The Basics of Flow Cytometry

Janine Bögli, Biozentrum, 11. June 2020C ore

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The Basics of Flow Cytometry

Janine Bögli, Biozentrum, 11. June 2020C ore

F ACS

F acility

The functions of theFACS Core Facility

Centralization of equipment and expertise

Sorter operationAdvice and

troubleshootingTrain users

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Why is Flow Cytometry important?… and what it is used for• 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

• Analyze thousands of cells in a short time

• Statistical information obtained quickly• Flexibility of data acquisition• Ability to re-analyze

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Publications citing 'flow cytometry'

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The use of flow in research has boomed since the mid-1980s

Why we use it…

Plus manyothers!

Overview

• What is Flow Cytometry• Fluorescence• The basics of a flow cytometer

• Fluidics• Optics• Electronics

• Data Display• How does flow cytometry data look like• Analysis and Gating

• Applications

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What is Flow Cytometry?

Cytometry

“kytos” Greek: Hollow/cell

Measurement

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The measurement canbe substrate-based…

What is Flow Cytometry?Flow

Cytometry

“kytos” Greek: Hollow/cell

Measurement

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…or flow-based.

What can Flow Cytometry do?

Analyze light signals to:• Enumerate particles in suspension• Evaluate 105 to 106 particles in less than 1 min• Detection of rare cell populations• Measure multiple parameters• Sort single particles for subsequent analysis

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Fluorescence

• Intrinsic fluorescence• Inheritent molecules within the cell

• Autofluorescence

• Extrinsic fluorescence• Added to the cells by investigators

• Includes dyes and fluorescent proteins

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What is Fluorescence?

• Excitation (Absorbance) with one color• Emission (Fluorescence) with a different color• Fluorophore: the part of the molecule that is fluorescent• Fluorochrome: the whole molecule (can have several

fluorophores)11

absorbedexcitationlight

emittedfluorescencelight

Jablonski DiagramFITC EmissionFITC Excitation

Fluorochromes

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Pacific Blue FITC PE APC

Make use of spectral viewers:https://www.thermofisher.com/us/en/home/life-science/cell-analysis/labeling-chemistry/fluorescence-spectraviewer.htmlhttp://www.bdbiosciences.com/sg/research/multicolor/spectrum_viewer/index.jsphttps://www.fpbase.org/

Fluorescent Probes

Cyan FPGreen FPYellow FPOrange FPRed FPmCherrymTomato

FITCPhycoerythrinAllophycocyaninPerCPAlexaFluor dyesPE-Cy5, PE-Cy7BV, BUV

DAPIHoechst dyesPropidium iodideAcridine OrangeTO-PRO-3DyeCycle dyesSYTOX dyes

Antibodies Fluorescent dyes Fluorescent proteins

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80’s to late 90’s

1-2 Lasers 3-4 Detectors

low dimensional data

Early 2010’s

3-5 Lasers 6-18 Detectors

higher dimensional data

Early 2000’s

1-3 Lasers 3-8 Detectors

mid dimensional data

Late 2000’s

2-4 Lasers 3-16 Detectors

mid-high dimensional data

Beyond 2015

4-9 Lasers 12-25+ Detectors

very high dimensional data

1980 20201990 2000 2010

What is inside the Flow Cytometer?

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The Many Parts of Flow

Basic components:Fluidics Optics Electronics

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Fluidics – Schematic Overview

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Sample injectionprobe (SIT)

Interrogation point

Flow cell

waste aspirator

Interior reservoir

Sample tube

Sample Flow

Sheath Flow

Sheath filter

Sheath tank

Illumination volumeIntercept

Laser intersection point

Fluidics – Hydrodynamic Focusing

Adapted from Purdue University Cytometry Laboratories

Sheathfluid

Injector Tip

sample

Focused Laser Beam

Forward ScatterSignal

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Fluidics – Laminar Flow • Sheath and sample fluids stream go in parallel through the

flow cell • The sample flows in the very center of the sheath• Sample and sheath fluids don’t mix

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We need to keep a smooth laminar flow!

no airno aggregates

Fluidics – Sample Differential

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The sample pressure determines the flow rate…

Low Medium High

Fluidics – Sample Differential

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…and thus the core diameter.

Low Medium High

Increased differential pressure will increase the sample flow rate, but will also increase the incidence of multiple cells

passing through the laser at the same time.

Fluidics – There is a Speed Limit

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With a higher flow rate we…

• …increase coincidences• …get no single-cell analysis• …get sub-optimal data• …lose data

BAD To increase aquisitionrates you have toconcentrate your

sample

Optics - Lasers

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• Laser light is coherent and generally of a single wavelength• Flow cytometers can have a single or up to 7 lasers (or more)

wikipedia

UV (325, 355, 375nm)

Violet (405nm)

Blue (488nm)

Green (532nm)

Yellow (561nm)

Red (633nm)

Optics – Laser Arrangement

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Optics – Light Scattering

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• Light scattering activity depends on:• cells optical density, it’s roughness and to some extend

to its size• Scattering is dependent on the primary laser light

Optics – Optical Arrangement

coherent light source (488nm)

flow cell

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sample stream

forward scatterdetector

Light scatters in all directions!

obscuration bar

Forward Scatter(FSC,FALS,FS)

• ~2-20° of the laserintercept

• Based on MieScatter

• Scatterproportional tothe square ofthe diameter ofthe cell

• Based on ‘sphericalparticles

Optics – Optical Arrangement

coherent light source (488nm)

flow cell

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sample stream

forward scatterdetector

obscuration bar

Side Scatter(SSC, SS, orthogonal scatter)

• orthogonal scattering (90°)

• darkfield• complexity and

granularity

side scattercollection lens

Optics – Optical Arrangement

coherent light source (488nm)

flow cell

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sample stream

forward scatterdetector

obscuration bar

Fluorescenceemission

• Collected with thesame lens as theSSC (15-150°)

• Intrinsic or extrinsicfluorescence

collection lens forSSC and fluorescence

Optics – Fluorescence

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488nm

FITC

Optics – Capture Fluorescence

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488nm

FITC

Optics – Capture Fluorescence

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488nm

GFP

Optics – Capture Fluorescence

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FITC /GFP

Stokes shift

Optics – Capture Fluorescence

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PE

Stokes shift

Optics – Capture Fluorescence

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488nm

FITC PE

Optics – Capture Fluorescence

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488nm

FITC PE

Optics – Optical Filters• Filters transmit light of a specific wavelength while reflecting

other wavelengths• There are three types of dichroic filters:

• Shortpass (SP) filters• Longpass (LP) filters• Bandpass (BP) filters

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Optics - Filter PropertiesLongpass filters transmit wavelengths above a cut-on wavelength

Bandpass filters transmit wavelengths in a narrow range around a specified wavelength

550LP

Emission light

> 550nmEmission light

510/20BP

500-520nm

LP

Emission light

550SP

< 550nm

Shortpass filters transmit wavelengths below a certain wavelength

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SP BP

Optics - Dichroic Mirror

With a specific angle the filter can be used as a dichroic mirror

Transmitted Light

Light Source

Filter placed at 45o

Reflected light

Detector E Detector D

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PEFITC

Optics –Instrument Configuration

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Know yourinstrument

configuration whenyou select for

fluorescent probes.

• Excitation spectrum: will determine whether we can use that fluorochrome

• Emission spectrum: will tell us which filter to use and whether we can combine the fluorochrome with others.

Detector E Detector D

PEFITC

Electronics - Overview

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PMT

VGain

• photons are filtered, collected, and multiplied• The current generated is converted to a voltage pulse• The voltage pulse is digitized• The values are stored

N photons in

e-e-

e-e-

e-

Photocathode Anode

Current Out

e-e-

e-

e-e-

e-

e-e-

e-

e-e-e-

Photoelectrons

Electronics – Light detection• Photomultipliers (PMTs) simply detect photons• Light needs to be optically filtered before• Photon energy is converted into a signal that is dependent

on:• Number of photons• Voltage applied to the PMT

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The measurement is only relative!

Controls, controls and controls!

PMT voltage setup is important

Electronics – Signal Conversion

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Laser

Transmitted Light Signal Photons

Detector (PMT, ADP, CCD)Electrons

Volts

Time

Area AHigh H

Width W

t2

t1

t0 t1

t3

t2 t3

t0 t1 t2 t3

t0 t1 t2 t3

Quantitative MeasureCurrent

Summary I: Channel Layout• Photon-distribution to detectors according to energy-levels

(wavelengths)• Optical elements provide separation of channels and

wavelength selection

Illumination Volume

PI, PerCP

FITC, Alexa 488, GFPPE

Flow cell

SSC

FSC

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What does a cytometer give us?

• Listmode file: correlated data file where each event is listedsequentially, parameter by parameter

• Flow cytometry standard (FCS 3.1)• Allows other software programs to recognize and analyze data• Data and header portion

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FSC SSC Time FL1 FL2 … FLnCell 1 50 20 0.001 45 2000 686

Cell 2 55 18 0.007 47 1867 600

…

Cell n 67 234 12.754 86 2134 765

PMT Voltages• Where are my negatives?

• Signals should be withinlinear range of the detector

• Other approaches exist: • Setting the voltage based

on your cells being 2.5*rSDof the electronic noise (see Trotter, Meinelt et al, BD Biosciences Tech Bulletin)

• Quality assurance forpolychromatic flowcytometry using a suite ofcalibration beads (seeStephen P Perfetto et al, Nature Protocols (2012))

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Low voltage

Mid voltage

High voltage

Data Display – Histogram

• The further to the right, the brighter the fluorescence• Good to display normal distributions• Show differences between samples/populations• Peak hight is a function of the CV (spread) and the number

of events

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Data Display – Dot Plot

Red

fluor

esce

nce

Green fluorescence

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Each event is plotted according to its value in the x and y dimensions

x

y

Q1 Q2Q3 Q4

FITC

APC

Data Display – Dot Plot

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• Bivariate plots show 2 dimensions

• They provide more graphical information and more data

CD8 (fluorescence 1 red)

CD

4 (fl

uore

scen

ce2

blu

e)

What we learn from Light Scattering

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Sid

e Sc

atte

rSSC

Forward Scatter FSCBigger

Mor

e G

ranu

lar

Live Cells

Bigger Cells or Aggregates

Dead Cells

Apoptotic Cells

Data Display - N-Dimensional Plots

• Can rotate them• For analysis only usually• Statistics are hard to determine on these

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…and 4+ parameters?

Data Display - N-Dimensional Plots

• We have to reduce the data• Dimensionality reduction and embedding methods• For this we define populations and gates

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New Tech - Spectral Flow Cytometry

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40 c

olor

s…

Gates

• A region (or region of interest ROI) can be drawn on• A histogram to define boundaries and calculate percentage of

positive• A bivariate plot to define populations and calculate percentages

• Gating should be based on some scientific basis• Proper controls are essential• Follow the density of the population

Gates• Gates can be combined and are used to isolate subsets of

data• Gates are usually hierarchical organized, like a family tree

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child grandchild

Why Gating is useful• Eliminates false positive events and cleans up your plots• Is used to isolate a subset of cells on a plot• We can use them to select a population for further study• Allows the ability to look at parameters specific to only that

subset

54From Annual Flow Cytometry course, University Zürich

Summary II - the Main Points• Keep a smooth laminar flow! Prevent air and aggregates.• To increase acquisition rates you have to concentrate your

sample.• The key to good results is good sample preparation.• Know your instrument configuration when selecting for

fluorescent probes.• Gating helps to define your cells of interest.• All measurements are relative, don’t forget the controls.• Include a viability dye and doublet

discrimination gating to eliminate false positive events.

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There is more…

Applications:• Major applicacions phenotyping and fluorescent proteins• Examples for special applications: RNAFlow & FRET

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Immunopheno-typing

DNA and RNA analysis

Cell deathFunctional analysis

Transduction / Transformation confirmation

Basic Use:

Application - Immunophenotyping

• Establish the presence or level of an antigen

• Use of labeled antibodies to identify cells of interest

• Detection of cell surfacemolecules as examplecluster of differentiation

Lymphocyte T or B????

only cells fromR1

SSC

FSCwww.med4you.at T-cell marker

B-ce

llm

arke

r57

Application - Intracellular staining

• Intracellular staining of cytokines, cytoskeleton, enzymes, transcription factors, signaling molecules

• Monitor signaling cascades

FixationPermeabilization

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Krutzik et al. 2011

Application - DNA Analysis• DNA content of individual cells informs about their ploidy • Suitable dyes: PI, 7-AAD, DAPI

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Adapted from Beckman-Coulter

From Bitesizebio

Application - Cell Death

Measurements of cell death:• Expression of proteins

involved in apoptosis• Activation of caspases• Changes in the

mitochondrial membrane potential

• Changes in the plasma membrane

• Cell shrinkage• Chromatin changes• DNA degradation

Normal HL60

apoptotic HL60

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Fluorescence Resonance EnergyTransfer Assays

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https://www.innovabiosciences.com/resources/applications/fret/

• protein-protein interactions with the help of fluorescently labeled proteins

• distance between the two proteins must be less than 10nm

• The emission spectrum of the donor fluorophore must overlap the absorption spectrum of the acceptor fluorophore

Application - RNA Flow Assay

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Gene-specific oligonucleotide probe set and branched DNA technology:

• Compare RNA and protein kinetics in the same cell• Parallel analysis of microRNA targets in combination with

antibody staining• Detect target-specific RNA for which flow cytometry

antibodies are nonexistent

Application - RNA Flow Assay

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Gene Expression

• Genes well characterized and can be cloned in frame with gene of interest

• Can be used to monitor rates of gene expression

• Commonly used as marker of transfection

• Level of intensity can be variable

• Great for cell sorting applications

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green fluorescence

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ores

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Application - Cell Sorting• Separating cells based on

properties measured in flow is also called Fluorescence-Activated Cell Sorting (FACS)

• High-speed cell sorting is based on droplet deflection

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Summary III

isac-net.orgwww.cyto.purdue.eduflowbook-wiki.denovosoftware.comlink.springer.com/content/pdf/10.1007/978-1-4939-7346-0.pdfwww.biozentrum.unibas.ch/research/groups-platforms/overview/unit/fcf/

janine.boegli@unibas.chReso

urce

s:

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• Basic principle of every conventional flow cytometer• Quantitative measurements of single cells• High dimensionality and Multiplexing• Diverse application range

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Controls in Flow Cytometry

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• Instrument specific controls• Fluorescence controls

• Compensation controls• Autofluorescence controls

• Experiment specific controls• Staining controls

• Non-specific binding controls• FMO controls• Secondary only controls

• Reference controls• Biological controls

Biological variabilitygreatest source of variation

Choose well or Compensate

Violet Blue Yellow/Green RedHoechst FITC PE APC-Cy6DAPI GFP PE tamdems APCCFP Alexa 488 PI Alexa 647Pacific Blue PerCP RFP TO-ProBrilliant Violet PerCP tandems mCherry Alexa 700

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• Spectral overlap is inherent in multicolor experiments• This will have impact on experimental design and detection

sensitivity• BUT we can do something about it

• Combine fluorochromes that do not overlap• Run the right controls and compensate

Compensation (1)

What we want…

Source: Tim Bushnell

Compensation (2)

…and what we get

Source: Tim Bushnell

Spillover

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100 22 …1 100 …… … 100

FITC

PEFITC

PE

How much FITC spills into others.

…

…How much fromothers spill into

FITC.

FITCdetector

PE detector

FITC signal

PE signal

Compensation (3)

• «Compensation»• Intra-laser compensation -

more intuitive aspect• Inter-laser compensation -

less intuitive but extremely important to consider, especially with tandems

• Conceptually very simple procedure

• Introduce a linear „correction coefficient“ for all parameters that receive the spill-over

• Look at a control samples stained with a single dye, ‘single stained controls’

Source: Tim Bushnell/BD