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

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

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 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.

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.

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

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.

Principles of Flow Cytometer

Immunobiology 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 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.

Energy State of Fluorescence during Excitation and

Emission

http://probes.invitrogen.com/resources/education/tutorials/4Intro_Flow/player.html

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

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 Cytometer

Operation• Laser excitation• PMT• Optical filtersData analysis• Electronic compensation

Curr. Protocol. Immunol. 2002 Chap. 5

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

Optical Filters Light Transmittance through Longpass, Shortpass

(Cutoff) and Bandpass (Center point) filters

www.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)?

http://micro.magnet.fsu.edu/primer/digitalimaging/concepts/photomultipliers.html

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

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

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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