Flow Cytometry Basics

James MarvinDirector, Flow Cytometry Core FacilityUniversity of Utah Health Sciences CenterOffice 801-585-7382Lab 801-581-8641jmarvin@cores.utah.edu

New Instrumentation

iCyte/Sony Eclipse-4 lasers-5 color detection-Electronic volume

BD FacsCanto-4 lasers-8 color detection

Seventeen-colour flow cytometry: unravelling the immune systemNature Reviews Immunology, 2004

“This ain’t your grandma’s flow cytometer”

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# of plots1136101521283645

Flow CytometryApplications

Immunophenotyping DNA cell cycle/tumor ploidy Membrane potential Ion flux Cell viability Intracellular protein staining pH changes Cell tracking and proliferation Sorting Redox state Chromatin structure Total protein Lipids Surface charge Membrane fusion/runover Enzyme activity Oxidative metabolism Sulfhydryl groups/glutathione DNA synthesis DNA degradation Gene expression Phagocytosis Microparticle analysis

The uses of flow in research has boomed since the mid-1980s, and is now the gold standard for a variety of applications

Section I

Background Information on Flow Cytometry

Experimental Design “One on One”

Instrumentation“Flow Basics”

Analysis“Data Analysis”

Presentation“Data Analysis”

• Sample Procurement• Sample preparation• Fix/Perm• Which Fluorophore• Controls• Isotype?• Single color• FMO

• Appropriate Lasers• Appropriate Filters• Instrument Settings• Lin vs Log• Time• A, W, H• Doublet

discrimination

• Interpretation• Mean, Median• % +• CV• SD• Signal/Noise• Gating

• Histogram• Dot Plot• Density Plot• Overlay• Bar Graph

Many components to a successful assay

What Is Flow Cytometry?

Flow ~ motionCyto ~ cellMetry ~ measure

Measuring both intrinsic and extrinsic properties of cells while in a moving fluid stream

Cytometry vs. Flow CytometryCytometry/Microscopy

Localization of antigen is possible

Poor enumeration of cell subtypes

Limiting number of simultaneous measurements

Flow Cytometry. No ability to determine

localization (traditional flow cytometer)

Can analyze many cells in a short time frame. (30k/sec)

Can look at numerous parameters at once (>20 parameters)

Section II

The 4 Main Components of a Flow Cytometer

What Happens in a Flow Cytometer?

Cells in suspension flow single filethrough a focused laser where they scatter

light and emit fluorescence that is filtered, measured, then converted to digitized

values that are stored in a file which can then be analyzed and interpreted

within specialized software.

Interrogation

Fluidics

Electronics

Interpretation

The Fluidics System“Cells in suspension flow single file”

Cells must flow one-by-one into the cytometer to do single cell analysis

Accomplished through a pressurized laminar flow system.

The sample is injected into a sheath fluid as it passes through a small orifice (50um-300um)

Sheath and Core

Sheath

Core

Hydrodynamic Focusing

V. Kachel, H. Fellner-Feldegg & E. Menke - MLM Chapt. 3

Notice how the ink is focused into a tight stream as it is drawn into the tube under laminar flow conditions.

PBS/SheathSample/cells/core

Laminar flowHydrodynamicFocusing

Laminar flow occurs when a fluid flows in parallel layers, with no disruption between the layers

Particle Orientation and Deformationa: Native human erythrocytes near the margin of the core stream of a short tube (orifice). The cells are uniformly oriented and elongated by the hydrodynamic forces of the inlet flow.

b: In the turbulent flow near the tube wall, the cells are deformed and disoriented in a very individual way. v>3 m/s.

V. Kachel, et al. - MLM Chapt. 3

What Happens in a Flow Cytometer (Simplified)

Cell flash.swf

Flow Cell- the place where hydrodynamically focused cells are delivered to the focused light source

Incoming Laser

Sample

Sheath Sheath

SampleCore

Stream

Low Differential High Differential or “turbulent flow”

Laser Focal Point

Gaussian- A “bell curved” normal distribution where the values and shape falls off quickly as you move away from central, most maximum point.

Low pressure

High pressure

FL30 1024 2048 3072 4096

Cou

nt

300280

260240

220200180

160140

120100

80

6040

200

68.70 19.16 9.56

G0/G1 CV= 2.42

G2/M

S phaseG0/G1

FL3

0 1024 2048 3072 4096

Cou

nt340320300280260240220200180160140120100

80604020

0

74.85 9.12 15.84

GO/G1 CV= 7.79

74.85 9.12 15.84

GO/G1 CV= 7.79

Fluidics Recap

Purpose is to have cells flow one-by-one past a light source.

Cells are “focused” due to hydrodynamic focusing and laminar flow.

Turbulent flow, caused by clogs or fluidic instability can cause imprecise data

What Happens in a Flow Cytometer?

Cells in suspension flow single filethrough a focused laser where they scatter

light and emit fluorescence that is filtered, measured, and converted to digitized values

that are stored in a file Which can then be read by specialized

software.

Interrogation

Fluidics

Electronics

Interpretation

InterrogationLight source needs to be focused on the

same point where cells are focused.

Light source 99%=Lasers

LasersLight amplification by stimulated emission of radiation

Lasers provide a single wavelength of light (monochromatic)

They can provide milliwatts to watts of power Low divergence Provide coherent light Gas, dye, or solid state

Coherent: all emmiting photons have same wavelength, phase and direction as stimulation photons

Light collection

Collected photons are the product of laser light scattering or bouncing off cells

488nm

Information associated with physical attributes of cells (size, granularity, refractive index)

Scatter FluorescenceVS Collected photons are

product of excitation with subsequent emission determined by fluorophore

350nm-800nm

Readout of intrinsic (autofluorescence) or extrinsic fluorescence (intentional cell labeling)

Forward Scatter

FSCDetector

Laser Beam

Original from Purdue University Cytometry Laboratories

.50-80

Forward Scatter

The intensity of forward scatter signal is often attributed to cell size, but is very complex and also reflects refractive index (membrane permeability), among other things

Forward Scatter=FSC=FALS=LALS FSC

Side Scatter

FSCDetector

CollectionLens

SSCDetector

Laser Beam

Original from Purdue University Cytometry Laboratories

Side Scatter

Laser light that is scattered at 90 degrees to the axis of the laser path is detected in the Side Scatter Channel

The intensity of this signal is proportional to the amount of cytosolic structure in the cell (eg. granules, cell inclusions, drug delivery nanoparticles.)

Side Scatter=SSC=RALS=90 degree Scatter

Why Look at FSC v. SSC Since FSC ~ size and SSC ~ internal structure, a

correlated measurement between them can allow for differentiation of cell types in a heterogenous cell population

FSC

SSC

Lymphocytes

Monocytes

Granulocytes

RBCs, Debris,Dead Cells

LIVE

Dead

FluorescenceEn

ergy

Absorbed exciting light

Emitted fluorescence

S0Ground State

Excited higher energy states

S1

S2

S3

•As the laser interrogates the cell, fluorochromes on/in the cell (intrinsic or extrinsic) may absorb some of the light and become excited•As those fluorochromes leave their excited state, they release energy in the form of a photon with a specific wavelength, longer than the excitation wavelength

Stokes shift- the difference in wavelength between the excitation and the emission

Optical Filters Many wavelengths of light will be emitted from a cell,

we need a way to split the light into its specific wavelengths in order to detect them independently. This is done with filters

Optical filters are designed such that they absorb or reflect some wavelengths of light, while transmitting other.

3 types of filters Long Pass filter Short Pass filter Band Pass filter

Long Pass Filters

Transmit all wavelengths greater than specified wavelength Example: 500LP will transmit all wavelengths

greater than 500nm

400nm 500nm 600nm 700nm

Tran

smitt

ance

Short Pass Filter

Transmits all wavelengths less than specified wavelength Example: 600SP will transmit all wavelengths

less than 600nm.

400nm 500nm 600nm 700nm

Tran

smitt

ance

Original from Cytomation Training Manual, Modified by James Marvin

Band Pass Filter

Transmits a specific band of wavelengths Example: 550/20BP Filter will transmit

wavelengths of light between 540nm and 560nm (550/20 = 550+/-10, not 550+/-20)

400nm 500nm 600nm 700nm

Tran

smitt

ance

Dichroic Filters

Can be a long pass or short pass filter Depending on the specs of the filter, some of the

light is reflected and part of the light is transmitted and continues on.

DichroicFilter

Detector 1

Detector 2

Spectra of Common Fluorochromes with Typical Filters

CompensationFluorochromes typically fluoresce over a

large part of the spectrum (100nm or more)A detector may “see” fluorescence from

more than 1 fluorochrome. (referred to as bleed over)

You need to “compensate” for this bleed over so that 1 detector reports signal from only 1 fluorochrome

Compensation-Practical Eg.

compensationexample.swf

Multi-laser Instruments and pinholes

Implications--Can separate completely overlapping emission profilesif originating off different lasers-Significantly reduces compensation

Spatial separation

Blue Laser Excitation

Blue and YellowLaser Excitation

585/

20 Y

ello

w

585/

20 B

lue

530/20 Blue 530/20 Blue

Interrogation Recap A focused light source (laser) interrogates a cell and

scatters light That scattered light travels down a channel to a detector FSC ~ size and cell membrane integrity SSC ~ internal cytosolic structure Fluorochromes on/in the cell will become excited by the

laser and emit photons These photons travel down channels and are steered and

split by dichroic (LP/SP) filters

What Happens in a Flow Cytometer?

Cells in suspension flow single file Through a focused laser where they scatter

light and emit fluorescence that is filtered, measured

and converted to digitized values that are stored in a file

Which can then be read by specialized software.

Interrogation

Fluidics

Electronics

Interpretation

Electronics

Detectors basically collect photons of light and convert them to an electrical current

The “electronics” must process that light signal and convert the current to a digitized value/# that the computer can graph

DetectorsThere are two main types of photo detectors

used in flow cytometry Photodiodes

Used for strong signals, when saturation is a potential problem (eg. FSC detector)

Photomultiplier tubes (PMT) Used for detecting small amounts of fluorescence

emitted from fluorochromes. Incredible Gain (amplification-up to 10million

times) Low noise

Detector names

Photoelectric Effect

Einstein- Nobel Prize 1921

pmt.swf

Photons -> Photoelectrons -> Electrons

cellflashelectronics.swf

Electric pulse generation

Measurements of the Pulse

Pulse HeightPulse Width

Pulse Area

Time

Mea

sure

d C

urre

nt a

t det

ecto

r

0

10

(Vol

ts)

Rel

ativ

e B

right

ness6.21 volts

1.23 volts

3.54 volts

101

102

103

104

1

ADCAnalog to Digital Conversion

Count

Does voltage setting matter?

Voltage=362 292 272 252 522

-Voltage doesn’t change sensitivity or laser power-All your doing is changing the amplification of the signal

-Caveat- there is large “sweet spot” of PMT voltage, outside of this range you may run the risk of non linear amplification

FSC SSC FITC PE APC APC-Cy7FCS Fileor

List Mode File

Electronics Recap Photons ElectronsVoltage pulseDigital # The varying number of photons reaching the

detector are converted to a proportional number of electrons

The number of electrons exiting a PMT can be multiplied by making more electrons available to the detector (increase Voltage input)

The current generated goes to a log or linear amplifier where it is amplified (if desired) and is converted to a voltage pulse

The voltage pulse goes to the ADC to be digitized The values are placed into a List Mode File

What Happens in a Flow Cytometer?

Cells in suspension flow single file pasta focused laser where they scatter light and

emit fluorescence that is collected, filtered and converted to digitized values that are

stored in a file Which can then be read by specialized

software.

Interrogation

Fluidics

Electronics

Interpretation

See you at Data Analysis

May 2nd 10am

AntibodyAntigen binding site

Immunophenotyping

roGFPRedox senstitive biosensor

Ca2+ Flux

Indo-1 Ca2+ freeEmission=500nm

Indo-1 Ca2+ boundEmission=395nm

Apoptosis

Live

Apoptotic

Annexin V

MTR

PI

FLIC

A

Cell cycle

Sorting

Last attached droplet