flow cytometry presentation

of 37 /37

Embed Size (px)

Transcript of flow cytometry presentation

  • By:Saberzadeh

  • 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

  • FACSCalibur1-2 Lasers, 3-4 Colors

    *

  • 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

  • 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.

    *

  • 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.

  • THE INSTRUMENT

    Modern flow cytometers can analyze several thousand particles every second.

    It can also actively separate and isolate particles having specified property.

  • 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

  • PRINCIPLEBeam of light of single wavelength is directed onto hydrodynamically focused stream of liquidDetectors 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.

  • 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.

  • Cells 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.

    HYDRODYNAMIC FOCUSING

  • 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 directionSide scatter: light scattered in very large angles.

  • Principles of Flow CytometerImmunobiology Janeway et. al.

    *

  • 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.

  • FLOURESCENCE IN FLOW CYTOMETRYMost 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.

  • Energy State of Fluorescence during Excitation and Emissionhttp://probes.invitrogen.com/resources/education/tutorials/4Intro_Flow/player.html

    *

  • Commonly Used Fluorochromes for FACS AnalysisFluorochromesEmission Max (nm)Laser (nm)

    APC-Cy7785 (infra red) 633PerCP-Cy5.5 695 (far red) 488

    *

  • 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.

  • 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

  • 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.

  • MEASURABLE PARAMETERSVolume and morphological complexity of cellsCell pigments such as chlorophyllCell cycle analysis, cell kineticsChromosomal analysisProtein modificationAntigensEnzymatic activityMembrane fluidity

  • Terminology of Flow CytometerOperationLaser excitationPMTOptical filters

    Data analysisElectronic compensation

    Curr. Protocol. Immunol. 2002 Chap. 5

    *

  • Laser Excitation

    488nm Blue LaserBasic laser which is equipped in almost all Flow CytometersGenerates FSC/SSCFluorochromes excited:FITC, Alexa488, GFP, CFSEPE and PE tandemsPIPerCP and PerCP tandems, 7-AAD

    *

  • Optical Filters Light Transmittance through Longpass, Shortpass (Cutoff) and Bandpass (Center point) filterswww.bdbiosciences.com

    *

  • 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)?

    *

  • FACS Instruments Generate Three Types of Data

    *

  • Excitation and EmissionUse the maximum excitation wavelengths to determine lasers that can be used to excite the fluorochromeUse the maximum emission wavelengths to determine filters and PMTs that can be used to measure the signal

    *Each fluorochrome can be further characterized by excitation and emission spectra. Read slide1st bullet: point to blue line in the graph indicating blue laser is good at exciting FITC2nd bullet: point to 530/30 filter and how it close to emission peak of the FITC and thats why an appropriate choice fo the filter.We will later in this class show you a great tool to visualize these curves for all fluorphores.

  • 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.

  • THANK YOU

    *

    **

    *

    *

    *

    *

    *

    *

    *

    *Each fluorochrome can be further characterized by excitation and emission spectra. Read slide1st bullet: point to blue line in the graph indicating blue laser is good at exciting FITC2nd bullet: point to 530/30 filter and how it close to emission peak of the FITC and thats why an appropriate choice fo the filter.We will later in this class show you a great tool to visualize these curves for all fluorphores.