MICR 304 Introduction to Flow Cytometry

OverviewWhat is flow cytometry?Development of flow cytometryComponents of FlowTypical applicationsFlow data

Flow CytomteryMeasurement (cytometry) of single cells in suspension that

pass by (flow) a laser beamNot 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 parametersSize (volume)Granularity (internal complexity)Fluorescence

Light scatter signals

Derived from fluorescent labels

Basic Outline of a Flow Cytometer

Fluidics

Optics Electronics

Commercial HistoryFirst commercial particle analyzer: Model A Coulter counter (1950)First commercial fluorescence analyzer: Partec (1969)First commercial cytometer, the Cytograph – the Cytofluorograph –

Kamentsky in 1970First commercial cell sorter: Becton & Dickinson FACS-1 (1974,

tradename) HertzenbergEpics series 1977-79 by CoulterFirst benchtop analyzers about 19813 Colors available 1985 and 4 colors in 1986First Benchtop Sorters 1992First commercial high-speed cell sorter: Cytomation MoFlo (1994)

Advantages of Flow Cytometry

Flexibility of the data acquisitionSpeed of measurement

Thousands of cells can be analyzed in secondsStatistical information immediately available Ability to reanalyze with new gates gives us new

information from old acquisitions

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

Measurements in Flow Cytometry Fluorescence

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!

Jablonski diagram illustrating the processes involved in the creation of an excited electronic singlet state by optical absorption and subsequent emission of fluorescence.

Fluorophore Excitation / AbsorbanceWavelength dependent

Fluorophore Emission / FluorescenceThe light given off

or emitted is at a longer wavelength – but lower energy

Human eye can “see” 380nm-680nm

Visible Light Region of the Electromagnetic Spectrum

Spectrum is often shown this way

Ethidium

PE

PI

FITC

600 nm300 nm 500 nm 700 nm400 nm

514488

Selected Laser LinesDyes

(FITC)

Where is Fluorescence in Flow Cytometry Coming from?

Intrinsic fluorescence 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

Y

Monoclonal AntibodiesImmunizationIsolation of B-cellsFusion 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 flow Cell

Down the stream

Into Wasteor Sort collection tubes

Through the tubing inside the instrument

Intersecting the laser

Following the Cytometer signal path

Cytometer

lens

computer

sort module

pulses

PMT’s

Cell

diff amps

linear amps

PD

log amps

signal processing

amplifiedsignals

Slide Courtesy of Joe Trotter, Director, Flow Cytometry Facility The Scripps Research Institute

Triggersignal

Stream

Laser

Histogram

IgM

IgD

StatisticsWhat types of statistics are we

interested in??Percentages of populationsHow bright those are – indicates how

MUCH antigen is presentDo those change?Is there a reaction to a stimulus?

Example MICR 304 S2008052108.020

FL1-H

FL2

-H

100

101

102

103

104

100

101

102

103

104

Overlay # FCS Filename Gate # of Events X Geometric Mean

1 052108.020 None 2819 312.62

1 052108.020 Gate 1 258 350.99

052108.019

FSC-H

SS

C-H

0 256 512 768 10240

256

512

768

1024

Overlay # FCS Filename Gate # of Events X Geometric Mean

1 052108.019 None 3828 185.34

1 052108.019 Gate 1 2552 307.66

1 052108.019 Gate 2 793 500.66

1 052108.019 Gate 3 48 568.17

1 052108.019 Gate 4 793 333.94

TUTORIALhttp://www.invitrogen.com/site/us/en/home/support/

Tutorials.html

AcknowledgementThis 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)