FLUORESCENCE

ACTIVATED CELL SORTER

PRESENTED BY

Sudhanshu Shekhar
[Link] (Biotech) IIIrd Sem
A7110709009
• Discovery: The Fluorescence Activated Cell Sorter (FACS)
was invented in the late 1960s by Bonner, Sweet, Hulett,
Herzenberg for cell sorting of viable cells at University of
Stanford, USA.

• The term FACS was coined by Len Herzenberg who won

the Kyoto Prize in 2006 for his work in flow cytometry.

• It is based on the Coulter principle which can be applied to

count and size the various cells that make up whole blood.
 What is fluorescence……???
Fluorescence is the emission of light by a substance that has
absorbed light or other electromagnetic radiation of a
different wavelength. Emitted light has a longer wavelength, and
therefore lower energy, than the absorbed radiation.

A wide range of fluorophores can be used as labels in flow cytometry.

These each have a characteristic peak excitation and emission
wavelength. Also, the emission spectra of the labels often overlap.
Consequently, the combination of labels which can be used depends
on the wavelength of the lamp(s) or laser(s) used to excite the
fluorochromes and on the detectors available.
Introduction: Fluorescence-activated cell sorting is a specialized
type of flow cytometry in which microscopic particles such as
cells and chromosomes has been counted and examined by
suspending them in a stream of fluid and passing them by an
electronic detection apparatus. It allows
simultaneous multiparametric analysis of the physical
and/or chemical characteristics of up to thousands of particles per
second.

It is a powerful tool which enumerated lymphocytes by detecting

and counting individual cells passing in a stream through a laser
beam.
• Fluorescence-activated cell sorter provides a
method for sorting a heterogeneous mixture of
biological cells into two or more containers, one cell
at a time, based upon the specific light scattering
and fluorescent characteristics of each cell.

• It is a useful scientific instrument, as it provides

fast, objective and quantitative recording of
fluorescent signals from individual cells as well as
physical separation of cells of particular interest.
 Multiparametric analysis
• Total DNA content (cell cycle analysis, cell kinetics, proliferation etc.
• Total RNA content
• DNA copy number variation (by Flow-FISH)
• Protein expression and localization
• Protein modifications, phospho-proteins
• Cell surface antigens (CD markers)
• Intracellular antigens (cytokines, secondary mediators)
• Nuclear antigens
• Apoptosis (measurement of DNA degradation)
• Cell viability
• Membrane fluidity
• Protein expression, localization and Protein modifications
• Characterizing multidrug resistance (MDR) in cancer cells
• Principle: A beam of light, usually laser light, of a
single wavelength is directed onto a
hydrodynamically focused stream of fluid.

• The purpose of fluidics system is to transport particles

in a fluid stream for laser beam interrogation, with
only one cell or particle moving through the laser
beam at any given moment. The flow of sheath fluid
accelerates the particles and restricts them to the
center of the sample core, in a process of
hydrodynamic focusing.
• Two light beams, one in line with the light beam
(Forward Scatter or FSC) and several perpendicular
to it (Side Scatter or SSC) is equipped with and one
or more fluorescent detectors.

• Each suspended particle from 0.2 to 150 µm

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 analyzing fluctuations
in brightness at each detector (one for each fluorescent
emission peak).

• Now it is 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).
 COMPONENTS
•A flow cell - liquid stream (sheath fluid), which carries and
aligns the cells so that they pass single file through the light
beam for sensing.
•An optical system - commonly used are measurement of
impedance (or conductivity) and optical systems - lamps
(mercury, xenon); high-power water-cooled lasers (argon,
krypton, dye laser); low-power air-cooled lasers (argon;
488 nm), red-HeNe (633 nm), green-HeNe, HeCd (UV); diode
lasers (blue, green, red, violet) resulting in light signals.
• A detector and Analogue-to-Digital
Conversion (ADC) system - which generates
FSC (Forward Scatter) and SSC (Side Scatter) as
well as fluorescence signals from light into
electrical signals that can be processed by a
computer
• An amplification system - linear or
logarithmic
• A computer for analysis of the signal
 Flow Cell

• Injector
• Tip
• Sheath
• fluid

• Fluorescence
• signals

• Focused laser
• beam
Fluorescence Activated
Cell Sorting
488 nm laser
FALS Sensor

Fluorescence detector

-
Charged Plates
+

Single cells sorted

into test tubes
 Forward Angle Light Scatter

Laser

FALS Sensor
 90 Degree Light Scatter

Laser FALS Sensor

90LS Sensor
 DATA ANALYSIS
Gating: The data generated by flow-cytometers can be
plotted in a single dimension, to produce a histogram,
or in two-dimensional dot plots or even in three
dimensions.

The regions on these plots can be sequentially

separated, based on fluorescence intensity, by creating
a series of subset extractions, termed "gates." Specific
gating protocols exist for diagnostic and clinical
purposes especially in relation to hematology.
Computational analysis: Recent progress on
automated population identification using
computational methods has offered an alternative
to traditional gating strategies. Automated
identification systems could potentially help
findings of rare and hidden populations.
Representative automated methods include
FLOCK in Immunology Database and Analysis
Portal (ImmPort).
 APPLICATIONS
• It has broad application in medicine (especially in
transplantation, hematology, tumor immunology and
chemotherapy, genetics and sperm sorting for sex pre-
selection).

Sperm sorting is a means of choosing what type of sperm

cell is to fertilize the egg cell. It can be used to sort out
sperm that are most healthy. The resultant 'sex-sorted'
spermatozoa are then able to be used in conjunction
artificial insemination or in-vitro fertilization (IVF) to
produce offspring of the desired sex.
• In protein engineering: Flow cytometry is used in to
identify cell surface-displayed protein variants with
desired properties by yeast display.

• In cell trafficking: Flow cytometry is widely utilized in

lymphocyte trafficking by using intravital microscopy
(IVM). IVM is a technique by which green fluorescent
protein GFP expressed on a CD45 to see all cells with
this receptor expressed, and we could compare levels
between before and after antigen stimulation
experiments, through counting lymphocytes and
extrapolating.
• In Blood Cancers and AIDS: Flow cytometry is
routinely used in the diagnosis of health disorders,
especially blood cancers and AIDS to count the
population of lymphocytes.

• Purification of organelles: Since the  organelle  is

surrounded by each type of organelle-
specific proteins, FACS are very useful in
purifying organelles and vesicles, particularly those
that have a similar size and density.
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