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Basic Principles in Flow
Cytometry
Prepared by Hector Nolla
Manager CRL Flow Cytometry Lab
University of California, Berkeley
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Flow Cytometry
Flow Cytometry is the technological process
that allows for the individual measurements
of cell fluorescence and light scattering.
This process is performed at rates ofthousands of cells per second.
This information can be used to individually
sort or separate subpopulations of cells.
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History Flow cytometry developed from microscopy. Thus
Leeuwenhoek is often cited in any discussion regardingits history.
F.T. Gucker (1947)build the first apparatus for detectingbacteria in a LAMINAR SHEATH stream of air.
L. Kamentsky (IBM Labs), and M. Fulwyler (Los AlamosNat. Lab.) experimented with fluidic switching andelectrostatic cell sorters respectively. Both described cellsorters in 1965.
M. Fulwyler utilized Pulse Height Analyzers toaccumulate distributions from a Coulter counter. Thisfeature allowed him to apply statistical analysis tosamples analyzed by flow.
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History
In 1972 L. Herzenberg (Stanford Univ.), developed a cell
sorter that separated cells stained with fluorescent
antibodies.The Herzenberg group coined the term
Fluorescence Activated Cell Sorter (FACS).
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Fluorescence Activation Process
(orImmunofluorescence)
FITCFITC
FITC
FITC
Antibodies recognize specific
molecules in the surface of
some cells
But not others
When the cells are analyzed by flow
cytometry the cells expressing the marker
for which the antibody is specific will
manifest fluorescence. Cells who lack the
marker will not manifest fluorescence
Antibodies are artificiallyconjugated to fluorochromes
Antibodies
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CellularParameters Measured by Flow
No reagents or probes
required (Structural)
Cell size(Forward Light
Scatter) Cytoplasmic grabularity(90
degree Light Scatter)
Photsynthetic pigments
Reagents are required. Structural
DNA content
DNA base ratios RNA content
Functional
Surface and intracellularreceptors.
DNA synthesis
DNA degradation(apoptosis)
Cytoplasmic Ca++
Gene expression
Intrinsic Extrinsic
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Flow Cytometry Applications
Immunofluorescence
Cell Cycle Kinetics
Cell Kinetics
Genetics
Molecular Biology
Animal Husbandry (and Human as well)
Microbiology
Biological Oceanography
Parasitology
Bioterrorism
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Flow cytometry integrates electronics,
fluidics, computer, optics, software, and
laser technologies in a single platform.
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Laser optics
Laser Beam
Flowchamber
Sheath
Sample
Y
X
Z
Y Z
X
Cells are presented
to the laser using
principles of
hydrodynamicfocusing
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PE FL
FITC FL
488nm Sct
Laminar Fluidic Sheath
Core
Sheath
Outer
Sheath
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Each cell generates a quanta of fluorescence
PE FL FITC FL 488nm Sct
Confocal LensDichroic Lenses
PhotomultiplierTubes
(PMTs)
Discriminating
Filters
Forward
Light
Scattering
Detector
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Negativecellsarealsodetected
PE FL FITC FL 488nm Sct
Confocal Lens
Dichroic LensesForward
Light
Scatter
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FlowCell
LaserBeam
FSSensor
Fluorescence
Pickup Lens
SSSensor
FL1Sensor525BP
FL2
Sensor575BP
FL3Sensor620BP
FL4Sensor
675BP
488DL
488BK
550DL
600DL
645DL
Optical Bench
Schematic
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From Fluorescence to Computer Display
Individual cell fluorescence quanta is picked up by thevarious detectors(PMTs).
PMTs convert light into electrical pulses.
These electrical signals are amplified and digitized using
Analog to Digital Converters (ADCs). Each event is designated a channel number (based on
the fluorescence intensity as originally detected by the
PMTs) on a 1 Parameter Histogram or 2 Parameter
Histogram.
All events are individually correlated for all the
parameters collected.
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Light Scattering, 2 Parameter Histogram
Forward Light Scatter (FLS)
90 degree
Light Scatter
Bigger
More
Granular
Live Cells
Bigger
CellsDead
Cells
Apoptotic
Cells
X Axis
Y Axis
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1 ParameterHistogram
1 2 3 4 6 7 150 160170 .. 190
Channel Number
Positive
Negative
BrighterDimmerCount
1
4
6
Fluorescence picked up from the FITC
PMT
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2 ParameterHistogram
FITC FL
PE FL
Negative
Population
Single Positive
FITC
Population
Single
Positive PIPopulation
Double PositivePopulation
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Gatingand Statistics
Data generated in flow cytometry is displayed using
MultiparamaterAcquisitionandDisplay software
platforms.
Histograms corresponding to each of the parameters ofinterest can be analyzed using statistical tools to
calculate percentage of cells manifesting specific
fluorescence, and fluorescence intensity.
This information can be used to look at fluorescenceexpression within subpopulations of cells in a sample
(gating).
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Flow Cytometry Data
SmallerRegion,
Live cells
mostly
Larger Region
includes all cells
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Running Samples
Prepare samples.
One sample should be completely negative. This sample
should be analyzed first. This sample is used for
adjusting the PMTs amplification voltage. Adjust the PMT Voltage until you can see a population
peak in the first decade of your 1 parameter and or your
two parameter plot.These samples are used for adjusting
Spectral Overlap.
Once the instrument settings are optimized, run samples
and collect data.
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UltrasonicTransducer
488nm Formard Light Scatter Detector
Collimated Light Path ThroughDichroic and Band Pass Filters
SS FL2FL1
FL4
FL3
Pulse Height
(0-10Volts)
Time(useconds)
Pressurized
1X
PBS(Sheath)
Pressurized Cell
Sample
Analog Data
PMTs
FlowCytometryandsorting
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