Flow cytometry and FACS

Chapter 7
Flowcytometry

• Technology that measures properties of single cells

• Measures light scatter, fluorescence and other properties of cells and
particles
• Can provide correlated data that links different population profiles

Principle
• The principle of flow cytometry is based on light scattering as the
result of particle size and sating properties which is facilitated by
– Specific fluorescence staing

– Hydrodynamically focused stream of particles

– Electrostatic particle separation for sorting

 Commercial Instruments
BC
BC

Luminex Bryte

Bay
Biosciences

Accuri

Millipore/Guava

BC

Amnis

BD BD Aria

BC
BD

BD Influx Apogee
Parts of the instrument
 Fluidics system
• One of the fundamentals of flow cytometry is the ability to consistently measure
properties of individual particles as they flow at high speed across focused laser
beams.
• When a solution of particles is injected into a flow cell, the particles are initially
randomly distributed
• To achieve measurement consistency the particles must first be aligned into a
single file before arriving at the laser interrogation points
• Precise particle alignment and consistent delivery is handled by fluidics
subsystem, including
– hydrodynamic focusing
– laminar flow
Hydrodynamic focusing

• Flow cells are designed to accelerate, focus and align particles.

• This is achieved by funneling (narrowing) the flow cell’s inner cross section

– The narrowest part at the point where particles exit on their way to the
lasers.
– Narrowing of the flow space confines the particles carried by the fluid
into a progressively smaller area with ever increasing velocities. Which is
the basis for hydrodynamic focusing.
– Soon after exiting the hydrodynamic focusing flow cell segment, a thin
core of single file particles surrounded and carried by sheath fluid,
travels at a constant velocity across the focused laser beams for
illumination and photon capture by the optics.
Laminar flow

• Characterized by fluid flowing in parallel layers with no mixing with

sample core. guarantee consistent, predictable motion and delivery
• When liquid runs laminar in a cytometer’s flow cell, it eventually
develops a parabolic velocity profile, where the liquid at the centre of
the pipe travels approximately at twice the average fluid velocity and
where liquid near the pipe’s walls has a velocity close to zero.
• This generates an inverse pressure gradient, with the lowest pressure at
the pipe’s centre and maximum pressure at the walls. Because of this,
particles are forced to relocate to the faster moving centre.
Optical system

Light source
• Lamps

• Xenon-Mercury

• Mercury

• Lasers

• Argon Ion (Ar)

• Krypton (Kr)

• Helium Neon (He-Ne)

• Helium Cadmium (He-Cd)

• Diodes

• Variety of wavelengths, cheap

Laser (Amplification by Stimulated Emission of Radiation)

• Laser light is coherent and monochromatic (same frequency and

wavelength)
• The band width is very narrow and formed line spectrum
• 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.
• The three important nature of LASER is
– Narrow width(monochromatic)

– Coherent

– Strong energy
 Laser ns

© J.Paul Robinson

Coherent Laser – 1999

(20 mw version)

07:32 AM
 Lasers

Noncoherent light

Coherent light
Diagram from Microsoft encylopedia

07:32 AM
Optical system….
Filters and PMT
PMT 5

PMT 4

Sample
PMT 3
Dichroic
Flow cell Filters
PMT 2

Scatter PMT 1

Sensor
Laser
Bandpass
Filters
Filters
• Dichroic filters
– Reflect light below a certain wavelength and pass light above that
wavelength
• Band Pass filters
– Block some wavelengths and pass others
• Short band pass filters allow wavelengths below the cutoff to
be passed
• Long band pass filters allow wavelengths above the cutoff to
be passed
 Coulter Optical System

Empty PMT PMTs

slot Dichroic filter slot

Light Scatter
Detector
Photos Source: J. Paul Robinson

07:32 AM ©1990-2010 J. Paul Robinson, Purdu Page 16

e University
Cytometer with more than one Lasers

488 nm
353 nm
325 nm 633 nm

UV\Beam Splitter He-Cd Laser 325/441

395 longPass
Argon Laser 353/488 nm
(High speed sorting)

633 Beam Splitter

He-Ne Laser 633 nm

Mirror Argon Laser 488 nm

Height Translators
Optical bench

© 1990-2010 - J. Paul Robinson, Purdue University

Interrogation point
Scattering light measurement
FSC vs SSC
Depends on the size and granularity

Eg. 3 – Differential analyzers

FSC vs SSC
Since FSC ~ size and SSC ~ internal structure, a correlated measurement
between them provides a basis for a simple differentiation between the
major populations

Granulocytes
Lymphocytes
SSC

Monocytes
RBCs, Debris,
Dead Cells
FSC
Gating

• Gating: electronically isolating a population for further analysis

• Gating strategies:
– FSC vs SSC: WBC differential (G, M, L) CD45 vs SSC
– CD45 is brighter on lymphocytes than monocytes, thus CD45 and
side scatter is more specific for lymphocytes than scatter alone
Fluorescence activated cell-sorting (FACS)
Cell sorting on FACS
Granularity vs fluorescence
Eg. 5- Differential analyzer
 Fluorescent Molecules

• Fluorescent probes
– Structural and functional dyes, fluorochrome-conjugated antibodies and
fluorescent proteins, are used in cytometry to directly label molecules
(proteins, DNA, other moieties)
– Fluorescent probes are useful in a wide range of applications including
identifying and quantifying distinct populations of cells, cell surface
receptors or intracellular organelles, cell sorting, immunophenotyping,
nucleic acid content, enzymatic activity, and apoptosis

• Fluorescence
– Property of a substance that results in a higher wavelength of light being
emitted when the substance is struck by a lower wavelength of light
Types Fluorescent Molecules

• Single fluorophores

– Typically synthesised or naturally occurring molecules that when excited by

an energy source undergo conformation changes and fluoresce.
– Single fluorophores include small synthetic molecules as FITC, organic
molecules derived from biological sources, such as phycoerythrin (PE) and
allophycocyanin (APC) and inorganic polymers such as Brilliant violet 421
and Brilliant violet 510.
– They have unique structures with specific excitation and emission
maximum
– Can be used as molecular blocks to build tandem fluorophores of extended
Stokes Shift.
Types Fluorescent Molecules
• Tandem fluorophore
– Tandems of two or more single fluorochromes “blocks” are covalently
linked. Typically, a small fluorochrome ‘added’ onto another larger
fluorochrome.
– When the laser excitable dye portion of the tandem (known as the
“donor”) is excited and reaches its maximal singlet state, all its energy
transfers to the second dye (the “acceptor”). The “acceptor” molecule,
whose absorption spectra overlaps with the emission spectra of the first
dye, produces the fluorescence emission.
– The process is called FRET (Forster resonance energy transfer). It’s a way to
increase the number of colors that can be analysed from a single laser
wavelength. Examples of tandem dyes are: PerCP-Cy5.5, PE-Cy7, APC-
Fire750 and Brilliant violet 650.
Types Fluorescent Molecules
• Fluorescent proteins (FPs)
– such as green fluorescent protein (GFP), mCherry or cyan fluorescent
protein (CFP) are typically used as surrogate indicators of endogenous
protein expression or protein over-expression.
• Fluorescent Dyes
– Synthetic chemicals that enter cells in various circumstances. Some
fluorescent dyes can cross the membrane of a live cell (membrane
permeable) such as Celltrace violet, CFSE, Hoechst 33342, JC1 and DRAQ9.
Other dyes are not able to enter live cells, and only cross the membranes
of compromised cells. These include nucleic acid binding dyes DAPI (A-T
regions), 7AAD and propidium iodide (G-C regions) that fluoresce once
bound to nucleic acids. These dyes are commonly used to distinguish
between live and dead cells, and to measure cell cycle.
 Common fluorophores for Ab conjugation
FLUOROCHROME Type of molecule Typical excitation Approximate
laser emission peak
Fluorescein Small organic 488 nm 518 nm
isotyocyanate (WL emitted by Argon
(FITC) laser)
AlexaFluor 488 Small organic 488 nm 518 nm
Phycoerythrin (PE) Protein 488 or 532 nm 574 nm

PE-Texas Red Protein tandem 488 or 532 nm 615 nm

PE-Cy5 Protein tandem 488 or 532 nm 665 nm
Peridinin Protein 488 or 532 nm 676 nm
chlorophyll protein
(PerCP)
PerCP-Cy5.5 Protein tandem 488 or 532 nm 695 nm
PE-Cy7 Protein tandem 488 or 532 nm 776 nm
Allophycocyanin Protein 633 nm 659 nm
(APC)
AlexaFluor 647 Small organic 633 nm 667 nm
AlexaFluor 700 Small organic 633 nm 718 nm
APC-Cy7 Protein tandem 633 nm 784 nm
Pacific Blue Small organic 405 nm 454 nm
AmCyan Protein 405 nm 487 nm
 Optics and Fluorescence Detection

• Scattered and emitted light originating from the laser interrogation

points is collected by optical lenses and delivered to the cytometer
detectors assembly (PMT).
• An array of light-dissecting optical filters block or reflect certain
wavelengths while transmitting (passing) limited bandwidths of photons
to each detector.
• There are three major classes of optical filters used in Cytometry:
– Long pass (LP) filters allow through light above a cut-off wavelength,

– Short pass (SP) filters transmit light below a cut-off wavelength

– Band pass (BP) filters transmit light within a specified narrow range of wavelengths.
Fluorescence Detection
Photomultiplier tube(PMT)
Result read out( Data Acquisition)
End of chapter 7