Page 1 © 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT BMS 631 - LECTURE 7 Flow Cytometry: Theory J. Paul Robinson Professor of

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© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT

BMS 631 - LECTURE 7Flow Cytometry: TheoryJ. Paul RobinsonProfessor of Immunopharmacology& Biomedical EngineeringPurdue University

Hansen Hall, B050Purdue UniversityOffice: 494 0757Fax 494 0517email: [email protected]

WEB http://www.cyto.purdue.edu

Detectors & Fluidics

3rd Ed. Shapiro 127-133

Notes:1. Material is taken from the course text: Howard M.

Shapiro, Practical Flow Cytometry, 3nd edition (1994), Wiley-Liss, New York.

2. RFM =Slides taken from Dr. Robert Murphy3. MLM – Material taken from Melamed, et al, Flow

Cytometry & Sorting, Wiley-Liss, 2nd Ed.

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Detectors

• Light must be converted from photons into volts to be measured

• We must select the correct detector system according to how many photons we have available

• In general, we use photodiodes for forward scatter and absorption and PMTs for fluorescence and side scatter

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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• At 900 nm the responsivity is about 0.5

amperes/watt, at 500 nm it is 0.28 A/W• Are usually operated in the photovoltaic mode (no

external voltage) (alternative is photoconductive mode with a bias voltage)

• Have no gain so must have external amps• quantum efficiency ()% = 100 x (electrons out/(photons in)

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PMT• Produce current at their anodes when photons impinge upon

their light-sensitive cathodes• Require external powersource• Their gain is as high as 107 electrons out per photon in• Noise 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• Spectral response of PMTs is determined by the composition of

the photocathode• Bi-alkali PMTs have peak sensitivity at 400 nm• Multialkali PMTs extend to 750 nm • Gallium Arsenide (GaAs) cathodes operate from 300-850 nm

(very costly and have lower gain)

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Signal Detection - PMTs

Cathode Anode

Dynodes

Photons in

AmplifiedSignal Out

EndWindow

• Requires Current on dynodes• Is light sensitive• Sensitive to specific wavelengths• Can be end`(shown) or side window PMTs

Secondary emission

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Photomultiplier tubes (PMT’s)

The PMTs in an Elite. 3 PMTs are shown, the other 2 have been removed to show their positions. A diode detector is used for forward scatter and a PMT for side scatter.

The Bio-Rad Bryte cytometer uses PMTs for forward and wide angle light scatter as well as fluorescence

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PMTs• 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

• There are side-window and end-window PMTs• While photodiodes are efficient, they produce

too small a signal to be useful for fluorescence

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Diode Vs PMT• Scatter detectors are frequently diode

detectors

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)

Sample stream

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Types of PMTs

Side Window

High voltage in

Signal out

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High Voltage on PMTs• The voltage on the PMT is applied to the dynodes• This increases the “sensitivity” of the PMT• A low signal will require higher voltages on the

PMT to measure the signal• When the voltage is applied, the PMT is very

sensitive and if exposed to light will be destroyed• Background noise on PMTs is termed “dark

noise”• PMTs generally have a voltage range from 1-

2000 volts• Changing the gain on a PMT should be linear

over the gain range• Changing the voltage on the PMT is NOT a linear

function of response

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Avalanche Photodiodes (APD’s)

• Combines the best features of PMTs and photodiodes• High quantum efficiency, good gain• Gain is 102-103 (much less than PMTs)• Problem with high dark current

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

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CCDs

• Charge Coupled devices (CCD) usually in our video cameras (also called charged transfer devices)

• light causes accumulation of electric charge in individual elements which release the charge at regular intervals

• Useful in imaging because they can integrate over time

• Not fast enough for flow cytometry application in general

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Summary so far….• Photodiodes can operate in two modes - photovoltaic

and photoconductive• PMTs are usually used for fluorescence measurements• Photodiodes are usually used for scatter• PMTS are sensitive to different wavelengths according

to the construction of the photocathode• PMTs are subject to dark current• Voltages and gain are not linear• Photodiodes are more sensitive than PMTs but because

of their low gain, they are not as useful for low level signals (too much noise)

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Flow Systems and Hydrodynamics

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

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Basics 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

Fluidics

Optics

Electronics

[RFM]

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Flow Cytometry:

The use of focused light (lasers) to interrogate cells delivered by a

hydrodynamically focused fluidics system.

Flow CellFlow Cell

FluorescenceFluorescencesignalssignals

Focused laserFocused laserbeambeam

Sheath fluid

Fluidics - Differential Pressure System

From C. Göttlinger, B. Mechtold, and A. Radbruch[RFM]

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Fluidics SystemsPositive Pressure Systems

• Based upon differential pressure between sample and sheath fluid. • Require balanced positive pressure via either air or nitrogen• Flow rate varies between 6-10 ms-1

+ + ++ + ++ + +

Positive Displacement Syringe Systems

• 1-2 ms-1 flow rate• Fixed volume (50 l or 100 l)• Absolute number calculations possible• Usually fully enclosed flow cells

100 l

Sample loop

Sample Waste

Flowcell3-way valve

Syringe

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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

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Fluidics

• Need to have cells in suspension flow in single file through an illuminated volume

• In most instruments, accomplished by injecting sample into a sheath fluid as it passes through a small (50-300 µm) orifice

[RFM]

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Fluidics

• When conditions are right, sample fluid flows in a central core that does not mix with the sheath fluid

• This is termed Laminar flow

[RFM]

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• Whether flow will be laminar can be determined from the Reynolds number

• When Re < 2300, flow is always laminar

• When Re > 2300, flow can be turbulent

Fluidics - Laminar Flow

Re d v

whered tube diameter

density of fluidv mean velocity of fluid

viscosity of fluid

[RFM]

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Fluidics

• The introduction of a large volume into a small volume in such a way that it becomes “focused” along an axis is called Hydrodynamic Focusing

[RFM]

FluidicsThe figure shows the 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.

From V. Kachel, H. Fellner-Feldegg & E. Menke - MLM Chapt. 3[RFM]

V. Kachel, H. Fellner-Feldegg & E. Menke - MLM Chapt. 3

Notice how the ink is focused into a tight stream as it is drawn into the tube under laminar flow conditions.

Notice also how the position of the inner ink stream is influenced by the position of the ink source.

[RFM]

Fluidics

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Fluidics

• How do we accomplish sample injection and regulate sample flow rate?– Differential pressure– Volumetric injection

[RFM]

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• Use air (or other gas) to pressurize sample and sheath containers

• Use pressure regulators to control pressure on each container separately

[RFM]

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• 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

[RFM]

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Fluidics - Volumetric Injection System

• Use air (or other gas) pressure to set sheath volume flow rate

• Use syringe pump (motor connected to piston of syringe) to inject sample

• Sample volume flow rate can be changed by changing speed of motor

• Control is absolute (under normal conditions)

[RFM]

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Syringe systems

• Bryte HS Cytometer

3 way valve

Syringe

Fluidics - Volumetric Injection System

H.B. Steen - MLM Chapt. 2

Page 33© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT

Hydrodynamic Systems

MicroscopeMicroscopeObjectiveObjective

WasteWasteFlowFlowCellCell CoverslipCoverslip

SignalsSignals

MicroscopeObjective

Waste

FlowCell Coverslip

SignalsSignals

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Fluidics - Particle Orientation and Deformation

• As cells (or other particles) are hydrodynamically focused, they experience different shear stresses on different points on their surfaces (an in different locations in the stream)

• These cause cells to orient with their long axis (if any) along the axis of flow

[RFM]

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Fluidics - Particle Orientation and

Deformation• The shear stresses can also cause

cells to deform (e.g., become more cigar-shaped)

[RFM]

Fluidics - Particle Orientation and Deformation

“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.”

Image fromV. Kachel, et al. – Melamed Chapt. 3[RFM]

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Fluidics - Flow Chambers

• The flow chamber– defines the axis and dimensions of

sheath and sample flow– defines the point of optimal

hydrodynamic focusing– can also serve as the interrogation

point (the illumination volume)

[RFM]

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Closed flow cells

Laser direction

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Coulter XL

Sample tubeSheath and waste system

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• Four basic flow chamber types– Jet-in-air

• best for sorting, inferior optical properties

– Flow-through cuvette• excellent optical properties, can be used for

sorting

– Closed cross flow• best optical properties, can’t sort

– Open flow across surface• best optical properties, can’t sort

[RFM]



Flow through cuvette (sense in quartz)

[RFM]



Closed cross flow chamber

[RFM]



Sample inSheath

Sheath in

Laser beam

Piezoelectriccrystal oscillator

FluorescenceSensors

Scatter Sensor

Core

Sheath

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Hydrodynamically focused fluidics

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Hydrodynamically focused fluidics

•Increase Pressure:•Widen Core•Increase turbulence

Signal



Flow Cell

Injector Tip

Fluorescencesignals


Sheath fluid

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What happens when the channel is blocked?

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Flow chamber blockage

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

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Bryte Fluidic Systems Detectors

• Sample Collection and hydrodynamics

Bryteb.mpg

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Fluorescence Detectors and Optical TrainBrytec.mpg

Shown above is the Bryte HS optical train - demonstrating how the microscope-like optics using an arc lamp operates as a flow detection system. First are the scatter detectors (left side) followed by the central area where the excitation dichroic can be removed and replaced as necessary. Behind the dichroic block is the arc lamp. To the right will be the fluorescence detectors.

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Flow CellFlow Cell

Injector Tip

FluorescenceFluorescencesignalssignals


Sheath fluid

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Sheath and waste systemsEpics Elite

Sheath Filter UnitLow PressureSheath and Waste bottles

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Fromlaser

Fluorescence collection lens, optical filters, dichroic filter, band pass filter

Beam shaping lens

reflector

J.Paul RobinsonProfessor of ImmunopharmacologySchool of Veterinary Medicine, Purdue University

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Lecture Summary

• Detection systems in flow cytometry• 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• There are many types of flow cells• Blockages must be properly cleared to obtain high

precision

WEB http://www.cyto.purdue.edu

Documents

Page 1 © 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT BMS 631 - LECTURE 7 Flow Cytometry: Theory J. Paul Robinson Professor of