Introduction to Flow Cytometry

Sheree Bailey
Flow Cytometry Facility
Flinders Medical Centre

Aims
To introduce you to the principles of flow
cytometry and fluorescence
To explain the components of a flow
cytometer
To provide an understanding of why we
use flow cytometry and of its advantages

What is Flow Cytometry?

A technology that simultaneously
measures multiple characteristics of single
cells at a rapid rate

Particle Characteristics
Its relative size (Forward Scatter - FSC)
Its relative granularity or internal
complexity (Side Scatter SSC)
Its relative fluorescence intensity (Green,
Orange, Red)
All of these characteristics are relative to
other particles within the sample

Properties of FSC and SSC

Forward Scatter - diffracted light
Relative to cell surface area
Detected parallel to the laser beam in the
forward direction

Side Scatter reflected and refracted light

Related to cell granularity and complexity

Flow Cytometers Analyse

Particles
Lysed Whole Blood

St Kilda Mangrove
Beads

B6
B4
B2

B5
B3

B1
V3
V2
V1

Supplied byJustin Seymour FU Biol Sci

Particles Analysed
Human and animal
cells
Mitochondria
Grapes
Pollen
Fish eggs
Bacteria

Nuclei
Oyster larvae
Plants
Nematodes
Fungi
Marine creatures
Red Wine

Flow Cytometry
Measures properties of cells (or particles)
in suspension
Sample is usually less than 100m in size
Measures properties of individual cells
among other populations in complex
mixtures

Flow Cytometry
Flow Sorting
Separates cells based
on properties
measured in flow
cytometry
Also called
FluorescenceActivated Cell
Sorting (FACS)

Flow Cytometry Analysers

Flow Cytometer Schematic

Cytometer Components
Fluidics
To introduce and focus the cells for
interrogation

Laser and Optics

To generate and collect the light signals

Electronics
To convert the optical signals to proportional
electronic signals and digitize them for
computer analysis

Fluidics Flow Analyser

Fluidics
Function
Puts the flow in flow cytometry
Focuses the sample stream
Controls the rate of which cells pass the laser

Control of the fluidics

Sheath regulator
Sample regulator
Air Pump
Flow cell

Fluidics Sample Pressure

The sample stream is
pressurised upward
through the flow cell
Particles are
interrogated by the
laser beam while still
in the flow cell

Fluidics - Hydrodynamic Focusing

Sheath and sample fluids
meet within the flow
chamber
Sample fluid is injected
into the centre of the
sheath fluid
As the fluids narrow, the
sample remains in the
centre of the sheath fluid

Fluidics - Hydrodynamic Focusing

Particles pass through
the laser bean one at a
time for interrogation
If one smooth stream is
injected into the centre
of another smooth
stream, they maintain
their positions and dont
mix.
laminar flow

Fluidics - Laminar Flow

sheath pressure narrows
sample stream - cells move
faster

sheath pressure widens

sample pressure widens

sample stream more cells
analysed per unit time
sample pressure narrows
sample stream less cells
analysed per unit time

sample stream - slows the

cells
Altering sample pressure does
The effect of altering sheath
not change cell velocity
pressure changes the cell
velocity through the laser beam

Fluidics - Laminar Flow

Why would velocity over per unit time
matter ?
If velocity is altered the cell spends more or
less time through the laser beam. Time spent
through the laser beam will alter the amount
of light from the cell
If core width is altered without velocity altered
more or less cells go though the beam at a
given time altering the precision

Optics
Excitation
A laser
Lenses to shape and focus the laser beam

Collection
a collection lens to collect light from the
particle/laser beam interaction
A system of prisms, angled mirrors and
optical filters to route specified wavelengths of
light to designated detectors

Lasers
Light Amplification by Stimulated
Emission of Radiation
Flow Cytometry Analysers use air-cooled
lasers of 10-40mW
Cells travelling through the laser beam
between 1-10seconds.
Amount of light detected is dependent
upon the intensity of illumination
Hence, an intense light source is required

Optics

Optical Filters
Longpass
Allows light of a longer wavelength to pass

Shortpass
Allows light of a shorter wavelength to pass

Bandpass
Allows light of a set wavelength to pass

Dichroic
Reflects a nominated wavelength(s) of light
while allowing other wavelengths to pass

Filters and Detectors

Detectors and Optical Filters

Collection Optics - Octagon

Detectors
Forward Scatter
Photodiode
Not very sensitive
May require a neutral density filter in front

Side Scatter
photomultiplier

Fluorescence channels
photomultiplier

Photomultipliers (PMTs)
Converts light photons to electrons
Require external voltage
Have gain, more electrons out than photons
go in

Photodiodes
Work by converting photons into electrons
Photodiodes do not require external voltage
They are small solar cells

They have no gain, small current produced

has to be amplified.
Adjusting the (gain) voltage on the FSC alters
the amplifier.

Electronics
Converts optical signals to proportional
electronic signals (voltage pulses)
Analyses voltage pulse height, area, or
width
Interfaces with computer for data transfer

Electronics Signal Processors

Two types of signal processors
Analog
Digital

Electronics - Signal Processing

As a particle crosses the laser beam it generates
an electrical pulse that is sent to the signal
processing electronics
Signal processor converts each signal to a
number
Signal characteristics are determined by size,
cell velocity, beam width and fluorochrome
Pulse processing measures different aspects
from the same signal

Peak height
Area
Width
Skew

Electronics

Pulse

Particle Characteristics
Its relative size (Forward Scatter - FSC)
Its relative granularity or internal
complexity (Side Scatter SSC)
Its relative fluorescence intensity (Green,
Orange, Red)
All of these characteristics are relative to
other particles within the sample

Where does the fluorescence

come from?
Intrinsic properties of the particles that
autofluoresce: phytoplankton
Dyes that bind to, or sequester to cell
compartments: DiOC6, Indo-1
Monoclonal antibodies labelled with
fluorochromes

What is Fluorescent Light ?

430-520nm

495nm

Fluorochromes
Fluorescence occurs when a molecule relaxes
to its ground state after being electrically excited
Electrons spin around the nucleus of a molecule
at a fixed distance the ground state
If the molecule absorbs photons of light that
excite the electron it moves to a vibrational state
Collisions with other molecules cause the
excited molecule to lose vibrational energy until
it reaches the lowest excited state

Fluorochromes
At the lowest excited state electrons
release some of the energy it gained as
either vibrations or heat
This causes it to go to the ground state
and release the rest of the energy as light.
But since there is now less energy in the
light, it has a longer wavelength
(fluorescence)

Stokes Shift
Difference in the wavelength that excites the
electrons and the light that is emitted is the
Stokes shift
Some fluorochromes have a small Stokes shift
(fluorescein) and others have a large Stokes
shift (phycoerythrin)
Both Fluorescein and PE can be excited at the
same wavelength, but the difference in Stokes
shift means they can be used simultaneously
This is the basis of multicolour flow cytometery

Wavelength

Fluorochromes
488nm

Alexa Fluor
488

FITC

PE
PerCP/Cy5.5
PE-Texas
Red
PE-Cy7

633nm
APC
APC-Cy7
Alexa Fluor
647

405nm
Cascade
Blue
Alexa Fluor
430
Pacific Blue

Fluorescence

Fluorescence

Data Analysis

Data Analysis

Review

Review
Principles of Flow Cytometry and Scatter
Properties
The components of a Flow Cytometer
Basis of fluorescence
Advantages of using Flow Cytometry