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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, Wiley-
Liss, 2nd edition.
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2002 J.Paul Robinson, Purdue UniversityBMS 631- Flow Cytometry lecture002.ppt
Page 2
Fundamentals of a Flow Cytometer
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Basics of Flow Cytometry
cells in suspension
flow in single-file through
an illuminated volume where theyscatter light and emit fluorescence
that is collected, filtered and
converted to digital values
that are stored on a computer
Fluidics
Optics
Electronics
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|| FLUIDICS ||Flow Systems and HydrodynamicsGetting the cells in the right place (at the right time)!
(Shapiro, pp 133-143 - 3rd edition)
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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
Flow Cell
Fluorescence
signals
Focused laser
beam
Sheath
fluid
The use of focused light (lasers)
to interrogate cells delivered by a
Hydrodynamically focused
fluidics system.
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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 andinside 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.
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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
Sample
Sheath fluidin
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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 samplevolume flowrate
Control is not absolute -
changes in friction
cause changes in sample
volume flow rate
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Fluidics Systems
1
00
l
Sample loop
Sample Waste
Flowcell3-way valve
Syringe
Positive Displacement Syringe SystemsA. 1-2 ms-1 flow rate
B. Fixed sample volume (50 l or 100 l)
C. Sheath fluid pressure is calibrated to getoptimal sample flow rate
D. Absolute number calculations possible
E. Usually fully enclosed flow cells
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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
Control is absolute (under normal
conditions)
Flow nozzle
Motor with
variable speed
Valve
Sheath inlet
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Fluidics on CellsAs 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)
In the turbulent flow near the tube wall,
the cells are deformed and disoriented in a
very individual way. v>3 m/s.
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.
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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 (
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Fluidics - Differential Pressure System
From C. Gttlinger, B. Mechtold, and A. Radbruch
Sample lineValve
Valve
Sheath container
Sample tube
VacuumWaste Line pressure
Filter
Sheath pressure
adjust
Sheath pressure
gauge
Sheath to
sample d/d
pressure
gaugeSample to sheath
to d/d pressure
gauge
Filter
Sheath lineFlow
chamber
Valve
Purge
line
Valve
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Technical Components
Fluidics Positive Pressure Systems (DPS)EPICS C (Coulter)
EPICS 5 & 7, Elite series
FacStar (B-D)
FacsVantage (B-D)
BruckerProfile (Coulter)
XL (Coulter)
FacScan (B-D)
Positive Displacement (Syringe Drive) SystemsBryte HS
Cytotron Absolute
FACS Caliber (B-D)
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Syringe systems
Bryte HS Cytometer
3 way valve
Syringe
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Fluidics - Volumetric Injection System
H.B. Steen - MLM Chapt. 2
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Hydrodynamic Systems
Flow Cell
Injector
Tip
Fluorescence
signals
Focused laserbeam
Sheath
fluid
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Hydrodynamically focused fluidics
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Hydrodynamically focused fluidics
Increase Pressure:
Widen Core
Increase turbulence
Signal
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Flow chamberIt 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)
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Flow chamber
Jet in air nozzle
Best for sorting
Piezoelectric crystal oscillator is
responsible for precise sorting
Inferior optical properties
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Flow chamber
Jet in air nozzleSieve/Fileters are essential
BD: smal filter made up of bundle of
Microcapillary tubes, < 40um internal
Diameter
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
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Flow chamber
Flow through cuvette
Excellent optical properties
It can be used for sorting
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Flow chamber
Closed cross flow chamber
Best optical properties
It cant sortIt can be used to visualize cell
distortion under flow
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Summary
Fluid flow may be Coaxial (common)
Axial (microscope based)
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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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Hydrodynamic Systems and Sorting
Sample inSheath
Laser beam
Piezoelectric
crystal oscillator
Fluorescence
Sensors
Scatter Sensor
Core
Sheath
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Hydrodynamic Systems and Sorting
This figure shows theprinciple of electrostatic
cell sorting based on Sweets
inkjet printer technology.
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Hydrodynamic Systems and SortingIn 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.
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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/syringedrive system
There are many types of flow cells
Blockages must be properly cleared to obtain high precision
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|| OPTICS ||Source, Filters, DetectorsGetting the cells interrogated !
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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
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Optics Principle
Low-order Hermite-Gaussian resonator modes
TEMmn
Where m and n are no. of nodes in x- and y- axes
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Optics: Light SourceNeed 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
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Illumination Sources
Lamps Xenon-Mercury
Mercury
Lasers
Argon Ion (Ar)
Krypton (Kr)
Helium Neon (He-Ne) Helium Cadmium (He-Cd)
YAG
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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
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LaserLight Amplification by Stimulated Emission ofRadiation
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)
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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.
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Laser Production
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 desiredlaser intensity, the partial reflector allows the laser light to pass through.
pumping energy
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Lasers
Coherent Enterprise laser - UV-visible
Air cooled laser (Argon)
A & K t L
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Argon & Krypton Lasers
Kr-Ar laser (488, 568, 647 nm lines) (Front)
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Dye Lasers
Dye lasers use a source laser known as the pump laser to excite another laserknown as the dye laser.
The dye laser consists of a dye mixed in a solvent which exhibits desirableproperties 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 over-
heated increasing the cost considerably
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Dye LasersA wavelength-dispersive
element (a prism or diffraction
grating) is placed inside the lasercavity.
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.
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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).
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Helium-Neon Lasers
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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
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Diode Lasers Small, efficient, cheap
Only red wavelengths available at reasonable prices (blue works, but stillproblems)
Mostly made ofGallium Aluminum Arsenide (GaAlAs)
Emission ratio is varied by changing the ratio of gallium to aluminum in thesemiconductor
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 and0.02% with water cooled argon lasers
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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
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Solid State Lasers
B i f L P d ti
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Basics of Laser Production
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Common Lasers
Lasing
Medium
Cooling Power Wavelength
He-Ne Ne gas No 0.1 W-50 mW 543
633, 594, 511
C L
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Common Lasers
Lasing
Medium
Cooling Power Wavelength
(nm)
Ar-Kr Ar and/or Kr Air cooling 8-200 mW 488
514.5
353 (UV)
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Lasers Hazards Laser light is very dangerous and should be treated as a
significant hazard
Water cooled lasers have additional hazards in that theyrequire high current and voltage in addition to the waterhazard
Dye lasers use dyes that can be potentially carcinogenic
Elite Cytometer with 4 Lasers
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y
Mirror
395 longPass
He-Cd Laser 325/441
Argon Laser 353/488 nm(High speed sorting)
He-Ne Laser 633 nm
Argon Laser 488 nm
633 Beam Splitter
UV\Beam Splitter
325 nm
353 nm633 nm
488 nm
Height TranslatorsOptical bench
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Goals of Light Collection
Maximum signal, minimum noise Maximum area of collection
Inexpensive system if possible
Easy alignment Reduced heat generation
Reduced power requirement
Ob i b & fi ld
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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 (whichis going in all directions)/scattering light
In a capillary or cuvett system, a field stop which is placed in the imageplane achieves the same result
Optical translators
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Optical translators
The laser beam remains parallel, but
horizontally translated.
This reduces the difficulty in aligning
the laser.
No cytometer should be without one!!!
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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
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Spectral Selection
Monochromators Vs Filters
Filters are reasonably inexpensive
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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 typicalcytometers
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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)
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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) isdetected 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)
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Optics: Scatter
Forward scatter tends to be more sensitive to surfaceproperties 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 withincells than forward scatter
It can be used to distinguish granulated cells from
non- granulated cells
O ti Fl
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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
Fl
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Fluorescence
Photon emission as an electron returns from an
excited state to ground state
Fluorescence
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Fluorescence
ENERGY
S0
S1
S2
T2
T1
ABS FL I.C.
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
IsC
IsC
PH
[Vibrational sublevels]
Jablonski Diagram
Vibrational energy levels
Rotational energy levels
Electronic energy levels
Singlet States Triplet States
3rd
Ed. Shapiro p 87
Three nonradiative
deactivation processes Internal conversion
(IC),
Intersystem crossing
(ISC) and
Vibrational relaxation
Fluorescence
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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 excitingwavelength
Fluorescence
Fl
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The wavelength ofabsorption is related to the sizeof the chromophores
Smaller chromophores absorbs light of shorter wavelength (higherenergy)
Larger chromophores absorb light oflonger the wavelength (lowerenergy)
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 (Largerchromophores)
e.g. UV light from sun - this causes the sunburn, not the redvisible light
Fluorescence
Fluorescence
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2002 J.Paul Robinson, Purdue University
Stokes Shift is the energy difference between the highest energy
peak of absorbance and the highest energy of emission
495 nm 520 nm
Stokes Shift is 25 nmFluorescein
molecule
Fluorescnec
eI
ntensity
Wavelength
Fluorescence
i f l l l
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Properties of Fluorescent Molecules
Large extinction coefficient at the region ofexcitation
High quantum yield
Optimal excitation wavelength
Photostability
Excited-state lifetime
Minimal perturbation by probe
Allophycocyanin (APC)
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Allophycocyanin (APC)
Protein 632.5 nm (HeNe)
Excitation Emisson
300 nm 400 nm 500 nm 600 nm 700 nm
600300 500 700400
457350 514 610 632488 Common Laser Lines
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Ethidium
PE
cis-Parinaric acid
Texas Red
PE-TR Conj.conjugate of Texas Red with
R-phycoerythrin
PI
FITC
600 nm300 nm 500 nm 700 nm400 nm
C l i
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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 ofwavelength
Optics: Filters
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Optics: FiltersClassification as per frequency filtration:
Long pass filters transmit wavelengths above a cut-onWavelength
Short pass filters transmit wavelengths below a cut-offWavelength
Band pass filters transmit wavelengths in a narrow rangearound a specified wavelength- (Band width can be specified)
Neutral Density filter is a non-discriminant intensityreducing filter
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Standard Long Pass Filters
Transmitted LightLight Source 520 nm Long Pass Filter
>520 nmLight
Transmitted LightLight Source
575 nm Short Pass Filter
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Standard Band Pass Filters
Transmitted LightWhite Light Source
63010 nm Band-Pass Filter
620 -640 nm Light
Neutral density filters (N D)
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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
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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 (usuallydeposited 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
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Optical filters
Classification as per function:
Interference filters:
Dichroic, Dielectric, Reflective filters.reflect the unwanted
Wavelengths
An optical filter consisting of multiple layers of evaporatedcoatings 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
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Construction of Interference Filters
Filter
components
Single Optical
filter
glue
Construction of Interference Filters
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Construction of Interference Filters
Optics Filter Properties
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Optics - Filter Properties
When a filter is placed at a 45o
angle to a light source, lightwhich 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
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Dichroics
They used to direct light in different spectral region todifferent 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
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Optical Filters
Dichroic Filter/Mirror at 450
Reflected light
Transmitted LightLight Source
Transmission determination
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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
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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
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Measuring Filter Properties
Filters must be measured at the angle they are going tobe used
Filters placed at 90o
have different properties whenthey are placed at 45o
Short pass and long pass filters
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TR
A
N
S
MI
S
S
I
O
N
WAVELENGTH
SP filter LP filter
cutoff cuton
T max T max
For Band-pass filters
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TR
A
N
S
MI
S
S
I
O
N
WAVELENGTH
T max
FWHM
Optical filters evaluation
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Optical filters evaluation
Use a population of appropriately stained particlesand identify which filters give the maximum signal.
Spectrofluoremeter and spectrophotometers can beused as tools for assessment of optical filters.
Optical filter evaluation
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p
optical filter(90o)slit/shutter
(to reduce the
amount of light at
collection point)
light source
detector
Monochromator
(select as per your filter)
Spectrofluorometer for Assessmentof Optical Filter Transmission
Optical filter evaluation
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p
light source
Monochromator / grating
beam splitter (45o)reference PMT
slit/shutter
Optical filter (45o
)
sample PMT
PMT
Monochromator / grating
Light loss by optics
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g y p
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
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Continued.
Glass can fluoresce highest when illuminated at UVwavelength.
For excitation > 450nm, you can use glass filters, < 450nm use
quartz or silica filters.
Plastic optical filters are unsatisfactory
Optics: Detectors
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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
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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 modewith 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
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Current is produced at anodes when photons impinge upon their light-
sensitive 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
currentIf very low levels of signal are available, PMTs are often cooled to reduce heat
effects
PMT
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Spectral Response:
Spectral response of PMTs is determined by the composition of thePhotocathode
Material Peak sensitivity at Comments
Bi-alkali PMTs 400 nm Higher gain
Multi-alkali PMTs 750 nm Highest gain
Gallium Arsenide
(GaAs) cathodes
300-850 nm very costly and have
lower gain
PMTs
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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
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g g
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
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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
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Signal Detection PMTs
Cathode Anode
Dynodes
Photonsin
AmplifiedSignal
Out
End
Window
Requires Current on dynodes
Is light sensitive
Sensitive to specific wavelengths
Shown here is an end window PMT
Secondary emission
Types of PMTs
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Side Window
High voltage in
Signal
out
Diode Vs PMT
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Diode Vs PMT
Scatter detectors are frequently diode detectors
Back of Elite forward scatter detector
showing the preampFront view of Elite forward scatter detector
showing the beam-dump and video camera
signal collector (laser beam and sample sheath
are superimposed)
Sample stream
Avalanche Photodiodes (APDs)
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Combines the best features of PMTs and photodiodes
To increase gain, we need to decrease the capacitance of diode
By decreasing surface area By increasing depletion zone
PHOTOVOLTAIC CELL
Avalanche Photodiodes (APDs)
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Add a thick depletion layer
To increase depletion zone
Apply reverse bias of high voltage
(upto 2000 V)
increase site for recombination
AVALANCHE PHOTODIODEPIN 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)
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Image From: http://micro.magnet.fsu.edu/primer/java/photomicrography/avalanche
Summary
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y 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 butbecause of their low gain, they are not as useful for low level
signals
Flow Cytometry Software? What for?
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Display flow cytometry data
(1D, 2D, and 3D displays)
Identification of cells of interest
Define a cluster
RegionMixed populations and noise Gating
Characterization of cells of interest
Intrinsic parameters (mean/median scatter and fluorescence intensities; positive/negativecells)
Cell counts (abundance)
Kinetics (evolution of a cell parameter with time)
Cell cycle analysis
Classical Data Analysis:
V i t f d t di l
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Various types of data displays
Frequency distribution
Dot plot
Density plot
Contour plot
Frequency distribution
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Frequency distribution
Histograms display the distributions of theEvents for one parameter.
Simplicity of the plot
No correlation with the other parameters Problem for cluster identification
Histogram overlay
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g y
Superimpose the data from several data files
Dot plot
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Dot plot
Displays correlated data from anytwo parameters.
Each dot corresponds to a particle(event) analyzed by the flow
cytometer.
Several events can occupy the samedot if they have the same parameterintensities.
No indication of the relative density of the events
Problem with large data files
Density and Contour plot
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Density plot:
Displays two parameters as a frequencydistribution. Color is used to code the different frequenciesof events.
Simulation of a 3D display with a " third " parameter beingthe number of events. Can clarify clusters
Contour plot:
Displays correlated data from any twoparameters, with contour lines joiningpoints of equal elevation (frequencydistribution).
3D Displays
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p y
2 parameters versus density 3 parameters displayed together
Danger!!!
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With Density plots and Contour plots some options like
-Resolution-Smoothing
can emphasize or hide clusters of cells.
64x64128x128256x256
Example : Changing Resolution
Particle (cell) Discrimination
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Particle (cell) Discrimination
Problem :
Very often, samples are heterogeneousthere 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?
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What is a Region?
A region can be defined as setof points carefully selected by theuser that determine an area on agraph.
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
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Different styles of regions
Polygon
Rectangle
Ellipse
Cluster discrimination Positive/Negative cell identification
Quadrants
Propidium iodideRed fluorescence
Greenfluoresc
ence
SYBRGreen)
Compromised
membranes
Damaged
membranes
Membrane integrity
E.coli
What is a Gate?
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A gate can be defined as oneor more regions combined usingBoolean (logic) operators (AND,NOT, OR)
Defines a subset of the data tobe displayed.
Used to compute statistics
and characterize the subset ofevents selected