Flow Cytometry Principles & practice of “Fluorescence Spectroscopy in Biological Diagnosis &...

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


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


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


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



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

Flow Cytometry Principles & practice of “Fluorescence Spectroscopy in Biological Diagnosis &...

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