Flow cytometry is a technique used to count, examine the complexity and shape, and measure properties of cells. Cells are passed through a column where a beam passes through them, detecting forward and side scattering. Forward scattering indicates cells without granules, while side scattering indicates cells with granules. Detected light signals are converted to electronic signals and analyzed by a connected computer. A flow cytometer consists of three main systems: fluidics to transport particles through a laser beam, optics to illuminate particles and direct light signals to detectors, and electronics to convert light signals to electronic signals for computer processing and possible particle sorting. Fluorescence is also detected using fluorescent compounds and lasers. Data is stored according to flow cytometry standard format and can be displayed

1. FLOWCYTOMETRY
Flow Cytometeryasthe name suggestsisa technique forcell counting, inter-complexity,shape and
measurementof differentpropertiesof the cell.Inthistechniquethere isacolumninwhich cellsare
placedcan be more granulatedor agranulated whenthese cellsare passedthroughthe columnthe
beamis passedthroughthe cell whichisdetectedbythe detector.Addingsaline solutiontothe column
givesthe directional focusing. The cellswhichhave nogranules,passingthroughthe columnshows
forwardscattering.While the cell withgranulesshow side scattering.These detectorsare connectedby
the computer.A flowcytometerismade upof three mainsystems:
 FLUIDICS- the fluidicssystemtransportsparticles inastreamto the laserbeamforinterrogation
 OPTICS-The opticssystemconsistsof laserstoilluminate the particlesinthe sample streamand
optical filterstodirectthe resultinglightsignalstothe appropriate detectors.
 ELECTRONICS-The electronicssystemconvertsthe detectedlightsignal intoelectronicsignals
that can be processedbythe computer.Thisissystemisalsocapable of initiatingsorting
decisionstocharge and deflectparticles.
A fluorescentcompoundabsorbslightenergyover arange of wavelengthsthatis characteristicforthat
compound.Thisabsorptionof lightcausesanelectroninthe fluorescentcompoundtobe raisedtoa
higherenergylevel.The excitedelectron quicklydecaystoitsgroundstate,emittingthe excessenergy
as a photonof light.This transitionof energyiscalledfluorescence. The argonionlaseriscommonly
usedinflowcytometrybecause the 488-nmlightthat itemitsexcitesmore thanone fluoro-chrome.
Lightsignalsare generatedasparticlespassthroughthe laserbeamina fluid stream.These lightsignals
are convertedtoelectronicsignals(voltages) byphotodetectorsandthenassignedachannel number
on a data plot.There are twotypesof photodetectorsinBDflow cytometers:photodiodesand
photomultipliertubes(PMTs).The photodiodeislesssensitivetolightsignalsthanthe PMTsand thus is
usedto detectthe strongerFSCsignal.PMTs are usedto detectthe weakersignalsgeneratedbySSCand
fluorescence.A voltage pulseiscreatedwhenaparticle entersthe laserbeamandstartsto scatterlight
or fluoresce.Once the lightsignals,orphotons,strike one sideof the PMT or the photodiode,theyare
convertedintoaproportional numberof electronsthatare multiplied,creatingagreaterelectrical
current.The electrical currenttravelstothe amplifierandisconvertedtoa voltage pulse.The highest
pointof the pulse occurswhenthe particle isinthe centerof the beamandthe maximumamountof
scatter or fluorescence isachieved.Asthe particle leavesthe beam, the pulse comesbackdowntothe
baseline
Data CollectionandDisplay
Once lightsignalshave beenconvertedtoelectronicpulsesandthenconvertedtochannel numbersby
the ADC, the data mustbe storedbythe computersystem.Flow cytometricdataisstoredaccordingto a
standardformat,the flowcytometrystandard(FCS) format,developedbythe SocietyforAnalytical
Cytology.Accordingtothe FCSstandard,a data storage file includesadescriptionof the sample
acquired,the instrumentonwhichthe datawascollected,the dataset,andthe resultsof dataanalysis.
A single cell analyzedforfourparameters(FSC,SSC,FITC,andPE fluorescence)generates8bytesof
data. Whenmultipliedbythe approximately10,000 events collectedforasingle sample,anFCSdata file
typicallycontains80kB of data. Once a data file hasbeensaved,cell populationscanbe displayedin
several differentformats.A single parametersuchasFSC or FITC (FL1) can be displayedasasingle

2. parameterhistogram,where the horizontal axisrepresentsthe parameter’ssignal valueinchannel
numbersandthe vertical axisrepresentsthe numberof eventsperchannel number. Eacheventis
placedinthe channel that correspondstoitssignal value.Signalswithidentical intensitiesaccumulate in
the same channel.Brightersignalsare displayedinchannelstothe rightof the dimmersignals. A subset
of data can be definedthroughagate.A gate isa numerical orgraphical boundarythatcan be usedto
define the characteristicsof particles toinclude forfurtheranalysisthisisknownasGating.