Overview
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What is flow cytometry?
Development of flow cytometry
Components of Flow
Typical applications
Flow data
Flow Cytomtery
 Measurement (cytometry) of single cells in suspension that
pass by (flow) a laser beam
 Not appropriate for analysis of cell clumps or tissues
 Discrete measurements from each cell in the sample,
providing a distribution rather than an average of the
measured characteristics in the cell sample
 Simultaneous measurement of multiple parameters
 Size (volume)
Light scatter signals
 Granularity (internal complexity)
 Fluorescence
Derived from fluorescent labels
Basic Outline of a Flow Cytometer
Fluidics
Optics
Electronics
Commercial History
 First commercial particle analyzer: Model A Coulter counter (1950)
 First commercial fluorescence analyzer: Partec (1969)
 First commercial cytometer, the Cytograph – the Cytofluorograph –
Kamentsky in 1970
 First commercial cell sorter: Becton & Dickinson FACS-1 (1974,
tradename) Hertzenberg
 Epics series 1977-79 by Coulter
 First benchtop analyzers about 1981
 3 Colors available 1985 and 4 colors in 1986
 First Benchtop Sorters 1992
 First commercial high-speed cell sorter: Cytomation MoFlo (1994)
Advantages of Flow Cytometry
 Flexibility of the data acquisition
 Speed of measurement
 Thousands of cells can be analyzed in seconds
 Statistical information immediately available
 Ability to reanalyze with new gates gives us new
information from old acquisitions
Measurements in Flow Cytometry

Light Scatter
 All objects passing through a laser beam in a cytometer will
scatter light
 Large objects will scatter more light in the forward direction
than small objects
 Forward Angle Light Scatter (FSC), roughly indicates size
•Forward light scatter, FALS , FS, FSC
 Side Scatter near 90° (SSC), structure dependent - “reflective”
qualities, or granularity of a particle
•SS, SSC, 90° light scatter
 Actual laser spot is obscured and the light at 2° - 20° off the
straight laser line is what is measured
Measurements in Flow Cytometry
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Fluorescence
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Excitation light energy is absorbed by fluorescent
molecule, and molecule is “excited”
As excited molecule returns to unexcited ground-state,
a specific wavelength is emitted.
Fluorescence emission is always of a longer wavelength
(lower energy ) than the excitation wavelength.
 The longer the wavelength the lower the energy
 The shorter the wavelength the higher the energy
e.g.. UV light from sun causes the sunburn not the
red visible light
Emission Accomplished!
 Fluorophore Excitation /
Absorbance
 Wavelength dependent
 Fluorophore Emission /
Fluorescence
 The light given off
or emitted is at a longer
wavelength – but lower
energy
Jablonski diagram illustrating the processes involved in the creation of an excited
electronic singlet state by optical absorption and subsequent emission of fluorescence.
Visible Light Region of the
Electromagnetic Spectrum
Human eyeis can
“see”
380nm-680nm
Spectrum
often
shown
this way
Selected Laser Lines
488 514
300 nm
400 nm
500 nm
600 nm
700 nm
Dyes
PI
Ethidium
PE
FITC
(FITC)
Where is Fluorescence in Flow
Cytometry Coming from?
 Intrinsic fluorescence
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Genuine feature of the cell
“autofluorescence”
tryptophan, tyrosine, pigment content, hemoglobin,
green fluorescent protein (GFP) - transfection assays
 static
 Extrinsic fluorescence
 Experimentally added to the cell
 Fluorescent probes/dyes - FITC, PE, PI, etc
 Static
 Kinetic
Common Applications
 Immunophenotyping
 Made possible with the advent of Monoclonal antibodies
 Large majority of the uses of flow
 Determination of cell surface antigens and after permeabilization for
intracellular stains
 Clinically important for disease prognosis and diagnosis
 The number of subsets of cells that can be recognized is growing
yearly.
 DNA quantification
 Intercalating dyes like propidium iodide (red fluorescent)
 Functional assays
 Calcium probes, probes for oxidative burst (DHR), membranes ,
phagocytosis assays, and many more
Monoclonal
Antibodies
 Immunization
 Isolation of B-cells
 Fusion with
metabolically
deficient myeloma
cell
 Selection
 Cloning by limited
dilution
Example: Lymphocyte Typing
Following the Sample
From the sample tube
Through the aspiration rod
Through the tubing inside the instrument
Through the flow Cell
Intersecting the laser
Down the stream
Into Waste
or Sort collection tubes
Following the
Cytometer signal path
diff amps
log amps
Stream
amplified
signals
PMT’s
lens
Laser
Cell
pulses
linear amps
PD
Cytometer
Trigger
signal
signal processing
computer
sort module
Slide Courtesy of Joe Trotter, Director, Flow Cytometry Facility The Scripps Research Institute
Histogram
IgM
IgD
Statistics
 What types of statistics are we
interested in??
 Percentages of populations
 How bright those are – indicates how
MUCH antigen is present
 Do those change?
 Is there a reaction to a stimulus?
Example MICR 304 S2008
052108.019
1024
052108.020
4
10
768
3
FL2-H
SSC-H
10
512
2
10
256
1
10
0
0
256
512
768
1024
0
10
0
10
FSC-H
Overlay #
FCS Filename
Gate
# of Events
X Geometric
Mean
Overlay #
FCS Filename
1
2
10
10
FL1-H
Gate
3
10
# of Events
4
10
X Geometric
Mean
1
052108.019
None
3828
185.34
1
052108.020
None
2819
312.62
1
052108.019
Gate 1
2552
307.66
1
052108.020
Gate 1
258
350.99
1
052108.019
Gate 2
793
500.66
1
052108.019
Gate 3
48
568.17
1
052108.019
Gate 4
793
333.94
TUTORIAL
 http://www.invitrogen.com/site/us/en/home/support/Tu
torials.html
Acknowledgement
 This lecture has been drawn from a Dakocytomation training
PowerPoint presentation
 Credit to Andrew Beernink (abeernink@novasite.com); Susan
DeMaggio MS BSMT(ASCP)Qcym (flocyte@cox.net)