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Transcript of flow cytometry presentation
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By:Saberzadeh
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INTRODUCTIONTechnique for counting and
examining microscopic particles.
Particles are suspended in fluid.
Very powerful tool for analyzing multiple parameters of cells in a heterogeneous population
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FACSCalibur
1-2 Lasers, 3-4 Colors
FACSCanto II
2-3 Lasers, 6-8 Colors Up to 8 Colors
LSR II
Up to 7 Lasers, 18 Colors
LSRFortessa
Up to 5 Lasers, 18 Colors
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Generaly:
Each cell is subjected to a laser beam.
The light reflected from each cell is captured.
Information is then interpreted statistically by a software
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HISTORY
Mark Fulwyler was the inventor of the forerunner of the modern flow cytometer.
This was developed in 1965
First fluorescence based flow cytometer was developed in 1986by Wolfgang Gohde.
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HISTORYThe flow cytometer was originally called
pulse cytophotometry.
In 1988, the name was officially changed to “flow cytometry” at the Conference of the American Engineering Foundation in Pensacola, Florida.
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THE INSTRUMENT
Modern flow cytometers can analyze several thousand particles every second.
It can also actively separate and isolate particles having specified property.
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COMPONENTSLiquid stream system: carries and aligns cells so that they pass
through a single file.
Measuring system: measure the impedence.
optical system: lamps, high power and low power lasers
Detector and analogue system
computer
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PRINCIPLEBeam of light of single wavelength is directed onto
hydrodynamically focused stream of liquid
Detectors are placed where the stream passed through the
light beam.
The light scattered by the cells are detected.
The amount of light scattered is measured.
This is analysed and results are interpreted.
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HYDRODYNAMIC FOCUSINGCells to be analyzed are suspended in liquid
and forced through a small aperture.A tube is used through which the sheath fluid
is pumped.Cells along with the fluid are forced through
this narrow aperture.Cells move in a single file or line.Laser hits each cell and data from each cell
can be read.
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1. Cells to be analyzed are suspended in liquid and forced through a small aperture.
2. A tube is used through which the sheath fluid is pumped.
3. Cells along with the fluid are forced through this narrow aperture.
4. Cells move in a single file or line.
5. Laser hits each cell and data from each cell can be read.
HYDRODYNAMIC FOCUSING
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SCATTERINGCells pass through the laser.
Light gets refracted or scattered in all angles.
2 types of scatter are analyzed:
Forward scatter: scatter in the forward direction
Side scatter: light scattered in very large angles.
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Principles of Flow Cytometer
Immunobiology Janeway et. al.
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FORWARD SCATTERForward scatter is the light that is scattered by the cell in
the forward direction.
Magnitude is proportional to the size of the cell.
The detector converts intensity of light into voltage or an electric pulse.
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FLOURESCENCE IN FLOW CYTOMETRY
Most common method of studying cellular characteristics.
Antibodies are tagged with a flourophore.
The antibody binds to cells.
When laser hits the flourophore, the molecule gets excited.
Signal is emitted which can be detected.
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Energy State of Fluorescence during Excitation and
Emission
http://probes.invitrogen.com/resources/education/tutorials/4Intro_Flow/player.html
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Commonly Used Fluorochromes for FACS Analysis
Fluorochromes Emission Max (nm)
Fluorescein isothiocyanate (FITC)
519 (green)
488
R-phycoerythrin (R-PE) 578 (orange-yellow) 488Propidium iodide (PI) 620 (red) 488
PerCP 678 (red) 488APC 660 (red) 633
Laser (nm)
APC-Cy7 785 (infra red) 633PerCP-Cy5.5 695 (far red) 488
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DETECTION OF FLOURESCENCEFluorescent t light is travels the same path as
side scatter.The light is directed through a series of
filters and mirrors.Particular wavelength of light is detected by
the appropriate detector.
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TWO COLOUR EXPERIMENTCells are labeled with two different fluorophores.The fluorophores must have compatible spectra.
Eg: AlexaFluor 488 ans Phycoerithrin (PE) have compatible spectra.
Both have an excitation peak at 480- 520 nm.The excitation peaks are far away so that discrete emission can be collected
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FLUORESCENCE ACTIVATED CELL SORTER(FACS)
It is a specialized form of cell sorting.
Provides a method for sorting heterogeneous mixture of biological material like cells based on light scattering and fluorescent characteristics.
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MEASURABLE PARAMETERSVolume and morphological complexity of cellsCell pigments such as chlorophyllCell cycle analysis, cell kineticsChromosomal analysisProtein modificationAntigensEnzymatic activityMembrane fluidity
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Terminology of Flow Cytometer
Operation• Laser excitation• PMT• Optical filtersData analysis• Electronic compensation
Curr. Protocol. Immunol. 2002 Chap. 5
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Laser Excitation
•488nm Blue Laser–Basic laser which is equipped in almost all Flow Cytometers–Generates FSC/SSC–Fluorochromes excited:
•FITC, Alexa488, GFP, CFSE•PE and PE tandems•PI•PerCP and PerCP tandems, 7-AAD
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Optical Filters Light Transmittance through Longpass, Shortpass
(Cutoff) and Bandpass (Center point) filters
www.bdbiosciences.com
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Emissions from fluorescent dyes bound to individual cells are detected by photomultiplier tubes (PMTs) which convert and multiply light signals (analog) as much as 100 million times into electronic signals (digital(
What is a Photomultiplier Tube (PMT)?
http://micro.magnet.fsu.edu/primer/digitalimaging/concepts/photomultipliers.html
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FSCLaser
FACS Instruments Generate Three Types of Data
Forward scatter (FSC) Approximate cell size
Side scatter (SSC) Cell complexity or granularity
Fluorescence To investigate cell structure and function
SSC
SSC
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Excitation and Emission
Use the maximum excitation wavelengths to determine lasers that can be used to excite the fluorochrome
Use the maximum emission wavelengths to determine filters and PMTs that can be used to measure the signal
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In order to properly analyze multicolor
flow cytometry experiments it is
necessary to employ a mechanism
called color compensation.
Specialized circuitry in the flow
cytometer is used to subtract a portion
of one detector's signal from another,
leaving only the desired signal. In the
above example, region A represents
unwanted FITC fluorescence appearing
in the FL2 detector.
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THANK YOU