Flow Cytometry
Flow Cytometry
• Cyto = Cell
• Metry = Measure
• Cytometry =measure cells
Two major techniques
FACS – fluorescence assisted cell sorting
MACS – magnetic assisted cell sorting
Basic Flow Cytometers
• Flow Cytometers are
machines that measure
multiple aspects of
single cells
• The cells are examined
by lasers
• They are interrogated
as they flow by in a
stream of fluid.
Uses of Flow Cytometers
• Medical Research (especially in cancer
research and immune functions)
• Medical Diagnoses (For detecting or
monitoring diseases like HIV/AIDS or for
monitoring transplants)
• Marine Biology
• Protein Engineering
• Pathology
Measurement about cells
• Size
• Shape (Granularity)
• Makeup (Surface proteins)
• Density
Three basic parts of FC
• Optics (where the cells are analyzed by
the lasers)
• Electronics (where light is translated
into electrons and the cells are sorted)
• Computer Analysis (where the data is
analyzed using software like Flowjo)
Cytometers work by examining fluorescent
markers
• The markers are like dyes and are
added to samples before they are run
through the cytometer.
• They bind to cells and give off light
when stimulated by a laser.
• Some common ones used are “FITC”,
“TRITC”, “NHS”, and “PE”.
• One should graph the relative amounts
of these markers to distinguish cells.
Luciferin
(derived from fireflies)
Fluorescein Isothiocyanate
(FITC)
[Link]
Amount of Yellow Markers
Amount of Blue Markers
• In real-world examples, the graphs will
look a lot more complicated.
• Most cytometry samples contain
thousands of cells, not just four.
Side Scatter (OrthSc)
Each one of these dots
represents a single cell!
Forward Scatter (ForSc)
Cell Sorting
The Fluidic System
Fluidic System
• In order to do analyses
in Flow Cytometers, the
machine needs to
analyze cells
individually
• To accomplish this,
cells are run through
the machine in fast-
moving stream of fluid
(hence the name flow
cytometry)
• This process is called
“Hydrodynamic
Focusing”
[Link]
• The sample fluid stream is directed
through the laser by the sheath fluid
• The sample fluid is always a higher
pressure than the sheath fluid
• The relative pressure in the sample fluid
controls the velocity of the stream as it
flows through the laser beam, or
interrogation point
[Link]
• A beam of laser of a single wavelength
is directed onto a hydrodynamically-
focused stream of liquid.
• A number of detectors are aimed at the
point where the stream passes through
the light beam.
• One in line with the light beam (Forward
Scatter or FSC) and many perpendicular
to it (Side Scatter or SSC) and one or
more fluorescence detectors
• Each suspended particle from 0.2 to 150
micrometers passing through the beam
scatters the ray, and fluorescent chemicals
found in the particle or attached to the
particle may be excited into emitting light at
a longer wavelength than the light source.
This combination of scattered and
fluorescent light is picked up by the
detectors, and by analysing fluctuations in
brightness at each detector (one for each
fluorescent emission peak), it is then
possible to derive various types of
information about the physical and chemical
structure of each individual particle.
• FSC correlates with the cell volume and
SSC depends on the inner complexity of
the particle (i.e., shape of the nucleus,
the amount and type of cytoplasmic
granules or the membrane roughness).
• This is because the light is scattered off
of the internal components of the cell.
• Flow cytometers form images of each
cell's fluorescence, scattered light, and
transmitted light.
Lasers for exitation
• Light Amplification by Stimulated
Emission of Radiation
• Lasers are almost always a coherent
light source (they have uniform
wavelength)
• Usually, multiple lasers are used in Flow
Cytometers to analyze cells
[Link]
Excitation
• Multiple Lasers are used
in Flow Cytometers to
excite the cells
• Fluorescent markers
absorb and reemit
different wavelengths
• Different types of cells
scatter different colored
light, this helps identify
what kind of cells they are
[Link]
Light Sorting
• Lenses collect emitted light and filters route
specific wavelengths into detectors
• It is important to keep this as exact as
possible to avoid overlapping colors
[Link]
Electrostatic Flow Sorting
• After the cells are
interrogated by the laser,
vibrations separate the
sample stream into
droplets containing either
one or zero cells, called
the “break-off point”
[Link]
• At the point at which the
stream breaks into droplets,
it passes through an
electrically charged ring
which charge cells based on
the results detected by the
cytometer’s laser and
detector system
• The cells then pass by
charged plates which sort
the cells based on the
charge that they have been
given
[Link]
Electronics System
So what happens after the cells
are interrogated by the laser and
sorted by type?
What does the electronics system
do?
• Converts the reflected
light into electrons
• Converts analog
signals to digital
• Performs
compensation
• Transfers data to the
computer
[Link]
The electronics system takes the
data from collection to computer
[Link]
Photomultipliers and
Photodiodes
• These are the
two types of
detectors that
convert
photons into
electrical
signals
• They control
sensitivity by
adjusting
voltage
[Link]
Analyzing Flow Cytometry
Data
Where does the data come from?
•Cell samples are collected for use in the
flow cytometer. Cells come from non-solid
sources, like blood.
•Sensors pick up the light emitted or
reflected by each particle as the laser hits
them.
•The sensors transmit the data to a
computer where it can be analyzed.
Flow Cytometry data can show medical
researches whether new cures are having
an affect, whether a person has AIDS or
not, whether a person is rejecting a
transplanted organ, and many other things
in other fields as well
Software to analyze the data
•By using software called FlowJo
•FlowJo allows to analyze raw data from
a flow cytometer graphically and
numerically.
Good Information Resources
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Image Resources
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