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Transcript of Introduction to Flow Cytometry IGC Workshop General Programme TimeTutorialRoomSpeaker Day 1...
Introduction to Flow Cytometry IGC Workshop
General Programme
Time Tutorial Room Speaker
Day 1
9h30-10h45 Fundamentals of Flow Cytometry Ionians Rui Gardner
11h00-13h00 Module 1 – Group 1Module 2 – Group 2 Analyzer room (Hands-on)
Day 2
9h30-10h45 Multicolor Analysis Ionians Rui Gardner
11h00-13h00 Module 1 – Group 3Module 2 – Group 1 Analyzer room (Hands-on)
Day 3
9h30-10h45 Applications in Flow Cytometry Ionians Claudia Bispo
11h00-13h00 Module 1 – Group 2Module 2 – Group 3 Analyzer room (Hands-on)
Day 4
9h30-11h00 Cell Sorting Ionians Rui Gardner
11h15-13h00 FlowJo Basic Training Giordano Bruno
Module 1 – Cell Cycle Analysis using FACScan (Cláudia Bispo)Module 2 – Multicolor Analysis using CyAn ADP (Rui Gardner)
What is Flow Cytometry?
Flow Cytometry uic
Fundamentals of Flow Cytometry
Rui GardnerIGC – April 2, 2013
Introduction to Flow Cytometry IGC Workshop
Overview
3
• 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
Resources
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Books:• Shapiro, H. “Practical Flow Cytometry”, 4ed, Online version.
http://PracticalFlowCytometry.com
Purdue Univ. Cytometry Labs: all you need to know about Cytometry!• http://www.cyto.purdue.edu
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
Tutorials:• http://www.invitrogen.com/site/us/en/home/support/Tutorials.html
Resources (Web Tools)
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BD Biosciences:http://www.bdbiosciences.com/research/multicolor/tools/index.jsp
Resources (Web Tools)
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Life Technologies:http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cell-Analysis/Cell-and-Tissue-Analysis-Technical-Resources.html
Resources (Web Tools)
7
eBioscience:http://www.ebioscience.com/resources.htm
Resources (Web Tools)
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Bioledgend:http://www.biolegend.com/webtoolstab
What is Flow Cytometry?
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Cytometry:Measurement of physical and chemical properties of cells.
Flow Cytometry:Characterization of cells flowing in a stream of fluid
Flow Cytometry is not equivalent to FACS: Fluorescence Activated Cell Sorting
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.
Key Advantages of Flow Cytometry
• Population data• Measure thousands of cells/particles per second• Measure multiple parameters simultaneously.
• Detection of extremely rare populations
• Cell sorting• High purity, speed, and yield.
0.02%
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
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Cells in suspensionflow in single file through…
Fluidics
an illuminated volume where theyscatter light and emit fluorescencethat is filtered, collected and…
Optics
converted to digital valuesthat are processed and analyzed in a computer.
Electronics
Flow Cytometers
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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
Flow Cytometers
Analyzers High Speed Cell Sorters
Fluidics
Interrogation point
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Simplified Schematics
SheathFlow
SheathFlow
SampleInjected
Laser
Flow Chamber
HydrodynamicFocusing
Flow Chamber in a BD FACSAria Cell Sorter Flow Chamber in a BD LSR Fortessa
SheathFlow
SheathFlow
SampleInjected
Laser
Flow Chamber
Flow Rate
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More cells passper unit time
SheathFlow
SheathFlowSample
Flow
Laser
High Flow Rate
Less cells passper unit time
SheathFlow
SheathFlowSample
Flow
Laser
Low Flow Rate
CyAn ADP
LSR Fortessa
FACSCalibur
FACScan
Flow Rate
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Low Flow Rate
Laser
Sheath
Sample
High Flow Rate
Laser
Sheath
Sample
100 101 102 103 104
FL2 Log Comp
0
776
1552
2328
3105
Counts
100 101 102 103 104
FL2 Log Comp
0
776
1552
2328
3105
Counts
100 101 102 103 104
FL2 Log Comp
0
776
1552
2328
3105
Counts
High Power
Lower Power
High Power
Lower Power
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.
Typical jet-in air cytometer
1 5 10 50 100 50010001
5
10
50
100
500
1000
P re s s u re p s i
vms
Exposure time
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v ~ 5-30m/s
Therefore exposure time ~ 10-7-10-5 s
𝐯=√ 2∆𝐏𝜌Cell velocity is proportional to Sheath Pressure
𝐯=√ 2∆𝐏𝜌 𝐹
Laser beam height
~ 5-20 µm
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.
Optics
Excitation - Lasers
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488 nm
Most common laser wavelengths available
442
Multiple Laser Systems
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Laser Interceptsin Flow Cell
Spatially Separated Laser Beams
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.
Emission - Light Scatter
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Light is reflected towards all directions
Low angle: Forward Scatter (FSC)High angle: Side Scatter (SSC)
Forward Scatter (FSC)
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The magnitude of Forward Scatter is roughly proportional to the size of the cell.
Side Scatter (SSC)
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Side Scatter is caused by granulosity and structural complexity inside the cell.
(2D) Scatter Plot
25
Optics(Fluorescence)
Fluorescence (1)
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Fluorophore Energy Levels
Low Energy
High Energy
Ground State
AbsorbedLight
Excited State
EmittedLight
Absortion
StokesShift
Emission
Fluorescence (2)
Fluorophore
Absortion
StokesShift
Emission
Low Energy
High Energy
1
2
3
1
2
3
Lower Wavelength
Higher Frequency
Higher Energy
Higher Wavelength
Lower Frequency
Lower Energy
Excitation-Emission Spectrum (2)
Emission maximaExcitation maxima
Each Fluorochrome has a specific Excitation and Emission Spectra
Excitation Spectrum (1)
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Excited
480 nm 520 nm 550 nm 590 nm
Excitation-Emission Spectrum (1)
Excitation max550 nm
Excitation
Emission
Excitation - Emission
Excitation at different wavelengthsdecreases 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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Optics(Fluorescent Markers)
Reactive and Conjugated Probes
Cascade Blue Pacific Blue Pacific Orange Alexa DyesLucifer yellow NBD R-Phycoerythrin (PE) PE-Cy5 conjugates PE-Cy7 conjugates PE-Texas RedPerCP TruRed PerCP-Cy5.5 conjugateFluorescein (FITC)BODIPY-FL TRITC X-Rhodamine XRITCLissamine Rhodamine B Texas Red Allophycocyanin (APC) APC-Cy7 conjugatesEtc…
Immunohistochemistry
First dye-coupled antibody using fluorescein isothiocyanate (FITC) patented by Joseph Burckhalter and Robert Seiwald, in 1960
Direct method Indirect method
Pictures adapted from wikipedia
Tandem Dyes
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PE-Alexa Fluor 700
PE-Cy5PE-TxRedPerCP-Cy5.5
PE-Cy7APC-Cy7
DNA and Cell Function Probes
Calcium Membrane pH Cell Tracker DNA MitocondriaFura 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
http://www.tsienlab.ucsd.edu/HTML/Images/IMAGE - PLATE - Beach.jpg
Roger Tsien, Nobel Lectures, 2009
Tsien Lab
What is Flow Cytometry?
Flow Cytometry uic
Introduction to Flow Cytometry IGC Workshop
Coffee Break…!