Flow Cytometry

Notes:
Material is taken from the course text: 1. Howard M. Shapiro, Practical Flow Cytometry, 3rd edition (1994), Wiley-Liss, New York. 2. Slides taken from Dr. Robert Murphy 3. Materials from Melamed, et al, Flow Cytometry & Sorting, WileyLiss, 2nd edition.

Fundamentals of a Flow Cytometer

2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt

Page 2

Basics of Flow Cytometry

Fluidics
cells in suspension

flow in single-file through

an illuminated volume where they

Optics

scatter light and emit fluorescence

that is collected, filtered and

Electronics

converted to digital values

that are stored on a computer

|| FLUIDICS || Flow Systems and Hydrodynamics

Getting the cells in the right place (at the right time)! (Shapiro, pp 133-143 - 3rd edition)

Hydrodynamics and Fluid Systems

Cells are always in suspension The usual fluid for cells is saline The sheath fluid can be saline or water The sheath must be saline for sorting Samples are driven either by syringes or by pressure systems

FLUIDICS
The use of focused light (lasers) to interrogate cells delivered by a Hydrodynamically focused fluidics system.
Flow Cell
Sheath fluid

Fluorescence signals

Focused laser beam

FLUIDICS
The introduction of a small volume into a large volume in such a way that it becomes focused along an axis is called Hydrodynamic Focusing
Requirement: Laminar Flow

Mapping between the flow lines outside and inside of a narrow tube as fluid undergoes laminar flow (from left to right).
The fluid passing through cross section A outside the tube is focused to cross section a inside.

Fluidics Systems
Positive Pressure Systems (or DPS):

Use air (or other gas) to pressurize sample and sheath containers
Use pressure regulators to control pressure on each container separately Based upon differential pressure between sample and sheath fluid Flow rate varies between 6-10 ms-1

Air in

Sheath fluid in

+++ +++ +++

Sample

Fluidics Systems
Sheath pressure will set the sheath volume flow rate (assuming sample flow is negligible) Difference in pressure between sample and sheath will control sample volume flow rate Control is not absolute changes in friction cause changes in sample volume flow rate

Fluidics Systems
Positive Displacement Syringe Systems A. 1-2 ms-1 flow rate
3-way valve

B. Fixed sample volume (50 l or 100 l)

Syringe

Flowcell

C. Sheath fluid pressure is calibrated to get optimal sample flow rate

D. Absolute number calculations possible E. Usually fully enclosed flow cells

100 l

Sample Sample loop

Waste

Fluidics Systems
Sheath volume flow rate is set by air/gas pressure Use syringe pump (motor connected to piston of syringe) to inject sample Sample volume flow rate can be changed by changing speed of motor
Sheath inlet Flow nozzle

Valve

Motor with variable speed

Control is absolute (under normal conditions)

Fluidics on Cells
As cells (or other particles) are hydrodynamically focused, they experience different shear stresses on different points on their surfaces (and in different locations in the stream) These cause cells to orient with their long axis (if any) along the axis of flow The shear stresses can also cause cells to deform (e.g., become more cigar-shaped)

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.

In the turbulent flow near the tube wall, the cells are deformed and disoriented in a very individual way. v>3 m/s.

Fluidics on Cells
Refractive Index Difference: Do exist between sheath fluid and core fluid. Affects excitation if cells are interrogated in flow chamber Not significant for eukaryotic cells Important for bacterial cells, particles (<5 um) and platelets

Noise: Usually in scatter channel It doesnt affect fluorescent output If noise is high, it can be checked whether it is due to RI difference or not

How?

Fluidics - Differential Pressure System

Sample line Flow chamber Valve
Sheath to sample d/d pressure gauge

Valve

Sheath line

Purge line Valve

Valve

Sample to sheath to d/d pressure gauge

Sample tube

Filter

Sheath pressure adjust

Sheath pressure gauge

Filter

Waste

Vacuum

Sheath container

Line pressure

From C. Gttlinger, B. Mechtold, and A. Radbruch

Technical Components
Fluidics
Positive Pressure Systems (DPS) EPICS C (Coulter) EPICS 5 & 7, Elite series FacStar (B-D) FacsVantage (B-D) Brucker Profile (Coulter) XL (Coulter) FacScan (B-D)
Positive Displacement (Syringe Drive) Systems Bryte HS Cytotron Absolute FACS Caliber (B-D)

Syringe systems
Bryte HS Cytometer

Syringe

3 way valve

Fluidics - Volumetric Injection System

H.B. Steen - MLM Chapt. 2

Hydrodynamic Systems
Flow Cell
Injector Tip
Sheath fluid

Fluorescence signals Focused laser beam

Hydrodynamically focused fluidics

Hydrodynamically focused fluidics

Signal

Increase Pressure: Widen Core Increase turbulence

Flow chamber
It defines the axis and dimensions of sheath and sample flow It defines the point of optimal hydrodynamic focusing It can also serve as the interrogation point (the illumination volume)

Four basic flow chamber types: 1. Jet-in-air 2. Flow-through cuvette 3. Closed cross flow 4. Open flow across surface (no more used)

Flow chamber
Jet in air nozzle
Best for sorting Piezoelectric crystal oscillator is responsible for precise sorting Inferior optical properties

Flow chamber
Sieve/Fileters are essential BD: smal filter made up of bundle of Microcapillary tubes, < 40um internal Diameter

Jet in air nozzle

Ortho: Hollow fiber filter

Guava: Nylon mesh of 40um internal diameter

Note: if both sheath and sample fluid are pressure driven, clogging occurs at filter leading to decrease in flow rate If the dye used is acridine orange/cyanine (neutral), the dye diffusion from center to sheath is affected leading to distortion in dye intensity

Flow chamber
Flow through cuvette
Excellent optical properties It can be used for sorting

Flow chamber
Closed cross flow chamber

Best optical properties It cant sort It can be used to visualize cell distortion under flow

Summary
Fluid flow may be Coaxial (common) Axial (microscope based)

What happens when the channel is blocked?

Flow chamber blockage

A human hair blocks the flow cell channel. Complete disruption of the flow results.

Hydrodynamic Systems and Sorting

Sample in
Sheath

Piezoelectric crystal oscillator

Fluorescence Sensors Laser beam

Sheath Core

Scatter Sensor

Hydrodynamic Systems and Sorting

This figure shows the principle of electrostatic cell sorting based on Sweets inkjet printer technology.

Hydrodynamic Systems and Sorting

In the previous figure, a stream of liquid intersects a laser beam (or multiple laser beams 1, 2, 3). The stream is vibrated by a piezo-electric crystal oscillator at frequencies from 10,000 to 300,000 Hz depending upon the orifice size, stream velocity, nature of the stream, and particle size Typically 3050,000 Hz is used to create droplets at the same frequency. Once a cell/particle is identified as desirable, a charge is placed on the stream that remains with the last drop (last attached drop) that leaves the stream. Using a computation method, this drop is sorted by being attracted toward a plate almost parallel with the stream and containing opposite charges in the vicinity of 5000 volts.

Lecture Summary
Critical aspects of flow systems Flow must be laminar (appropriate Reynolds #) When Re < 2300, flow is always laminar Samples can be injected or flow via differential pressure/syringe drive system There are many types of flow cells Blockages must be properly cleared to obtain high precision

|| OPTICS || Source, Filters, Detectors

Getting the cells interrogated !

Optics Principle

Signal peaks when Cell/Particle is at the center of laser beam As cell leaves the beam, intensity (measured by voltage) returns to zero Time of flight (TOF): Cell/particle journey through beam Area: The quantity usually measured that indicates fluorescent intensity

Optics Principle

Low-order Hermite-Gaussian resonator modes

TEMmn Where m and n are no. of nodes in x- and y- axes

Optics: Light Source

Need to have a light source focused on the same point where cells have been focused (the illumination volume) Two types of light sources

Lasers Arc-lamps
An optical channel is a path that light can follow from the illuminated volume to a detector

Optical elements provide separation of channels and wavelength selection

Illumination Sources
Lamps Xenon-Mercury Mercury Lasers Argon Ion (Ar) Krypton (Kr) Helium Neon (He-Ne) Helium Cadmium (He-Cd) YAG

Optics - Light Sources

Arc-lamps Provide mixture of wavelengths that must be filtered to select desired wavelengths Excitation filters are mandatory Provide milliwatts of light Inexpensive, air-cooled units Incoherent

Laser
Light Amplification by Stimulated Emission of Radiation Laser light is coherent and monochromatic this means the emitted radiation is in phase with and propagating in the same direction as the stimulating radiation ION lasers use electromagnetic energy to produce and confine the ionized gas plasma which serves as the lasing medium. Lasers can be continuous wave (CW) or pulsed (where flash lamps provide the pulse) Laser efficiency is variable - argon ion lasers are about 0.01% efficient (1 W needs 10KW power)

Spot Illumination - Lasers

Advantages are that the pathway is easier to define (you know where the light is going !!) It is usually monochromatic light so excitation filters are not needed Brighter source of light than arc lamps (higher radiance) Spot size (d) can be calculated:
d=1.27 . ( f/D)
where D is the beam diameter in mm and f is the focal distance from the lens OR

d= 4/3 . F .

(F= f/aperture diameter= focal number)

For a spherical lens with focal length 125 mm and laser of 515 nm, spot size is 55 um and it is 61 um at 458 nm.

Laser Production
pumping energy

The emitted photons reflect to and fro between the pair of reflectors and during this process they cause electrons to emit photons. When the number of photons increases above the criteria for desired laser intensity, the partial reflector allows the laser light to pass through.

Lasers
Coherent Enterprise laser - UV-visible Air cooled laser (Argon)

Argon & Krypton Lasers

Kr-Ar laser (488, 568, 647 nm lines) (Front)

Dye Lasers
Dye lasers use a source laser known as the pump laser to excite another laser known as the dye laser. The dye laser consists of a dye mixed in a solvent which exhibits desirable properties such as excitation and emission.

The lasing medium is a fluorescent dye (e.g. Rhodamine 6G) which is dissolved in an organic solvent such as ethanol or ethylene glycol Advantage: The laser can be tuned, usually by a rotatable filter or prism Disadvantage: The dye must be circulated and cooled to prevent it being bleached or overheated increasing the cost considerably

Dye Lasers
A wavelength-dispersive element (a prism or diffraction grating) is placed inside the laser cavity. The dispersive element is aligned so light at one wavelength is reflected back along the cavity axis Other wavelengths are dispersed. This ensures that the laser will oscillate only at the selected wavelength.

Helium-Neon Lasers
He-Ne - low power, no cooling needed Cheap, mostly emit red light at 633 nm Output power 0.1 W to 50 mW Lines available include green (543nm) and red (633 nm, 594nm or 611 nm).

Helium-Neon Lasers

Helium-Cadmium Lasers
He-Cd laser 5-200mW power usually at 325 nm (UV) or 441 nm (blue) Air cooled Uses cadmium vapor as the lasing medium Major problem is noise (plasma noise between 300-400 kHz) Good for ratio measurements (calcium) because power fluctuations dont matter here since these lasers do have power fluctuation problems.
He-Cd laser

Diode Lasers
Small, efficient, cheap Only red wavelengths available at reasonable prices (blue works, but still problems) Mostly made of Gallium Aluminum Arsenide (GaAlAs) Emission ratio is varied by changing the ratio of gallium to aluminum in the semiconductor
Disadvantage: Lack of fluorescent probes to be excited at 650-900 nm Poor beam profiles for diode lasers Advantage: Noise levels are generally 0.05% or less compared to 1% for air cooled argon and 0.02% with water cooled argon lasers

Solid State Lasers

Neodynymium-YAG (Yttrium aluminum garnet) lasers Lasing medium is a solid rod of crystalline material pumped by a flash lamp or a diode laser 100s mWs at 1064 nm By adjusting energy, one can produce 532 nm or 355 nm Disadvantage Noisy Reasonably expensive

Solid State Lasers

Basics of Laser Production

Common Lasers
Lasing Medium He-Ne Ne gas Cooling Power Wavelength No 0.1 W-50 mW 543 633, 594, 511

Common Lasers
Lasing Medium Ar-Kr Ar and/or Kr Cooling Power Wavelength (nm) 488 514.5 353 (UV) Air cooling 8-200 mW

Lasers Hazards
Laser light is very dangerous and should be treated as a significant hazard Water cooled lasers have additional hazards in that they require high current and voltage in addition to the water hazard Dye lasers use dyes that can be potentially carcinogenic

Elite Cytometer with 4 Lasers

353 nm 325 nm

488 nm 633 nm

UV\Beam Splitter 395 longPass

He-Cd Laser 325/441 Argon Laser 353/488 nm

(High speed sorting)

633 Beam Splitter

He-Ne Laser 633 nm

Mirror

Argon Laser 488 nm

Optical bench

Height Translators

Goals of Light Collection

Maximum signal, minimum noise Maximum area of collection Inexpensive system if possible Easy alignment Reduced heat generation Reduced power requirement

Obscuration bars & field stops

Obscuration bar is placed along the path of the illuminating beam before the collection of forward scatter Highly necessary even if the spot size is 20um (stream dia < 20um). It blocks the direct light but allows some of the fluorescence signal (which is going in all directions)/scattering light In a capillary or cuvett system, a field stop which is placed in the image plane achieves the same result

Optical translators
No cytometer should be without one!!!

The laser beam remains parallel, but horizontally translated.

This reduces the difficulty in aligning the laser.

The point of a good optical system is to obtain a good Signal Vs Noise

Good optical filters Remove as much excitation signal as possible Collect as much fluorescence as possible (use concave spherical mirrors)

Spectral Selection
Monochromators Vs Filters Filters are reasonably inexpensive

Lecture Summary
Excitation light sources and their properties Each light source has unique utility Optical components together with light source creates an optical system The general nature of optical systems in typical cytometers

Optics: Scatter
When a laser light source is used, the amount of light scattered in the forward direction (along the same axis that the laser light is traveling) is detected in the forward scatter channel The intensity of forward scatter is proportional to the size, shape and optical homogeneity of cells (or other particles)

Optics: Scatter
When a laser light source is used, the amount of light scattered to the side (perpendicular to the axis that the laser light is traveling) is detected in the side or 90o scatter channel The intensity of side scatter is proportional to the size, shape and optical homogeneity of cells (or other particles)

Optics: Scatter
Forward scatter tends to be more sensitive to surface properties of particles (e.g., cell ruffling) than side scatter It can be used to distinguish live from dead cells Side scatter tends to be more sensitive to inclusions within cells than forward scatter

It can be used to distinguish granulated cells from non- granulated cells

Optics: Fluorescence

The fluorescence emitted by each fluorochrome is usually detected in a unique fluorescence channel
The specificity of detection is controlled by the wavelength selectivity of optical filters and mirrors

Fluorescence
Photon emission as an electron returns from an excited state to ground state

Fluorescence
Jablonski Diagram
Singlet States Triplet States
Vibrational energy levels Rotational energy levels Electronic energy levels

S2

ENERGY

S1
ABS FL I.C.

Three nonradiative deactivation processes Internal conversion (IC), T2 Intersystem crossing (ISC) and Vibrational relaxation T1

IsC

PH IsC

S0

[Vibrational sublevels]

ABS - Absorbance S 0.1.2 - Singlet Electronic Energy Levels FL - Fluorescence T 1,2 - Corresponding Triplet States I.C.- Nonradiative Internal Conversion IsC - Intersystem Crossing PH - Phosphorescence

3rd Ed. Shapiro p 87

Fluorescence
Chromophores are components of molecules which absorb light They are generally aromatic rings

Extinction coefficient of chromophore decides the absorbtion Excitation Spectrum Intensity of emission as a function of exciting wavelength

Fluorescence
The wavelength of absorption is related to the size of the chromophores Smaller chromophores absorbs light of shorter wavelength (higher energy) Larger chromophores absorb light of longer the wavelength (lower energy)

The energy content absorption is related to the wavelength The shorter the wavelength the higher the energy (Smaller chromophores) The longer the wavelength the lower the energy (Larger chromophores)
e.g. UV light from sun - this causes the sunburn, not the red visible light

Fluorescence
Stokes Shift is the energy difference between the highest energy peak of absorbance and the highest energy of emission

Fluorescnece Intensity

Fluorescein molecule

Stokes Shift is 25 nm 495 nm 520 nm

2002 J.Paul Robinson, Purdue University

Wavelength

Properties of Fluorescent Molecules

Large extinction coefficient at the region of excitation High quantum yield Optimal excitation wavelength Photostability Excited-state lifetime Minimal perturbation by probe

Allophycocyanin (APC)
Protein
300 nm 400 nm 500 nm 600 nm
632.5 nm (HeNe)

700 nm

Excitation

Emisson

350
300 nm

457 488 514

400 nm 500 nm

610 632
600 nm 700 nm

Common Laser Lines

PE-TR Conj.
conjugate of Texas Red with R-phycoerythrin

Texas Red PI

Ethidium PE FITC

cis-Parinaric acid

Conclusions
Dye molecules must be close to but below saturation levels for optimum emission Fluorescence emission is longer than the exciting wavelength The energy of the light increases with reduction of wavelength

Optics: Filters
Classification as per frequency filtration: Long pass filters transmit wavelengths above a cut-on Wavelength Short pass filters transmit wavelengths below a cut-off Wavelength Band pass filters transmit wavelengths in a narrow range around a specified wavelength - (Band width can be specified) Neutral Density filter is a non-discriminant intensity reducing filter

Standard Long Pass Filters

520 nm Long Pass Filter Light Source Transmitted Light

>520 nm Light

Standard Short Pass Filters

Light Source
575 nm Short Pass Filter Transmitted Light

<575 nm Light

Standard Band Pass Filters

63010 nm Band-Pass Filter

White Light Source

Transmitted Light

620 -640 nm Light

Neutral density filters (N.D)

Attenuation of the light without discrimination of the wavelength

The N.D filters could be reflective or absorptive type.

They are partially silvered mirrors. They can be used as splitters

Beam Splitters
Absorptive N.D filters can not be used here; simply because of the heat, they will melt. Common cover slips can be used as beam splitters if small portion of the light is wanted, up to 5%
A half-silvered mirror. This is a plate of glass with a thin coating of aluminum (usually deposited from aluminum vapor) The thickness of the aluminum coating such that part, typically half, of light incident at a 45 degree angle is transmitted, and the remainder reflected. Instead of a metallic coating, a dielectric optical coating may be used.

Optical filters
Classification as per function:

Interference filters: Dichroic, Dielectric, Reflective filters.reflect the unwanted Wavelengths

An optical filter consisting of multiple layers of evaporated coatings on a substrate, whose spectral properties are the result of wavelength interference rather than absorption.

Absorptive filters: Colour glass filters..absorb the unwanted wavelengths

Construction of Interference Filters

Filter components
glue

Single Optical filter

Construction of Interference Filters

Optics - Filter Properties

When a filter is placed at a 45o angle to a light source, light which would have been transmitted by that filter is still transmitted but light that would have been blocked is reflected (at a 90o angle) Used this way, a filter is called a dichroic filter or dichroic mirror

Dichroics
They used to direct light in different spectral region to different detectors. They are interference filters , long pass or short pass. "dichroic" Di- is Greek for two, and -chroic is Greek for color - from Greek dikhroos, bicolored

Optical Filters
Dichroic Filter/Mirror at 450

Light Source

Transmitted Light

Reflected light

Transmission determination
Constructive and destructive interference occurs between reflections from various layers Transmission determined by : thickness of the dielectric layers number of these layers angle of incidence light on the filters

Expression of effective transmission

Bandpass filters characterized by their Tmax and FWHM (Full Width at Half Maximum) Notch filters are band pass filters in the upside down position Long pass and Short pass filters: characterized by their Tmax and cut-on, cut-off wavelength.

Measuring Filter Properties

Filters must be measured at the angle they are going to be used

Filters placed at 90o have different properties when they are placed at 45o

Short pass and long pass filters

T R A N S M I S S I O N
SP filter LP filter

T max

T max

cutoff

cuton

WAVELENGTH

For Band-pass filters

T R A N S M I S S I O N

T max

FWHM

WAVELENGTH

Optical filters evaluation

Use a population of appropriately stained particles and identify which filters give the maximum signal. Spectrofluoremeter and spectrophotometers can be used as tools for assessment of optical filters.

Optical filter evaluation

light source slit/shutter (to reduce the amount of light at collection point) optical filter (90o)

detector

Monochromator (select as per your filter)

Spectrofluorometer for Assessment of Optical Filter Transmission

Optical filter evaluation

reference PMT
beam splitter (45o) light source

slit/shutter Monochromator / grating sample PMT PMT

Monochromator / grating

Optical filter (45o)

Light loss by optics

In dichroics More on the in line arrangement PMTs In optical filters

The thicker the glass the less light transmitted.

Problems with glass - UV light will not pass In UV light system use minimum optics.

Continued.

Glass can fluoresce highest when illuminated at UV wavelength. For excitation > 450nm, you can use glass filters, < 450nm use quartz or silica filters. Plastic optical filters are unsatisfactory

Optics: Detectors
Light must be converted from photons into volts to be measured The correct detector system must be selected according to how many photons available In general, photodiodes are used for forward scatter while PMTs are used for fluorescence and side scatter

Silicon Photodiodes
A silicon photodiode produces current when photons impinge upon it (example are solar cells) Does not require an external power source to operate Peak sensitivity is about 900 nm Usually operated in the photovoltaic mode (no external voltage), (alternative is photoconductive mode with a reverse bias voltage) No gain so must have external amplifiers
Characterization:

Responsivity: Output current / Input power (A/W) Quantum efficiency : ( )% = 100 x (electrons out)/(photons in)

PMT
Current is produced at anodes when photons impinge upon their lightsensitive cathodes Require external power source PARAMETERS: Gain: Number of electrons out per photon in (it is as high as 107 ) It has logarithmic relationship with the applied voltage Noise: It can be generated from thermionic emission of electrons - this is called dark current If very low levels of signal are available, PMTs are often cooled to reduce heat effects

PMT
Spectral Response: Spectral response of PMTs is determined by the composition of the Photocathode
Material Peak sensitivity at Comments

Bi-alkali PMTs
Multi-alkali PMTs Gallium Arsenide (GaAs) cathodes

400 nm
750 nm 300-850 nm

Higher gain
Highest gain very costly and have lower gain

PMTs
Sensitivity: Output signal/Input power It is dependent upon applied voltage, cathode material, ambient temperature Keeping other parameters constant, higher the voltage, higher is the sensitivity of the PMT Keeping voltage constant cathodes can determine the sensitivity

High Voltage on PMTs

The voltage on the PMT is applied to the dynodes PMTs generally have a voltage range from 2 - 2000 volts A low signal will require higher voltages on the PMT to measure the signal When high voltage is applied, the PMT is very sensitive and if exposed to light will be destroyed Background noise on PMTs is termed dark noise that increases with voltage

Conclusion about PMT

High voltage regulation is critical because the relationship between the high voltage and the PMT gain is non-linear (almost logarithmic) PMTs must be shielded from stray light and magnetic fields

Room light will destroy a PMT if connected to a power Supply

TYPES: There are side-window and end-window PMTs according to the convenience of use

Signal Detection - PMTs

Cathode Secondary emission Anode

Photons in

Amplified Signal Out

End Window

Dynodes

Requires Current on dynodes Is light sensitive Sensitive to specific wavelengths Shown here is an end window PMT

Types of PMTs
Side Window

Signal out High voltage in

Diode Vs PMT
Scatter detectors are frequently diode detectors
Sample stream

Back of Elite forward scatter detector showing the preamp

Front view of Elite forward scatter detector showing the beam-dump and video camera signal collector (laser beam and sample sheath are superimposed)

Avalanche Photodiodes (APDs)

Combines the best features of PMTs and photodiodes

PHOTOVOLTAIC CELL

To increase gain, we need to decrease the capacitance of diode

By decreasing surface area

By increasing depletion zone

Avalanche Photodiodes (APDs)

To increase depletion zone

Add a thick depletion layer

Apply reverse bias of high voltage (upto 2000 V) increase site for recombination

PIN PHOTODIODE AVALANCHE PHOTODIODE

Advantage: High quantum efficiency, Good gain (102-103 much less than PMTs but higher than photodiodes) Function = PIN+Amplifier

Disadvantage High cost Problem with high dark current

Avalanche Photodiodes (APDs)

Image From: http://micro.magnet.fsu.edu/primer/java/photomicrography/avalanche

Summary
Photodiodes can operate in two modes - photovoltaic and photoconductive Photodiodes are usually used for scatter Avalanche PDs like PMTs are subject to dark current Voltages and gain are not linear Photodiodes are equivalent or more sensitive than PMTs but because of their low gain, they are not as useful for low level signals

Flow Cytometry Software? What for?

Display flow cytometry data (1D, 2D, and 3D displays)

Identification of cells of interest Define a cluster Region Mixed populations and noise Gating

Characterization of cells of interest Intrinsic parameters (mean/median scatter and fluorescence intensities; positive/negative
cells)

Cell counts (abundance)

Kinetics (evolution of a cell parameter Cell cycle analysis

with time)

Classical Data Analysis: Various types of data displays

Frequency distribution Dot plot Density plot

Contour plot

Frequency distribution

Histograms display the distributions of the Events for one parameter.

Simplicity of the plot No correlation with the other parameters Problem for cluster identification

Histogram overlay

Superimpose the data from several data files

Dot plot
Displays correlated data from any two parameters. Each dot corresponds to a particle (event) analyzed by the flow cytometer. Several events can occupy the same dot if they have the same parameter intensities.

No indication of the relative density of the events Problem with large data files

Density and Contour plot

Density plot:

Displays two parameters as a frequency distribution. Color is used to code the different frequencies of events.

Contour plot: Displays correlated data from any two parameters, with contour lines joining points of equal elevation (frequency distribution). Simulation of a 3D display with a " third " parameter being the number of events. Can clarify clusters

3D Displays

2 parameters versus density

3 parameters displayed together

Danger!!!
With Density plots and Contour plots some options like -Resolution -Smoothing can emphasize or hide clusters of cells.

Example : Changing Resolution

256x256

128x128

64x64

Particle (cell) Discrimination

Problem : Very often, samples are heterogeneous there are events which are not of interest (other cells, debris, electronic noise).

Several clusters of interest mixed together

Solution : Discriminate the cells of interest. Need to exclude the unwanted events from the analysis.

What is a Region?
A region can be defined as set of points carefully selected by the user that determine an area on a graph. Several regions can be defined on the same graph.

Isolate the cluster(s) of interest Better discrimination of the cluster(s) using color

Different styles of regions

E.coli

Quadrants
Membrane integrity

Green fluorescence SYBRGreen )

Rectangle Polygon Ellipse

Damaged membranes

Compromised membranes

Propidium iodide Red fluorescence

Cluster discrimination

Positive/Negative cell identification

What is a Gate?
A gate can be defined as one or more regions combined using Boolean (logic) operators (AND, NOT, OR)

Defines a subset of the data to be displayed.

Used to compute statistics

and characterize the subset of events selected