Principles Principles of of Flow CytometryFlow Cytometry

DrDr. Uta Rabenhorst. Uta RabenhorstGroupGroup of PD of PD DrDr. Martin . Martin ZörnigZörnig

Flow CytometryFlow Cytometry

Staining withAntibodies,dyes...

Expression ofFluorescenceMarkers...

Flow Cytometry is a high-throughput method (up to 10.000events/sec) for the measurement of multiple parameters

• Flow = Fluid• Cyto = Cell• Metry = Measurement

Flow Cytometry - Typical Applications

• Analysis:– Measurement of Cell Cycle, Apoptosis– Antibody Staining and Measurement of Marker Expression,

Phospho-Proteins...

• Cell Sorting:– Purification of sub-populations in mixed cell samples (e.g.

GFP-positive cells after transduction, stem cells...)

• Advantages of Flow Cytometry:– Single cell detection– Simultaneous measurement of multiple parameters

≠

FACS: Fluorescence-Activated Cell Sorting

FSC Lin

Sca1 CD150

SS

C

Mac

1

c-K

it

CD

48

⇒⇒

⇐

Which Which Parameters of Parameters of the Particles can bethe Particles can bemeasured by Flow Cytometrymeasured by Flow Cytometry??

their relative size (Forward Scatter - FSC) their relative granulocity/internal complexity

(Side Scatter - SSC) their specific fluorescence (FL1, FL2, FL3, FL4…)

and the respective relative fluorescence intensities

chromosomes blood cells protozoans

Forward and Forward and Sideward ScatterSideward Scatter

Forward Scatter light (FSC) -Diffraction proportional to cell surface (cell size) measured along the axis of the incoming light

Side Scatter light (SSC) - Refraction and Reflection proportional to cell complexity or cell granulocity measured perpendicular to the axis of the incoming light

Side Scatter light (488 nm):Cell complexity/granulocity

Forward Scatter light (488 nm):Cell size

Light source (488 nm)

An An example for example for FSC/SSC:FSC/SSC:Blood CellsBlood Cells

debris

neutrophilesgranulocytes

lymphocytes

Forward Scatter Light (FSC)

0 200 400 600 800 1000

020

040

060

080

010

00

monocytes

Side

Sca

tter

Lig

ht (S

SC)

How is fluorescent lightHow is fluorescent lightgenerated & measured?generated & measured?

The fluorochrome absorbs a specific amount of laserlight energy

The fluorochrome releases the absorbed energy: The difference in wavelength between the absorbed and the

emitted light is called Stoke´s shift

Fluorescent signal is quantified by different detectorspositioned 90° to the axis of incoming laser light

λ = 488 nm(blue)

energy of emittedfluorescence light

Antibody

energy ofincoming (laser)

light fluoresceinmolecule

λ ≅ 519 nm(green)

HO

CO2H

O

C

Absorption and Emission Absorption and Emission Spectra Spectra ofoftypical Fluorochromestypical Fluorochromes

wave length(nm)

R-PE APC

FITCPerCP

PI

400 450 500 550 600 650 700

1000

800

600

400

200

0

Red laser 635 nmBlue laser 488 nm

700400 450 500 550 600 650

1000

800

600

400

200

0

R-PE

APC

FITC PerCPPI

Fluorescence IntensityFluorescence Intensity

The amount of emitted fluorescent light is proportionalto the number of bound fluorochrome molecules

relative intensity (amount) of fluorescence

num

ber

of e

vent

s (=

cou

nts)

FITC

FITC

FITC

100 101 102 103 104FITC

FITC

FITCFITC

FITC

FITC

FITC

A Flow A Flow Cytometer Cytometer consists of three componentsconsists of three components

Liquid system Transport and hydrodynamic focusing of cells on the

point of measurement

Optical system Excitement Detection

Electronic system Conversion of optical into electrical (digital) signals for

subsequent computer analysis

Liquid system of the BD Liquid system of the BD FACSCaliburFACSCalibur™™

sample

Air pump

Pressureregulator

Filter forsheath fluid

Flow cell

wastecontainer

Sheath fluidcontainer

Air filter

Regulatorfor probepressure

Sample Flow in the Flow cell:Sample Flow in the Flow cell:Hydrodynamic FocusingHydrodynamic Focusing

Sheath liquid generates a laminarflow

Reduction of the diameter withinthe flow cell leads to anacceleration and tapering of thesheath and the sample flow(hydrodynamic focussing). Bothflows do not mix!

The distance between twoadjacent cells within the sampleflow is increasing, so that each cellpasses the laser beam individually.

Laser light is scattered andfluorescence is emitted. Thesesignals are detected by suitabledetectors.

sample(cell suspension)

Laser Beam

SheathSheath

Sample Flow in the Flow cell:Sample Flow in the Flow cell:Hydrodynamic FocusingHydrodynamic Focusing

low sampleconsumption~12 µl/min

high sampleconsumption~ 60 µl/min

laminarflow

laminarflow

low sample pressure high sample pressure

sheathflow

sheathflow

sheathflow

sheath flowsample sample

Laser

Interrogation Point

Laser

The optics IThe optics I

The excitation optics consists of: air-cooled Lasers (Light Amplification

by Stimulated Emission of Radiation) 488 nm blue Laser 633 nm red Laser 405 nm violet Laser

prisms (Beam Expander) to form and focus the laserbeam (lenses)

The optics IIThe optics II

The detection optics consists of: a converging lense which collects the emitted light

perpendicular to the axis of the incoming light a system of mirrors and filters (beam splitters) which

divert the specific wave lengths from the emitted lightto the corresponding detectors

460 500 540460 500 540 460 500 540

SP 500SP 500LP 500LP 500 BP500/80BP500/80

long pass short pass band pass

The optics: older ModelsThe optics: older Models

http://www.bdbiosciences.com/support/training/itf_launch.jsp

The optics: new ModelsThe optics: new Models

http://www.bdbiosciences.com/support/training/itf_launch.jsp

Electronic system IElectronic system I

• Converts optical signals (photons) intoelectronic signals (voltage pulses)

• Voltage pulse is analysed:– Height– Area– Width

• Electronic signals are digitalised and sent tothe computer for analysis

Electronic system IIElectronic system II

http://www.bdbiosciences.com/support/training/itf_launch.jsp

Electronic system IIIElectronic system III

Optical electronicalsignal

Electronical digital signal

Threshold

• Dot plot

• Densityplot

Different possibilities to present FACS dataDifferent possibilities to present FACS data

• Histogram

M1M2

• Contour plot

Instrument SettingsInstrument Settings

Detectors/Amplifiers Sensitivity: proportion optical : electronical signal

Threshold• only signals with an intensity greater or equal to the threshold value

will be processed an sent to the computer

Compensation

Fluorescence emission spectraFluorescence emission spectra

650 700500 600

FL3670 LP

FL1530/30

FL2585/42

rela

tive

inte

nsity

FITC PE PerCP

wave length (nm)

550

FL4661/16

APC

1. Laser 1. Laser 1. Laser2. Laser

FITC FITC fluorescence overlapfluorescence overlap

650 700500 550 600

rela

tive

inte

nsity

wave length (nm)

FL2

FL1

FL1530/30

FL2585/42

CompensationCompensation ( (11))

Compensation allows to correctfor spectral overlap.

Compensation needs to beapplied whenever more than one(adjacent) fluorescence ismeasured and analyzed.

The settings for compensationhave to be defined after the PMTvoltage settings for eachfluorescence were found and setfix (functional depen-dency ofboth).

Compensation (Compensation (22))

Compensation values depend on thefluorochromes used.

Compensation values also depend on theindividual Flow Cytometer with its specificoptical properties (because of the functionaldependency on the PMT voltage settings).

Compensation is applied during acquisition andis not possible afterwards during softwareanalysis (not true for newer software [Divasoftware]).

General Rule for FACS Experiments

• Always prepare appropriate controls!!!– e.g. unstained cells, single stains, for complicated protocols

Fluorescence minus one (FMO) control

• Basic principles– Laminar flow, hydrodynamic focussing,

simultaneously measured parameters onsingle cell level

• Instrument components– Liquid -, Optical - and Electronic System

• Instrument settings– Detectors, Threshold, Compensation

SummarySummary