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Webinar Series Science WHAT CYTOMETRY CAN DO FOR YOU
The Pros and Cons of Image and Flow Cytometry 30 October, 2012
Sponsored by:
Participating Experts:
Brought to you by the Science/AAAS Custom Publishing Office
Webinar Series Science WHAT CYTOMETRY CAN DO FOR YOU
The Pros and Cons of Image and Flow Cytometry 30 October, 2012
Paul Gokhale, Ph.D. University of Sheffield Sheffield, UK
Bill Telford, Ph.D. National Cancer Institute, NIH Bethesda, MD
Liz Roquemore, Ph.D. GE Healthcare Cardiff, UK
What Cytometry Can Do For You: The Pros and Cons of Image
and Flow Cytometry
Bill Telford
National Cancer Institute National Institutes of Health
Bethesda, MD USA
First, you need a biological sample…
…usually labeled with a fluorescent marker
You need to move these cells in a linear stream through a focused light source.
You need a light source to excite the fluorescent molecular on or in the cell.
Usually a CW or quasi-CW laser, good short- and long-term stability, low noise
PMT
You need a filter that only admits light from the fluorescent probe.
You need a sensitive light detector (usually a photomultiplier tube).
You need electronics that can convert analog fluorescent signals to digital ones.
Finally, you need a computer to process the digital signals and display the data
What goes into a traditional flow cytometer?
Fas
expr
essi
on
CD8 expression
Fas expression
CD8 expression
What do we analyze by flow cytometry? Three main things…
Low molecular weight (LMW) fluorophores like fluorescein. Higher molecular weight phycobiliproteins like phycoerythrin. Phycobiliprotein-LMW dye comjugates (tandems) Quantum nanoparticles.
Modern flow cytometers can distinguish 10 or more fluorescent probes simultaneously for detailed interrelational study of the immune system.
PE anti-Fas
fluorescein anti-CD8
Fluorescent probes for cell surface and intracellular labeling
Usually antibodies or other proteins tagged with a fluorochrome.
What do we analyze by flow cytometry?
PE-C
D45
FITC-CD3
monocytes and macrophages
lymphocytes
monocytes and macrophages
B cells
basophils
T cells side
sca
tter
forward scatter
We can analyze many cellular parameters simultaneously, and relate them to each other
What do we analyze by flow cytometry?
Expressible fluorescent proteins are another common tool in biomedical science. Rather than attaching these probes onto the surface of the cell, we actually insert the gene for these proteins into the DNA of the cell. The cell’s own genetic machinery produces the protein, which fluoresces upon excitation. Fluorescent proteins are used as markers of gene expression, and for cell tracking. Green Fluorescent Protein (GFP) is the most famous example, and the most widely used.
What do we analyze by flow cytometry?
Fluorescent probes of cell function
We also have a large variety of physiological fluorescent probes that specifically associate with different parts of the cell (cell membrane, DNA, mitochondria, etc.), and probes that can measure enzymes, detect signaling molecules and measure the flow of ions concentration inside and outside of cells. These probes can measure the viability or functionality of the cells, allow us to measure the amount of DNA, and can allow us to track specific cells against a background of unlabeled cells. Hundreds or more are available.
The limitations of flow cytometry
Flow cytometers can detect very low levels of fluorescence on individual cells. However, they only collect raw fluorescence pulses from each cell. We can analyze this data by integrating these pulses (area), or by measuring their height or width. But that is the extent of analysis possible with traditional flow cytometry. Information about the interior structure and morphology of cells is usually lost. Newer instrument technology allows the collection of cell imagery along with the raw fluorescence data. Image cytometry has become a powerful adjunct technology to flow cytometry. Most image cytometers are based on traditional microscope systems, and collect data from cells fixed to a horizontal slide or plate. However, they collect cytometric data along with images (usually from the images themselves). Images and cytometric data are then correlated. Imagery becomes a parameter.
time
fluo
resc
ence
WIDTH HEIGHT
AREA
1. You need an excitation light source (laser, lamp, or LED)
2. Optics to transmit the excitation light to the microscope
5. Dichroics and filters to separate the signals, and detectors (PMT, photodiode or CCD)
3. An integrated microscope system for visualizing the cells
4. A moveable stage to present the sample.
True image cytometers are capable of collecting and analyzing both images and cytometry data, and correlating the two through relational databasing. High-content analysis.
What is an image cytometer?
Field image mouse EL4 cells quercetin 16 h
Event thresholding… a key aspect of image cytometry
viable cell
apoptotic cell
labeled with PI
PI fluorescence
Field image mouse EL4 cells no treatment
labeled with PI
Like flow cytometers, image cytometers “trigger” on a particular characteristic (such as scatter or fluorescence) to identify objects as cells. Like flow cytometers (and unlike microscopy), analysis is therefore automated and event-based, and can have a significant rate of cellular throughput.
Image cytometry of apoptotic cells
fluorescein PPL caspase 3
fluorescein PPL caspase 3 7-
AA
D
7-A
AD
A
lexa
Flu
or 6
47
anne
xin
V A
lexa
Flu
or 6
47
anne
xin
V
7-AAD
7-AAD
no treatment
camptothecin 6 h
no treatment
camptothecin 6 h
Compucyte iCys analysis of apoptotic EL4 cells
viable early apoptotic advanced apoptotic
viable early apoptotic advanced apoptotic
Compucyte iCys field scans
untr
eate
d in
duce
d
subcontoured
subcontoured
number of spots/cell
number of spots/cell
num
ber
of c
ells
nu
mbe
r of
cel
ls
untreated
induced
Compucyte iCys field scans
Image cytometry of intracellular morphology A powerful advantage of image cytometry is the ability to interpret cellular morphology data as a cytometric parameter. Subcellular localized fluorescence, cytoplasmic > nuclear translocation, etc. can be measured and quantified.
Analysis of autophagy in U2OS cells by measuring the translocation and localization of GFP-LC3 to autophagosomes.
Slide- or plate-based image cytometers
BD Pathway 850
Compucyte iCys
Slide-based image cytometers are the most common. Some use lasers and PMTs like traditional cytometers, some use lamps and CCD cameras. Some are configured for high-throughput analysis. As high-content systems, they correlate and archive both image and cytometric data. The imagery is often of high quality, and is usually the source of the cytometric data.
GE IN Cell 2000 /6000 Analyzers
Thermo Scientific Cellomics ArrayScan VTI
Perkin-Elmer Opera/
Operetta
Stream-based image cytometers The Amnis ImageStream and FlowSight systems capture cellular images from a stream, rather than a fixed surface.
Brightfield Phi Phi Lux Annexin V Draq5 Composite
Again, direct correlation between cytometry and imagery.
Cyntellect Celigo
Special purpose image cytometers
Some image cytometers do not archive their image data, but still derive cytometric data from imagery. They are often designed for specific applications, like viability, apoptosis or basic immunolabeling. Although the imagery is not archived, intracellular features can still be quantified. Lower cost than more advanced systems. Often useful for small sample sizes. Can have rapid throughput.
Beckman-Coulter Vi-Cell
GE Life Sciences Cytell
TTL Labtech Accumen Explorer
Sponsored by:
Participating Experts:
Brought to you by the Science/AAAS Custom Publishing Office
Webinar Series Science WHAT CYTOMETRY CAN DO FOR YOU
The Pros and Cons of Image and Flow Cytometry 30 October, 2012
Paul Gokhale, Ph.D. University of Sheffield Sheffield, UK
Bill Telford, Ph.D. National Cancer Institute, NIH Bethesda, MD
Liz Roquemore, Ph.D. GE Healthcare Cardiff, UK
High Content Analysis Pros and Cons
Paul Gokhale, Ph.D.
Centre for Stem Cell Biology University of Sheffield
Level of detail
Throughput, stats
Flow cytometry High Content Microscopy
What assays is High Content Analysis suitable for ?
Morphology Fluorescent intensity
Fluorescent distribution
Cell behaviour over time
Higher-order structures
Blebbing Neurite outgrowth
Differentiation
Cell cycle Marker expression
Mitosis
Nuclear translocation e.g. NOTCH
DNA damage Co-localisation
Cell Cycle Lineage tracing
Migration
‘Virtual’ cells Colony morphology
Whole embryo Tissue distribution
What factors affect High Content assays?
• Plate thickness: 6 well T/C plates 800-1200~ μm. 96 or 384 nearer 200μm. • Resolution required: Subcellular structure would typically need 20x High NA lenses more difficult to use on T/C plates (may need to use slides) • Well size: Can you see enough of biology in 1 well?
10x lens require ~ 100 fields to cover 1 well of a 24 well plate c.f 4x ~ 20 fields.
• Colours Maximum typically = 4 (e.g. Sedat quad sets) reporters may need specific filter sets.
Pros and Cons of High Content Analysis
Flow Cytometry High Content Analysis
Acquisition
Throughput Large No’s of cells Large No’s of conditions
Parameters Up to 12 colours Typically 4 colour
Sensitivity Highly sensitive Less sensitive due to plates, optics (+ detection)
Sub-cellular imaging
Imaging flow cytometry
Routine
Data analysis
Single cell data
Collects single cell data Requires image segmentation High density causes problems
Time-series Only by sampling Possible
Cell-cell relations
Context lost Position information extractable
Image acquisition
Segmentation
(pre-processing)
Classification & Analysis
The High Content Analysis process
Segmentation and erosion of nuclei
Separation and extraction of a region of interest from an image
Labelling of extracted features from a region
of interest Feature extraction
Classification of features
Extracting the data:- segmentation
Nuclei segmentation Mitochondrial segmentation
(MitoTracker Red)
Subcellular features
Different segmentation methods are used to segment different types of features
Larger scale features Whole cell features
Colony segmentation (using DNA counterstain) Cytosol segmentation
Colony
Nuclear segmentation ‘dilated’ to pick out colonies vs cells
Imaging and analysis with IN Cell Analyzer system and Investigator software
Nuclear area
Morphological information for free Intensity, morphology & spatial data – all from the same sample
39 / Cytometry Webinar
10/31/2012
Addition of an S-Phase sensor Hoechst (DNA content) & BrdU (S-phase)
4
4.2
4.4
4.6
4.8
5
5.2
5.4
5.6
4.6 4.7 4.8 4.9 5 5.1 5.2
Log DNA Content
Log
BrdU
Inco
rpor
atio
n
G1
G2+M
S
Log Hoechst Intensity (DNA)
Log
Cy5
-αBr
dU I
nten
sity
40 / Cytometry Webinar
10/31/2012
G1/S Sensor
Addition of a dynamic phase reporter Detect uncoupling of DNA replication & phase transition
Dynamic phase reporter
+ Cpd A
41 Cytometry Webinar
10/31/2012
High Content Analysis Solutions Emerging trends Complete solutions – acquisition, analysis, interpretation; reagents Speed without compromising image quality Confocality only when you need it sCMOS & LED technology Flexibility for wider range of applications
42 Cytometry Webinar
10/31/2012
What about more routine assays?
Cell count & viability Daily cell culture Assay development
Cell health Membrane integrity Apoptosis
Cell phenotype Membrane integrity Apoptosis
T Cell CD8
Ease of Use & Accessibility Image Cytometers
43 Cytometry Webinar
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43 Cytometry Webinar
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Cell Count & Viability Routine assessment of cultures
Dead
Total Membrane permeant DNA dye labels total cell population
Plasma membrane impermeant DNA dye labels dead cells only
Scatter Plot Reporting % Dead & Live Cells
Sub-Population Analysis
44 Cytometry Webinar
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44 Cytometry Webinar
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Cell Count & Viability
Camptothecin (Log uM)
Effect of Topo I inhibition on Jurkat Cell Viability
HeLa (Adherent)
Benchmark (cells/ml)
Viable Count/ml (CytellTM)
Benchmark (cells/ml)
Jurkat (Suspension)
Viable Count/ml (CytellTM)
Correlation with benchmark cytometer
45 Cytometry Webinar
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45 Cytometry Webinar
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Immuno-Phenotyping Analysis of T-Cell enriched PBMCs
CD4 % CD8 % CD3 Cytell 54 37 96 Flow 60 38 94
54.1% CD4 +
36.9% CD8 +
95.7% CD3 +
* M. Bofill et al. Clin. exp. Immunol. (1992) 88, 243-252
Comparison to published values*
Comparison to flow cytometer results
CD4 % CD8 % Ratio
Cytell 54.1 36.9 1.47 Ref mean 43.6 29.5 1.48 Ref Range
27-61 14-46 0.66-3.52
TM
TM
46 Cytometry Webinar
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Considerations for routine assays Ease of Use, Accessibility, Speed
Level of expertise & location in lab Expert users only or open to all? Central facility or on any bench-top? Compact “plug and play” systems & ready-to-go applications increasingly available.
Pre-developed vs. user-defined protocols Pre-developed protocols can save time and expertise; freedom to develop protocols gives flexibility for more applications
Flexibility for dyes Open vs. closed system? Number of excitation and emission channels? Matching to common dyes?
Speed Consider time per sample from loading through to data reporting, not just acquisition time. Can multiple samples be run in parallel? Is additional time needed for set-up, calibration, cleaning regimes, etc.?
providing additional information from adherent as well as suspension cell types and becoming increasingly easy to use and accessible
Match the image cytometry solution to your needs from high-content (depth & breadth) to ready-access (compact & affordable)
For detailed phenotyping, functional studies and screens, look for High Content Analysis systems that give you the power to extract a wide range and number of cell measurements, while offering flexibility for present & future applications.
For more routine cell monitoring and characterization, consider compact and affordable image cytometers that will bring the capability to your bench-top easily and without the need for expert training. Flexibility to use your own dyes and develop your own protocols is important.
48 Cytometry Webinar
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48 Cytometry Webinar
10/31/2012
Image Cytometry Solutions GE Healthcare
IN Cell Analyzer 2000 Flexible wide field HCA with on-board image restoration
IN Cell Analyzer 6000 Laser-based confocal HCA with sCMOS detection & variable aperture technology
Cytell Image Cytometer Compact bench-top solution for rapid cell characterization
