The Evolution of Cytometry: From Cytometry to Cytomics
• Providing better instrument performance: • Increasing technical capabilities • Lowering size and cost • Increasing user-friendliness
• Providing more biological information: • Accelerating data acquisition • Improving data management and data mining
• Exploring new parameters and fields of application
Providing new and better instrument performance
• Increasing the number of parameters/cell:
• Polychromatic flow cytometry
• Accelerating the sample analysis rate:
• High-Throughput Flow Cytometry
• Accelerating the data acquisition rate:
• Acoustic-Focusing Cytometry
• Expanding the information content:
• Multispectral Image-in-Flow Cytometry
• Mass-Spectrometry Flow Cytometry
Providing new and better instrument performance
Inreasing the rate of samples: High-Troughput Cytometry
The HTFC™ Screening System performs high-throughput, multiplexed, single-cell
analysis on cells or particles in suspension:
• Reads a 96-well plate in 3 minutes, or a 384-well plate in 12 minutes
• Samples from 1 µl to 1 ml
• No sample goes to waste
• With 4 fluorescent channels and 2 light scatter measurements
• Multiuser share data and collaborate with the server-based HyperCytPRO software
• Utilizes suspension cells like blood cells, bone marrow, stem cells or non-adherent
cells lines to perform screening campaigns that require a relevant cell type
Increasing cell acquisition rate: Acoustic focusing in flow cytometry
• Acoustic focusing cytometry uses ultrasound waves (over 2 MHz) to position cells into a single focused line along the central axis of a flow channel without high velocity or high volumetric sheath fluid.
• The acoustic focusing actually concentrates the cells in the center of the fluid with sound energy. This means considerable flexibility in the sample concentration. The end result is more accurate and precise data.
• Acoustic focusing separates the alignment of cells from the particle flow rate. This allows to increase or decrease flow without disrupting the focus of cells in the capillary.
• Cells are hydrodinamically focused into a core stream and orthogonally illuminated for both darkfield and fluorescence imaging. The cells are simultaneously transilluminated for brightfield imaging.
• Light is collected with an objective lens and projected on a charge coupled detector (CCD).
• Prior to projection on the CCD, light is passed through a spectral descomposition system that directs different spectral bands to different lateral positions across the detector.
Multispectral Image-in-flow Cytometry: Linking Form and Function
• 1,000 cells per second (vs. 100 cells/sec)
• 12 images per cell (vs. 6 images per cell)
• Up to 5 lasers (405 / 488 / 561 / 592 / 658 vs. 405 / 488 / 658)
• Multiple magnifications (60X / 40X / 20X vs. 40X only)
• Fully automated 96 well plate handling (vs. single tube only)
• RGB brightfield system (full color vs. single color)
• Expanded ASSIST self-calibration routine
Multispectral Image-in-flow Cytometry: Linking Form and Function
Internalization & phagocytosis
Cell signaling
Shape change & chemotaxis
Surface and intracellular co-localization
Cell-cell interaction
Cell cycle & mitosis
DNA damage and repair
Cell death & autophagy
Stem cell biology
Microbiology
Parasitology
Multispectral Image-in-flow Cytometry: Linking Form and Function
Mass Flow Cytometer (CyTOF): A new multiparametric dimension
• A time-of-flight mass spectrometer (TOF-Ms) uses the differences in transit time through a drift region to separate ions of different masses.
• The ions from the ion source are accelerated by an electric field pulse.
• The accelerated particles then pass into a field-free drift tube that is about a meter in length.
• In TOF-Ms all ions are accelerated to the same kinetic energy. • Their velocities are inversely proportional to the square roots of their masses. • The lighter ions arrive at the detector earlier than the heavier ions, of lower velocity.
Mass Flow Cytometry (CyTOF): A new multiparametric dimension
Mass Flow Cytometry (CyTOF): A new multiparametric dimension
Mass Flow Cytometry (CyTOF): A new multiparametric dimension
Mass Flow Cytometry (CyTOF): A new multiparametric dimension
Mass Flow Cytometry (CyTOF): A new multiparametric dimension
Mass Flow Cytometry (CyTOF): A new multiparametric dimension
Mass Flow Cytometry (CyTOF): A new multiparametric dimension
Enhanced data management
• Managing increasing data per single cell
• Managing data from large experiments
• Mining the data with bioinformatic approaches
Management of increasing data per single cell
Management of increasing data per single cell
Enhanced data management: HTS Flow cytometry
200+ params/cell population statistics object values
Tabular Data
Imag
e G
alle
ry
Workspace
uni + bivariates flexible gating click dot to view cell custom parameters
Enhanced data management: Imaging Flow Cytometry
1 2 2 2
2 43 18 1
2 37 121 2
2 3 3 2 Area = 753 Aspect Ratio = 0.84 Centroid X = 43 Intensity = 153,119
Enhanced data management: Bioinformatic tools in Flow Cytometry
1. Calculate EC50/IC50
2. Array according to human LC50
3. Define toxicityclasses
4. Hyerarchical Cluster Analysis 1 2 3 4 5
Enhanced data management: Bioinformatic tools in Flow Cytometry
Enhanced data management: Bioinformatic tools in Flow Cytometry
Enhanced data management: Bioinformatic tools in Flow Cytometry
Enhanced data management: Bioinformatic tools in Flow Cytometry
Thank you for your aTTenTion, and…
…unTil xxiind cenTury!
www.uv.es/oconnor/IT-LIVER
