principles of flow cytometry - goethe university frankfurt€¦ · flow cytometry staining with...
TRANSCRIPT
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