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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