flow cytometry principles & practice of “fluorescence spectroscopy in biological diagnosis...
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Flow Cytometry Flow Cytometry Flow Cytometry Flow Cytometry
Principles & practicePrinciples & practice of of “Fluorescence“Fluorescence Spectroscopy in Spectroscopy in
Biological Diagnosis & Biological Diagnosis & Research”Research”
Dr.HekmatimoghaddamDr.Hekmatimoghaddam
Assistant professor of pathologyAssistant professor of pathology
DefinitionsDefinitionsDefinitionsDefinitions
Flow CytometryFlow Cytometry– Measuring properties of cells in flowMeasuring properties of cells in flow
Flow SortingFlow Sorting– Sorting (separating) cells based on Sorting (separating) cells based on
properties measured in flowproperties measured in flow– Also called Also called Fluorescence-Activated Fluorescence-Activated
Cell Sorting (FACS)Cell Sorting (FACS)
Basics of Flow CytometryBasics of Flow CytometryBasics of Flow CytometryBasics of Flow Cytometry
•Cells in suspension
•flow in single-file through
•an illuminated volume where they
•scatter light and emit fluorescence
•that is collected, filtered and
•converted to digital values
•that are stored on a computer
FluidicsFluidics
OpticsOptics
ElectronicsElectronics
FluidicsFluidicsFluidicsFluidics
Need to have cells Need to have cells in suspension in suspension flow flow in single file through an illuminated in single file through an illuminated volumevolume
In most instruments, accomplished In most instruments, accomplished by injecting sample into a by injecting sample into a sheath sheath fluid fluid as it passes through a small as it passes through a small (50-300 µm) (50-300 µm) orificeorifice
Flow Cell
Injector Tip
FluorescenceFluorescencesignalssignals
Focused laserFocused laserbeambeam
Sheath fluid
FluidicsFluidicsFluidicsFluidics
When conditions are right, sample When conditions are right, sample fluid flows in a central core that does fluid flows in a central core that does not mix with the sheath fluidnot mix with the sheath fluid
This is termed This is termed Laminar flowLaminar flow
FluidicsFluidicsFluidicsFluidics
The introduction of a large volume The introduction of a large volume into a small volume in such a way into a small volume in such a way that it becomes “focused” along an that it becomes “focused” along an axis is called axis is called Hydrodynamic Hydrodynamic FocusingFocusing
Fluidics - Differential Fluidics - Differential Pressure SystemPressure System
Fluidics - Differential Fluidics - Differential Pressure SystemPressure System
Use air (or other gas) to pressurize Use air (or other gas) to pressurize sample and sheath containerssample and sheath containers
Use pressure regulators to control Use pressure regulators to control pressure on each container pressure on each container separatelyseparately
Fluidics - Differential Fluidics - Differential Pressure SystemPressure System
Fluidics - Differential Fluidics - Differential Pressure SystemPressure System
Sheath pressure will set the Sheath pressure will set the sheath sheath volume flow rate volume flow rate (assuming sample (assuming sample flow is negligible)flow is negligible)
Difference in pressure between Difference in pressure between sample and sheath will control sample and sheath will control sample sample volumevolume flow rate flow rate
Control is not absolute - changes in Control is not absolute - changes in friction cause changes in sample friction cause changes in sample volume flow ratevolume flow rate
Fluidics - Differential Pressure Fluidics - Differential Pressure SystemSystem
Fluidics - Differential Pressure Fluidics - Differential Pressure SystemSystem
C. Göttlinger, B. Mechtold, and A. Radbruch
Fluidics - Particle Fluidics - Particle Orientation and Orientation and
DeformationDeformation
Fluidics - Particle Fluidics - Particle Orientation and Orientation and
DeformationDeformation“a: Native human erythrocytes near the margin of the core stream of a short tube (orifice). The cells are uniformly oriented and elongated by the hydrodynamic forces of the inlet flow.
b: In the turbulent flow near the tube wall, the cells are deformed and disoriented in a very individual way. v>3 m/s.”
V. Kachel, et al. - MLM Chapt. 3
Fluidics - Flow ChambersFluidics - Flow ChambersFluidics - Flow ChambersFluidics - Flow Chambers
H.B. Steen - MLM Chapt. 2
Flow through cuvette (sense in quartz)
Flow Cell
Injector Tip
FluorescenceFluorescencesignalssignals
Focused laserFocused laserbeambeam
Sheath fluid
OpticsOpticsOpticsOptics
Need to have a light source focused Need to have a light source focused on the same point where cells have on the same point where cells have been focused (the illumination been focused (the illumination volume)volume)
Two types of light sourcesTwo types of light sources– LasersLasers– Arc-lampsArc-lamps
Optics - Light SourcesOptics - Light SourcesOptics - Light SourcesOptics - Light Sources
LasersLasers– can provide a single wavelength of light can provide a single wavelength of light
(a (a laser linelaser line) or (more rarely) a mixture ) or (more rarely) a mixture of wavelengthsof wavelengths
– can provide from milliwatts to watts of can provide from milliwatts to watts of lightlight
– can be inexpensive, air-cooled units or can be inexpensive, air-cooled units or expensive, water-cooled unitsexpensive, water-cooled units
– provide provide coherent coherent lightlight
Optics - Light SourcesOptics - Light SourcesOptics - Light SourcesOptics - Light Sources
Arc-lampsArc-lamps– provide provide mixture mixture of wavelengths that of wavelengths that
must be must be filteredfiltered to select desired to select desired wavelengthswavelengths
– provide milliwatts of lightprovide milliwatts of light– inexpensive, air-cooled unitsinexpensive, air-cooled units– provide provide incoherentincoherent light light
Optics - Forward Scatter Optics - Forward Scatter ChannelChannel
Optics - Forward Scatter Optics - Forward Scatter ChannelChannel
When a laser light source is used, the When a laser light source is used, the amount of light amount of light scatteredscattered in the in the forwardforward direction (along the same axis that the direction (along the same axis that the laser light is traveling) is detected in laser light is traveling) is detected in the the forward scatter channelforward scatter channel
The intensity of forward scatter is The intensity of forward scatter is proportional to the proportional to the sizesize, , shapeshape and and optical homogeneity optical homogeneity of cells (or other of cells (or other particles)particles)
Forward Angle Light Scatter
FALS Sensor
Laser
Optics - Side Scatter Optics - Side Scatter ChannelChannel
Optics - Side Scatter Optics - Side Scatter ChannelChannel
When a laser light source is used, the When a laser light source is used, the amount of light amount of light scatteredscattered to the to the sideside (perpendicular to the axis that the (perpendicular to the axis that the laser light is traveling) is detected in laser light is traveling) is detected in the the side or 90side or 90oo scatter channel scatter channel
The intensity of side scatter is The intensity of side scatter is proportional to the proportional to the internal structure internal structure and granularity and granularity of cells (or other of cells (or other particles)particles)
90 Degree Light Scatter
FALS Sensor
90LS Sensor
Laser
Optics - Light ScatterOptics - Light ScatterOptics - Light ScatterOptics - Light Scatter
Forward scatter tends to be more Forward scatter tends to be more sensitive to sensitive to surface properties surface properties of particles of particles (e.g., cell ruffling) than side scatter(e.g., cell ruffling) than side scatter– can be used to distinguish can be used to distinguish livelive from from deaddead
cellscells Side scatter tends to be more sensitive to Side scatter tends to be more sensitive to
inclusions within inclusions within cells than forward cells than forward scatterscatter– can be used to distinguish can be used to distinguish granulated granulated cells cells
from from non-granulated non-granulated cellscells
Laser
Fluorescence Detectors
Fre
q
Fluorescence
FALS Sensor
Fluorescence detector(PMT3, PMT4 etc.)
Optics - Filter PropertiesOptics - Filter PropertiesOptics - Filter PropertiesOptics - Filter Properties
Long pass filters Long pass filters transmit wavelengths transmit wavelengths aboveabove a a cut-oncut-on wavelength wavelength
Short pass filters Short pass filters transmit transmit wavelengths wavelengths belowbelow a a cut-offcut-off wavelength wavelength
Band pass filters Band pass filters transmit wavelengths transmit wavelengths in a narrow in a narrow rangerange around a specified around a specified wavelengthwavelength– Band width Band width can be specifiedcan be specified
Standard Long Pass Standard Long Pass FiltersFilters
Standard Long Pass Standard Long Pass FiltersFilters
Transmitted LightTransmitted LightLight SourceLight Source520 nm Long Pass Filter520 nm Long Pass Filter
>520 nm >520 nm LightLight
Transmitted LightTransmitted LightLight SourceLight Source575 nm Short Pass Filter575 nm Short Pass Filter
<575 nm <575 nm LightLight
Standard Short Pass FiltersStandard Short Pass FiltersStandard Short Pass FiltersStandard Short Pass Filters
Standard Band Pass FiltersStandard Band Pass Filters
Transmitted LightWhite Light Source
630 nm BandPass Filter
620 -640 nm Light
Optics - Filter PropertiesOptics - Filter PropertiesOptics - Filter PropertiesOptics - Filter Properties
When a filter is placed at a When a filter is placed at a 4545oo angle angle to a light source, light which would to a light source, light which would have been transmitted by that filter have been transmitted by that filter is still transmitted but light that is still transmitted but light that would have been blocked is reflected would have been blocked is reflected (at a 90(at a 90oo angle) angle)
Used this way, a filter is called a Used this way, a filter is called a dichroic filter dichroic filter or or dichroic mirrordichroic mirror
Dichroic Dichroic Filter/MirrorFilter/Mirror
Dichroic Dichroic Filter/MirrorFilter/Mirror
Filter placed at 45o
Reflected light
Transmitted LightLight Source
Optics - Filter LayoutOptics - Filter LayoutOptics - Filter LayoutOptics - Filter Layout
To To simultaneouslysimultaneously measure more measure more than one scatter or fluorescence from than one scatter or fluorescence from each cell, we typically use each cell, we typically use multiple multiple channels channels (multiple detectors)(multiple detectors)
Design of multiple channel layout Design of multiple channel layout must considermust consider– spectral propertiesspectral properties of fluorochromes of fluorochromes
being usedbeing used– proper order proper order of filters and mirrorsof filters and mirrors
EthidiumEthidium
PEPE
cis-Parinaric acidcis-Parinaric acid
Texas RedTexas Red
PE-TR Conj.PE-TR Conj.
PIPI
FITCFITC
600 nm300 nm 500 nm 700 nm400 nm457350 514 610 632488Common
Laser Lines
PMT
PMT
PMT
PMT
DichroicFilters
BandpassFilters
Example Channel Layout for Laser-based Flow Cytometry
Laser
1
2
3
4
Flow cell
Optics - DetectorsOptics - DetectorsOptics - DetectorsOptics - Detectors
Two common detector typesTwo common detector types– PhotodiodePhotodiode
used for strong signals when saturation is a used for strong signals when saturation is a potential problem (e.g., forward scatter potential problem (e.g., forward scatter detector)detector)
– Photomultiplier tube (PMT)Photomultiplier tube (PMT) more sensitive than photodiode but can be more sensitive than photodiode but can be
destroyed by exposure to too much lightdestroyed by exposure to too much light
Summary of Part 1Summary of Part 1Summary of Part 1Summary of Part 1
•Cells in suspension
•flow in single-file through
•an illuminated volume where they
•scatter light and emit fluorescence
•that is collected, filtered and
•converted to digital values
•that are stored on a computer
FluidicsFluidics
OpticsOptics
ElectronicsElectronics
Typical Research Cytometer Typical Research Cytometer (Coulter 753) (1980s)(Coulter 753) (1980s)
Lasers
Fluidics
Computers
Detectors
Laser Power Supply
$200-300,000
Typical Clinical Cytometer
$90-120,000
Computer SystemDetector &Mechanical Fluidics
Clinical Applications OfClinical Applications OfFlow Cytometric AnalysisFlow Cytometric Analysis
FlowCytometric(immunophenotypic) FlowCytometric(immunophenotypic) Classification Of LeukemiasClassification Of Leukemias
ImmunophenotypingImmunophenotyping
CD2CD2CD4CD4
Log FITC
1000 100 10 1
11
0
.1
Cou
nt
3
0 7
00
1
50
ImmunophenotypingImmunophenotyping
Log FITC Fluorescence (CD8).1 1 10 100 1000
Log
PE
Flu
ores
cenc
e (C
D4)
.1 1
10
100
1 2
3 4
45% 2%
26%
CD4/CD8 Quadstats
27%
The Cell CycleThe Cell Cycle
G1
MG2
S G0
Quiescent cells
GG22
MM GG00
GG11
ss
0 200 400 600 800 1000
GG00GG11
ss GG22 MM
DNA AnalysisDNA Analysis
DNA content
Count
2N2N 4N4N
Normal Cell Cycle Normal Cell Cycle
0 200 400 600 800 1000
PI Fluorescence
Coun
ts0
7
5 1
50
22
5 3
00
DNA AnalysisDNA Analysis
Aneuploid peakAneuploid peak
DNA index 1.21DNA index 1.21
log Thiazole Orange.1 1000 100 10 1
Count
0
15
0
11
2
75
3
7
RMI = 0RMI = 0
log Thiazole Orange.1 1000 100 10 1
Count
0
15
0
11
2
75
3
7
RR11RR22RR33RR44
RMI = 34RMI = 34
Reticulocyte Analysis
RR11RR22RR33RR44
90 Degree Scatter0 200 400 600 800 1000
Forw
ard
Sca
tter
0 2
00 4
00 6
00 8
0010
00
8
15
20
30
40
50
100
200
1000
Lymphocytes
Monocytes
Neutrophils
Side Scatter Projection
Fo
rwa
rd S
catte
r P
roje
ctio
n
Light Scatter Gating
Scale