Transcript BMS 631 - LECTURE 1 Flow Cytometry: Theory J.Paul Robinson

BMS 631 - LECTURE 7
Flow Cytometry: Theory
J. Paul Robinson
Professor of Immunopharmacology& Biomedical Engineering
Purdue University
Detectors & Fluidics
Hansen Hall, B050
Purdue University
Office: 494 0757
Fax 494 0517
email: [email protected]
WEB http://www.cyto.purdue.edu
Notes:
1.
2.
3.
Material is taken from the course text: Howard M.
Shapiro, Practical Flow Cytometry, 3nd edition (1994),
Wiley-Liss, New York.
RFM =Slides taken from Dr. Robert Murphy
MLM – Material taken from Melamed, et al, Flow
Cytometry & Sorting, Wiley-Liss, 2nd Ed.
3rd Ed. Shapiro 127-133
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
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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
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
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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)
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
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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)
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
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Signal Detection - PMTs
Secondary emission
Cathode
Anode
Amplified
Signal
Out
Photons
in
End
Window
Dynodes
• Requires Current on dynodes
• Is light sensitive
• Sensitive to specific wavelengths
• Can be end`(shown) or side window PMTs
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
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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
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
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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
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
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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)
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
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Types of PMTs
Side Window
Signal
out
High
voltage in
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
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High Voltage on PMTs
•
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•
•
•
•
•
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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
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
Page 10
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/
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
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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
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
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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)
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
Page 13
Flow Systems and Hydrodynamics
Getting the cells in the right place (at the right
time)! (Shapiro, pp 133-143 - 3rd ed)
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
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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
[RFM]
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
Page 15
Flow Cytometry:
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
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
Page 16
Fluidics - Differential Pressure
System
[RFM]
From C. Göttlinger, B. Mechtold, and A. Radbruch
Fluidics Systems
Positive 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
Syringe
Fixed volume (50 l or 100 l)
Absolute number calculations possible
Usually fully enclosed flow cells
Flowcell
100 l
•
•
•
•
3-way valve
Sample
Waste
Sample loop
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
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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
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
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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]
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
Page 21
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]
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
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Fluidics - Laminar Flow
• Whether flow will be laminar can be
determined from the Reynolds number
Re

d v

where
d  tube diameter
  density of fluid
v  mean velocity of fluid
  viscosity o f fluid
• When Re < 2300, flow is always laminar
• When Re > 2300, flow can be turbulent
[RFM]
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
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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]
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
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Fluidics
The 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.
[RFM]
From V. Kachel, H. Fellner-Feldegg & E. Menke - MLM Chapt. 3
Fluidics
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]
V. Kachel, H. Fellner-Feldegg & E. Menke - MLM Chapt. 3
Fluidics
• How do we accomplish sample
injection and regulate sample flow
rate?
– Differential pressure
– Volumetric injection
[RFM]
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
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Fluidics - Differential Pressure
System
• Use air (or other gas) to pressurize
sample and sheath containers
• Use pressure regulators to control
pressure on each container separately
[RFM]
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
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Fluidics - Differential
Pressure System
• 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]
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
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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]
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
Page 30
Syringe systems
• Bryte HS
Cytometer
Syringe
3 way valve
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
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Fluidics - Volumetric Injection System
H.B. Steen - MLM Chapt. 2
Hydrodynamic
Systems
Signals
Flow
Cell
Coverslip
Signals
Flow
Cell
Microscope
Objective
Waste
Coverslip
Microscope
Objective
Waste
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
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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]
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
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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]
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
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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.”
[RFM]
Image fromV. Kachel, et al. – Melamed
Chapt. 3
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]
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
Page 37
Closed flow cells
Laser
direction
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
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Coulter XL
Sample tube
Sheath and waste system
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
Page 39
Fluidics - Flow Chambers
• 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]
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
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Fluidics - Flow Chambers
Flow through
cuvette (sense
in quartz)
[RFM]
H.B. Steen - MLM Chapt. 2
Fluidics - Flow Chambers
Closed cross
flow chamber
[RFM]
H.B. Steen - MLM Chapt. 2
Hydrodynamic
Systems
Sample in
Sheath
Piezoelectric
crystal oscillator
Sheath in
Fluorescence
Sensors
Laser beam
Scatter Sensor
Sheath
Core
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
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Hydrodynamically focused fluidics
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
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Hydrodynamically focused fluidics
Signal
•Increase Pressure:
•Widen Core
•Increase turbulence
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
Page 45
Hydrodynamic Systems
Injector
Tip
Flow
Cell
Sheath
fluid
Fluorescence
signals
Focused laser
beam
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
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What happens when the channel is
blocked?
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
Page 47
Flow chamber blockage
A human hair blocks the flow
cell channel. Complete
disruption of the flow results.
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
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Bryte Fluidic Systems
Detectors
Bryteb.mpg
• Sample Collection and hydrodynamics
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
Page 49
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.
Fluorescence Detectors and Optical Train
Brytec.mpg
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
Page 50
Flow Cell
Injector
Tip
Sheath
fluid
Fluorescence
signals
Focused laser
beam
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
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Sheath and waste systems
Epics Elite
Sheath Filter Unit
Low Pressure
Sheath and
Waste bottles
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
Page 52
Fluorescence collection lens, optical filters,
dichroic filter, band pass filter
J.Paul Robinson
Professor of Immunopharmacology
School of Veterinary Medicine, Purdue University
From
laser
reflector
Beam shaping lens
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
Page 53
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
© 1990-2002 J.Paul Robinson, Purdue University BMS 631 – LECTURE007.PPT
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