L5 Flowcytometry and Data Analysis

7/30/2019 L5 Flowcytometry and Data Analysis

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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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Filter

components

Single Optical

filter

glue



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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



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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



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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



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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

L5 Flowcytometry and Data Analysis

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Transcript of L5 Flowcytometry and Data Analysis