Flow Cytometry Basic TrainingA Look Inside the BoxHimani Anand Molecular & Cellular Endocrinology LabNIHFW, New DelhiOctober 2013

Section IBackground Information on Flow Cytometry

What Is Flow Cytometry?Flow ~ cells in motionCyto ~ cellMetry ~ measureMeasuring properties of cells while in a fluid stream

Cytometry vs. Flow CytometryCytometryLocalization of antigen is possiblePoor enumeration of cell subtypesLimiting number of simultaneous measurements

Flow Cytometry.Cannot tell you where antigen is.Can analyze many cells in a short time frame.Can look at numerous parameters at once.

Uses of Flow CytometryIt can be used forImmunophenotypingDNA cell cycle/tumor ploidyMembrane potentialIon fluxCell viabilityIntracellular protein stainingpH changesCell tracking and proliferationSortingRedox stateChromatin structureTotal proteinLipidsSurface chargeMembrane fusion/runoverEnzyme activityOxidative metabolismSulfhydryl groups/glutathioneDNA synthesisDNA degradationGene expressionThe use of flow in research has boomed since the mid-1980s

Background Info SummaryFlow Cytometry is a quickly expanding technologyHas continually increased in popularity since the mid 1980s.Gives us the ability to analyze many properties of many cells in very little time

Section IIThe 4 Main Components of a Flow Cytometer

What Happens in a Flow Cytometer?Cells in suspension flow single file pasta focused laser where they scatter light and emit fluorescence that is filtered and collectedthen converted to digitized values that are stored in a file Which can then be read by specialized software.

What Happens in a Flow Cytometer (Simplified)

The Fluidics SystemCells in suspension flow single fileYou need to have the cells flow one-by-one into the cytometer to do single cell analysisAccomplished through a pressurized laminar flow system.The sample is injected into a sheath fluid as it passes through a small orifice (50um-300um)

Fluidics SchematicSampleTube

How The Flow Cell WorksThe cells from the sample tube are injected into the sheath streamFlow in a flow cell is laminar.Hydrodynamic focusing pushes the cells to line up single file along their long axis.The shape of the flow cell provides the means for hydrodynamic focusing.

The Flow CellSheathThe introduction of a large volume into a small volume in such a way that it becomes focused along an axis is called Hydrodynamic Focusing.Original from Purdue University Cytometry Laboratories, Modified by James Marvin

Incoming LaserSampleSheathSheathSheathSampleSampleCoreStreamLow DifferentialHigh DifferentialLaser Focal Point

Sample DifferentialDifference in pressure between sample and sheathThis will control sample volume flow rateThe greater the differential, the wider the sample core. If differential is too large, cells will no longer line up single fileResults in wider CVs and increase in multiple cells passing through the laser at once. No more single cell analysis!

Fluidics RecapPurpose is to have cells flow one-by-one past a light source.Cells move out of tube because there is slightly greater pressure on the sample than on the sheathCells are focused due to hydrodynamic focusing and laminar flow.

What Happens in a Flow Cytometer?Cells in suspension flow single file pasta focused laser where they scatter light and emit fluorescence that is filtered, collectedand converted to digitized values that are stored in a file Which can then be read by specialized software.

InterrogationLight source needs to be focused on the same point where cells are focused.

Light sourceOn all flow lab instruments-Lasers

LasersLight amplification by stimulated emission of radiation

Lasers can provide a single wavelength of light (monochromatic)They can provide milliwatts to watts of powerAlso provide coherent lightAll help to create a stable and reliable signalCoherent: all emmiting photons have same wavelength, phase and direction as stimulation photons

Light ScatterWhen light from a laser interrogates a cell, that cell scatters light in all directions.The scattered light can travel from the interrogation point down a path to a detector.

Forward ScatterLight that is scattered in the forward direction (along the same axis the laser is traveling) is detected in the Forward Scatter Channel.The intensity of this signal has been attributed to cell size, refractive index (membrane permeability)Forward Scatter=FSC=FALS=LALS

Forward ScatterOriginal from Purdue University Cytometry Laboratories

Side ScatterLaser light that is scattered at 90 degrees to the axis of the laser path is detected in the Side Scatter ChannelThe intensity of this signal is proportional to the amount of cytosolic structure in the cell (eg. granules, cell inclusions, etc.)Side Scatter=SSC=RALS=90 degree Scatter

Side ScatterOriginal from Purdue University Cytometry Laboratories

Why Look at FSC v. SSCSince FSC ~ size and SSC ~ internal structure, a correlated measurement between them can allow for differentiation of cell types in a heterogenous cell population

Fluorescence ChannelsAs the laser interrogates the cell, fluorochromes on/in the cell (intrinsic or extrinsic) may absorb some of the light and become excitedAs those fluorochromes leave their excited state, they release energy in the form of a photon with a specific wavelength, longer than the excitation wavelengthThose photons pass through the collection lens and are split and steered down specific channels with the use of filters.

Fluorescence DetectorsOriginal from Purdue University Cytometry Laboratories, Modified by James Marvin

FiltersMany wavelengths of light will be scattered from a cell, we need a way to split the light into its specific wavelengths in order to detect them independently. This is done with filtersOptical filters are designed such that they absorb or reflect some wavelengths of light, while transmitting other.3 types of filtersLong Pass filterShort Pass filterBand Pass filter

Long Pass FiltersTransmit all wavelengths greater than specified wavelength Example: 500LP will transmit all wavelengths greater than 500nm

Original from Cytomation Training Manual, Modified by James Marvin

Short Pass FilterTransmits all wavelengths less than specified wavelengthExample: 600SP will transmit all wavelengths less than 600nm.Original from Cytomation Training Manual, Modified by James Marvin

Band Pass FilterTransmits a specific band of wavelengthsExample: 550/20BP Filter will transmit wavelengths of light between 540nm and 560nm (550/20 = 550+/-10, not 550+/-20)Original from Cytomation Training Manual, Modified by James Marvin

Dichroic FiltersCan be a long pass or short pass filterFilter is placed at a 45 angle to the incident lightPart of the light is reflected at 90 to the incident light, and part of the light is transmitted and continues on.

DetectorsThere are two main types of photo detectors used in flow cytometryPhotodiodesUsed for strong signals, when saturation is a potential problem (eg. FSC detector)Photomultiplier tubes (PMT)More sensitive than a Photodiode, a PMT is used for detecting small amounts of fluorescence emitted from fluorochromes.

Photodiodes and PMTsPhoto Detectors usually have a band pass filter in front of them to only allow a specific band width of light to reach itTherefore, each detector has a range of light it can detect, once a filter has been placed in front of it.

Interrogation RecapA focused light source (laser) interrogates a cell and scatters lightThat scattered light travels down a channel to a detectorFSC ~ size and cell membrane shapeSSC ~ internal cytosolic structureFluorochromes on/in the cell will become excited by the laser and emit photonsThese photons travel down channels and are steered and split by dichroic (LP/SP) filtersSpecific wavelengths are then detected by PMTs that have a filter in front of them

What Happens in a Flow Cytometer?Cells in suspension flow single file pasta focused laser where they scatter light and emit fluorescence that is collected, filtered and converted to digitized values that are stored in a file Which can then be read by specialized software.

ElectronicsDetectors basically collect photons of light and convert them to currentThe electronics must process that light signal and convert the current to a digitized value/# that the computer can graph

What Happens in the PMTA voltage is applied to the detector which makes electrons available for the photons to pick upAs the number of photons increase, more and more electrons are picked up yielding a greater current output from the detectorAlso, as the voltage applied to the detector increases the same amount of photons will have a greater current output

Electronics SchematicDetector

PMT Voltage Input150V-999VCurrent out Photons inLinear AmplificationLogAmplificationorVoltageTimeVoltageOriginal from Becton Dickinson Training manual, Modified by James MarvinPhotons

ThresholdWhen the laser interrogates an object, light is scattered.If the amount of light scattered surpasses a threshold, then the electronics opens a set window of time for signal detectionThe threshold can be set on any parameter, but is usually set on FSC

Threshold

Photons In ~ Voltage OutDetectorPhotons converted to no. of electronsPMT Voltage Input150V-999VCurrent out Photons inLinear AmplificationLogAmplificationorVoltageTimeVoltageOriginal from Becton Dickinson Training manual, Modified by James Marvin

The Voltage PulseAs the cell passes through the laser, more and more light is scattered until the cell is in the center of the laser (maxima)As the cell leaves the laser, less and less light is scattered After a set amount of time, the window closes until another object scatters enough light to be triggered.

The Pulse

Electronics RecapThe varying number of photons reaching the detector are converted to a proportional number of electronsThe number of electrons exiting a PMT can be multiplied by making more electrons available to the detector (increase Voltage input)The current generated goes to a log or linear amplifier where it is amplified (if desired) and is converted to a voltage pulseThe voltage pulse goes to the ADC to be digitizedThe values are placed into a List Mode File

What Happens in a Flow Cytometer?Cells in suspension flow single file pasta focused laser where they scatter light and emit fluorescence that is collected, filtered and converted to digitized values that are stored in a file Which can then be read by specialized software.

InterpretationOnce the values for each parameter are in a list mode file, specialized software can graphically represent it.The data can be displayed in 1, 2, or 3 dimensional formatCommon programs includeCellQuestFlowjoWinMDIFCS Express

Types of PlotsSingle Color HistogramFluorescence intensity (FI) versus countTwo Color Dot PlotFI of parameter 1 versus FI of Parameter 2Two Color Contour PlotFI of P1 versus FI of P2. Concentric rings form around populations. The more dense the population, the closer the rings are to each otherTwo Color Density PlotFI of P1 versus FI of P2. Areas of higher density will have a different color than other areas

PlotsContour PlotDensity PlotGreyscale DensityDot Plotwww.treestar.com

GatingIs used to isolate a subset of cells on a plotAllows the ability to look at parameters specific to only that subset Can use boolean logic to include or exclude multiple gates

Gating Example

Important Points on AnalysisWhat kind of data are you looking for?How much fluorescence?What percent are positive?How much more positive is x than y?What is the ratio between param1 and param2What kind of statistics are availableMFI (geometric or arithmetic)%-agesCVMedianAnything you can do with a list of numbers

Everythings RelativeThe relative bin numbers are just thatrelative.Saying your cells have a mean fluorescence intensity of 100 means absolutely nothing until you compare it to a negative.The fact that everything is relative allows you to compare 2, 3, or 20 samples using the same instrument settings.

What Happens in a Flow Cytometer?Cells in suspension flow single file pasta focused laser where they scatter light and emit fluorescence that is collected, filtered and converted to digitized values that are stored in a file Which can then be read by specialized software.

ReferencesNumerous References available in the Flow LabCytometryCurrent Protocols in Flow CytometryMany more reference books availablePurdue University Cytometry Laboratories website: http://www.cyto.purdue.edu/Dr. Robert Murphy, Carnegie Mellon University- Basic Theory 1 and 2 powerpoint slidesThe Scripps Research Institute Flow Cytometry Core Facility: http://facs.scripps.edu/

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