Transcript Introduction to Flow Cytometry

Introduction to Flow Cytometry
IGC Workshop
General Programme
Time
Tutorial
Room
Speaker
9h30-10h45
Fundamentals of Flow Cytometry
Ionians
Rui Gardner
11h00-13h00
Module 1 – Group 1
Module 2 – Group 2
Analyzer room
(Hands-on)
9h30-10h45
Multicolor Analysis
Ionians
Rui Gardner
11h00-13h00
Module 1 – Group 3
Module 2 – Group 1
Analyzer room
(Hands-on)
9h30-10h45
Applications in Flow Cytometry
Ionians
Claudia Bispo
11h00-13h00
Module 1 – Group 2
Module 2 – Group 3
Analyzer room
(Hands-on)
9h30-11h00
Cell Sorting
Ionians
Rui Gardner
11h15-13h00
FlowJo Basic Training
Giordano Bruno
Day 1
Day 2
Day 3
Day 4
Module 1 – Cell Cycle Analysis using FACScan (Cláudia Bispo)
Module 2 – Multicolor Analysis using CyAn ADP (Rui Gardner)
What is Flow Cytometry?
Introduction to Flow Cytometry
IGC Workshop
Flow Cytometry
uic
Fundamentals of Flow Cytometry
IGC – April 2, 2013
Overview
• Definitions: What is flow cytometry and what does it measure?
• Fluidics: Hydrodynamic focusing, Flow Rate...
• Optics: Light, fluorescence, emission and detection...
• Electronics: pulse, data acquisition...
• Analysis: Tips on Data handling
3
Resources
Purdue Univ. Cytometry Labs: all you need to know about Cytometry!
• http://www.cyto.purdue.edu
Books:
• Shapiro, H. “Practical Flow Cytometry”, 4ed, Online version.
http://PracticalFlowCytometry.com
Tutorials:
• http://www.invitrogen.com/site/us/en/home/support/Tutorials.html
History of Flow Cytometry:
• Shapiro, H. (2007) “Cytometry and Cytometers: Development and Growth”,
in Flow Cytometry with Plant Cells (Eds, J. Dolezel, J. Greilhuber, J. Suda),
WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Resources (Web Tools)
BD Biosciences:
http://www.bdbiosciences.com/research/multicolor/tools/index.jsp
5
Resources (Web Tools)
Life Technologies:
http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cell-Analysis/Cell-and-Tissue-Analysis-Technical-Resources.html
6
Resources (Web Tools)
eBioscience:
http://www.ebioscience.com/resources.htm
7
Resources (Web Tools)
Bioledgend:
http://www.biolegend.com/webtoolstab
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What is Flow Cytometry?
Cytometry:
Measurement of physical and chemical properties of cells.
Flow Cytometry:
Characterization of cells flowing in a stream of fluid
What is Flow Cytometry used for?
Flow cytometry can be used to count large numbers of cells or particles based
on size, internal compexity, phenotype, cellular state, cell function, DNA
content, gene expression, and to quantify these same cellular properties at a
single-cell level.
Flow Cytometry is not equivalent to FACS: Fluorescence Activated Cell Sorting
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Key Advantages of Flow Cytometry
• Population data
• Measure thousands of cells/particles per second
• Measure multiple parameters simultaneously.
• Detection of extremely rare populations
0.02%
• Cell sorting
• High purity, speed, and yield.
Flow Cytometry does not...
• Locate where a certain component is within a cell
• Measure distribution of cellular components
• Measure cell morphology
Flow Cytometer in a nutshell
Fluidics
Optics
Electronics
converted
digital
values
Cells intosuspension
an
illuminated
volume
where they
thatflow
arelight
processed
in single
file through…
scatter
and emit
fluorescence
and is
analyzed
a computer.
that
filtered,incollected
and…
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Flow Cytometers
FACSscan (BD)
FACSCalibur (BD)
CyAn ADP (BC)
LSR Fortessa (BD)
FACSCanto (BD)
EC800 (Sony-iCyt)
Sony Spectral Analyzer (Sony)
CyFlow Cube (Partec)
MacsQuant (Miltenyi Biotec)
Guava (Merck-Millipore)
Gallios (BC)
Accuri C6 (BD)
Attune (Life Technologies)
etc
MoFlo Legacy (BC)
FACSAria (BD)
MoFlo XDP (BC)
MoFlo Astrios (BC)
Influx (BD)
FACSJazz (BD)
FACS Vantage (BD)
EPICS ALTRA (BC)
Avalon (Propel Labs)
Synergy sy3200 (sony-iCyt)
SH800 (Sony)
etc
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Interrogation point
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Simplified Schematics
Flow Chamber
Sample
Injected
Laser
Sheath
Flow
Sheath
Flow
Hydrodynamic
Focusing
Sheath
Flow
Sheath
Flow
Laser
Sample
Injected
Flow Chamber in a BD FACSAria Cell Sorter
Flow Chamber in a BD LSR Fortessa
Flow Rate
CyAn ADP
Low Flow Rate
Sheath
Flow
Sample
Flow
Sheath
Flow
High Flow Rate
Sheath
Flow
Sample
Flow
Sheath
Flow
LSR Fortessa
Laser
FACScan
FACSCalibur
Laser
Less cells pass
per unit time
More cells pass
per unit time
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Flow Rate
High Flow Rate
Low Flow Rate
Sheath
Sheath
Lower Power
Laser
Laser
Lower Power
High Power
High Power
Sample
Sample
3105
3105
2328
1552
Counts
Counts
2328
1552
776
776
0
100
101
102
FL2 Log Comp
103
104
0
100
101
102
FL2 Log Comp
103
104
Samples should be run at the lowest flow rate (differential pressure) as possible.
If increasing flow rate has:
• No impact on population distribution: Use high flow rate.
• Changes population distribution: Use low flow rate and increase cellular concentration.
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Exposure time
Typical jet-in air cytometer
Cell velocity is proportional to Sheath Pressure
1000
v ~ 5-30m/s
100
vms
𝐯=
500
2∆𝐏
𝜌
50
10
5
Laser beam height
𝐯=
2∆𝐏
𝜌𝐹
1
1
~ 5-20 µm
5 10
50 100
5001000
Pressure psi
Therefore exposure time ~ 10-7-10-5 s
Typically, in sorters, sheath pressure can be reduced to increase exposure time, and therefore
sensitivity and resolution of acquisition.
Sheath pressure in Analyzers is usually fixed.
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Excitation - Lasers
442
488 nm
Most common laser wavelengths available
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Multiple Laser Systems
Spatially Separated Laser Beams
Laser Intercepts
in Flow Cell
FACSAria III (BD Biosciences)
FACSAria III (BD Biosciences)
Time delay between lasers must be determined for each instrument for a given sheath
pressure.
Changes in Sheath Pressure will impact measurements.
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Emission - Light Scatter
Light is reflected towards all directions
Low angle: Forward Scatter (FSC)
High angle: Side Scatter (SSC)
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Forward Scatter (FSC)
The magnitude of Forward Scatter is roughly
proportional to the size of the cell.
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Side Scatter (SSC)
Side Scatter is caused by granulosity and
structural complexity inside the cell.
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(2D) Scatter Plot
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Fluorescence (1)
Excited State
Absorbed
Light
High Energy
Emitted
Light
Ground State
Low Energy
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Fluorescence (2)
High Energy
2
2
1
1
3
3
Low Energy
Excitation-Emission Spectrum (2)
Excitation maxima
Emission maxima
Each Fluorochrome has a specific Excitation and Emission Spectra
Excitation Spectrum (1)
480 nm
520 nm 550 nm
590 nm
Excited
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Excitation-Emission Spectrum (1)
Excitation
Excitation max
550 nm
Emission
Excitation - Emission
Excitation at different wavelengths
decreases intensity of emission.
Fluorescence Charts
Fluorescence Spectra Viewers (Web Tools)
Spectra Viewers:
• BD: http://www.bdbiosciences.com/research/multicolor/spectrum_viewer/
• Life Technologies: http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/
•
•
•
•
Cell-Analysis/Labeling-Chemistry/Fluorescence-SpectraViewer.html
eBioscience: http://www.ebioscience.com/resources/fluorplan-spectra-viewer.htm
Bioledgend: http://www.biolegend.com/spectraanalyzer
UArizona: http://www.mcb.arizona.edu/ipc/fret/
Evrogen: http://www.evrogen.com/spectra-viewer/viewer.shtml
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Reactive and Conjugated Probes
Cascade Blue
Pacific Blue
Pacific Orange
Alexa Dyes
Lucifer yellow
NBD
R-Phycoerythrin (PE)
PE-Cy5 conjugates
PE-Cy7 conjugates
PE-Texas Red
PerCP
TruRed
PerCP-Cy5.5 conjugate
Fluorescein (FITC)
BODIPY-FL
TRITC
X-Rhodamine
XRITC
Lissamine Rhodamine B
Texas Red
Allophycocyanin (APC)
APC-Cy7 conjugates
Etc…
Immunohistochemistry
Direct method
Indirect method
First dye-coupled antibody using fluorescein isothiocyanate
(FITC) patented by Joseph Burckhalter and Robert Seiwald,
in 1960
Pictures adapted from wikipedia
Tandem Dyes
PE-Alexa Fluor 700
PE-Cy5
PE-TxRed
PerCP-Cy5.5
PE-Cy7
APC-Cy7
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DNA and Cell Function Probes
Calcium
Membrane
pH
Cell Tracker
DNA
Mitocondria
Fura 2
FM 1-43
SNAFLE
CMRA
DAPI
Nonyl Acridine O
Indo 1
FM 4-64
SNARF
CMTPX
HOECHST
MitoSox
Bapta/dye
DiO
BCECF
BMQC
TOPRO3
Mitotracker
Calcium Green
ANNINE 6
pHRodo
DMFDA
PI
Rhodamine 123
Fluo 4
Di4ANNEPS hq
lysosensor
CMTMR
DRAQ5
MitoProbe
Fluorescent Proteins (1)
GFP was first noted by Shimomura et al., in the jelly fish Aequoria
Victoria, in 1962 and cloned 30 years later
Fluorescent Proteins (2)
San Diego beach scene drawn with living
bacteria expressing 8 different colors of
fluorescent proteins
Tsien Lab
Roger Tsien, Nobel Lectures, 2009
http://www.tsienlab.ucsd.edu/HTML/Images/IMAGE - PLATE - Beach.jpg
What is Flow Cytometry?
Introduction to Flow Cytometry
IGC Workshop
Flow Cytometry
uic
Coffee Break…!