INTRODUCTION TO FLOW CYTOMETRY - Biozentrum: · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION...

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DEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility 17. October 2012

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Page 1: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

D E PA R T E M E N T B I O Z E N T R U M

INTRODUCTION TO FLOW CYTOMETRY

Janine Zankl FACS Core Facility 17. October 2012

Page 2: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

Overview or The Many Parts of Flow

• What is Flow Cytometry? • Fluidics • Light Scattering • Fluorescence • Data presentation • Interpreting the data • Applications • Sorting

Flow Basics

Motivation and Experiments

Data Analysis

Page 3: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

Cellular Parameters

Lymphocytes Basophils

Monocytes

Eosinophils

Neutrophils

Granulocytes

Page 4: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

Cellular Parameters

Page 5: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

What Is Flow Cytometry?

• Flow ~ cells in motion • Cyto ~ cell • Metry ~ measure • Measuring properties of cells while in a fluid stream

Page 6: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

History and uses of Flow Cytometry

It can be 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

0100020003000400050006000700080009000

10000

Publications citing 'flow cytometry'

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

Page 7: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

Flow Cytometer

Page 8: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

What Happens in a Flow Cytometer?

that is collected, filtered and converted

Cells in suspension

flow in single-file

through an illuminated

volume where they scatter light

and emit fluorescence

to digital values that are stored on a computer

http://flow.csc.mrc.ac.uk/?page_id=5

2. Optics

3. Electronics

1. Fluidics

Page 9: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

Fluidics – Hydrodynamic Focusing

• When conditions are right, the sample fluid flows in a central core that does not mix with the sheath fluid

• The introduction of a large volume into a small volume in such a way that it becomes “focused” along an axis is called Hydrodynamic Focusing

Sheath fluid

Injector Tip

sample

Fluorescence Signals

Adapted from Purdue University Cytometry Laboratories

Page 10: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

Light Scattering

Forward Scatter FSC small angle scattering (0.5-5°) (roughly) size and shape (blue)

FSC Intensity

coherent light source (488nm, blue)

Page 11: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

Light Scattering

Forward Scatter FSC small angle scattering (0.5-5°) roughly size and shape (blue)

FSC Intensity

coherent light source (488nm, blue)

Page 12: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

Light Scattering

coherent light source (488nm, blue)

Side Scatter SSC large angle scattering (15-150°), darkfield, complexity and granularity (blue)

SSC Intensity

Page 13: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

Light Scattering

coherent light source (488nm, blue)

Side Scatter SSC large angle scattering (15-150°), darkfield, complexity and granularity (blue)

SSC Intensity

Page 14: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

Histogram

Forward Scatter FSC

0 10 100 1000 10000

Coun

t

smaller bigger

Page 15: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

Dot Plot

Side

Sca

tter

SSC

Forward Scatter FSC

Bigger

Mor

e G

ranu

lar

Live Cells

Bigger Cells or Aggregates

Dead Cells

Apoptotic Cells

Page 16: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

Light Scattering in Whole Blood

Page 17: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

Colours

• Antibodies • Fluorescent dyes • Fluorescent proteins

NEW HAT? NO FACS

ANTIBODY!

Page 18: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

Fluorescence

absorbed excitation

light

emitted fluorescence

light

Page 19: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

Fluorescence

coherent light source (488nm)

green and blue fluorescence

Fluorescence emission lower energy than source light (green)

Fluorescence Detetor

Page 20: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

Filter Properties

Long-pass filters transmit wavelengths above a cut-on wavelength

Band-pass filters transmit wavelengths in a narrow range around a specified wavelength

550LP

Emission light > 550nm

Emission light

510/20BP

500-520nm

BP LP

Page 21: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

Fluorescence

coherent light source (488nm)

green fluorescence intensity

Fluorescence emission lower energy than source light (green)

Fluorescence Detetor

Bandpassfilter

Page 22: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

More Fluorescent Parameters…

Page 23: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

Dichroic Mirror

At an other angle the filter can be used as a dichroic filter or dichroic mirror

Transmitted Light

Light Source

Filter placed at 45o

Reflected light

Detector E Detector B

Page 24: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

Channel Layout of a laser-based Flow Cytometer

Illumination Volume

PI, PerCP

FITC, Alexa 488, GFP

PE

Flow cell

SSC

FSC

• Photon-distribution to detectors according to energy-levels (wavelengths)

• Optical elements provide separation of channels and wavelength selection

Page 25: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

Compensation

• Fluorochromes typically fluoresce over a large part of the spectrum • Overlap between the colors emitted by different fluorochromes • Mathematical compensation is used to reduce overlapping results

overlapping emmission spectra wrong positive signal

Page 26: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

Compensation

• Fluorochromes typically fluoresce over a large part of the spectrum • Overlap between the colors emitted by different fluorochromes • Mathematical compensation is used to reduce overlapping results

green fluorescence

yello

w f

luor

esce

nce

uncompensated

correctly compensated

green fluorescence

yello

w f

luor

esce

nce

Page 27: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

Microscopy vs Flow Cytometry

Microscopy • Localization of antigen is possible • Poor enumeration of cell subtypes • Limiting number of simultaneous

measurements

Flow Cytometry • Can not tell you where antigen is • Fast • Subpopulation analysis • Multiparameter analysis

Page 28: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

N-Dimensional Plots

Page 29: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

Gating

• Is used to isolate a subset of cells on a plot

• Allows the ability to look at parameters specific to only that subset

green

green red both

red

apoptotic

dead

Mixed T cell population

3% double pos. 60% double pos.

Page 30: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

Important Points on Analysis

• Visualization

• What kind of data are you looking for?

• How much fluorescence? • What percent are positive? • How much more positive is x

than y? • What is the ratio between

green and red fluorescence?

• What kind of statistics are available

• MFI (geometric or arithmetic) • %-ages • CV • …

Contour Plot Pseudo-colour Density

Greyscale Density Dot Plot

www.treestar.com

Page 31: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

Applications of Flow Cytometry

Cell function

DNA and RNA analysis

Cell death Sorting and cell isolation

Immunopheno-typing main

applications

Page 32: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

Application: Immunophenotyping

• Detection of cell surface molecules as example cluster of differentiation

Lymphocyte T or B????

only cells from R1

SSC

FSC

www.med4you.at

T-cell marker

B-ce

ll m

arke

r

Page 33: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

Application: Clinical phenotyping

Blood from Patient

Normal blood sample

www.med4you.at

Page 34: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

Application: Clinical phenotyping

www.med4you.at

Blood from Patient

Normal blood sample

Page 35: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

Application: Cell function

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

Permeabilization

Page 36: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

Application: DNA Analysis

• DNA content of individual cells gives information about their ploidy • Suitable dyes: PI, 7-AAD, DAPI • Combination with light scatter or immunofluorescence

Page 37: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

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

Time in hours Time in hours

Page 38: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

Application: Cell Expression

FSC SSC

SSC

DAPI

dead cells

Coun

t Ye

llow

Flu

ores

cenc

e In

tens

ity

Green Fluorescence Intensity

Page 39: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

Principle of Sorting

• Sorting (separating) cells based on properties measured in flow is also called Fluorescence-Activated Cell Sorting FACS

Page 40: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

Application: Sorting

green fluorescence

red

fluor

esce

nce

dead cells viable cells

analysis sort

green fluorescence

yello

w fl

uore

scen

ce culture

sort

Page 41: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

Advantages and Disadvantages

₊ Distinct cell populations defined by size and granularity ₊ Dead cells may be gated out for analysis ₊ Detection of weakly expressed surface antigens ₊ Multicolor analysis may be performed

₋ Sterility issues: antibiotics sufficient to control microbial contamination ₋ Viability of fragile cells : larger nozzle, 4°C, appropriate buffer

Page 42: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

What are the services in our facility

Analysis • BD Canto II • Introduction 1h • Booking / usage by yourself • Advice if requested

Sorting • BD AriaIII • Appointment for experiment

discussion • Check cells for fluorescence • Prepare the cells and let them sort

Page 43: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

Summary

isac-net.org www.cyto.purdue.edu

flowbook-wiki.denovosoftware.com www.bdbiosciences.com/research/multicolor/spectrum_viewer/index.jsp

www.invitrogen.com/site/us/en/home/support/Research-Tools/Fluorescence-SpectraViewer.html

Links:

[email protected]

Page 44: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

Electronics – Voltage Pulse

Laser Beam

Transmitted Light

Detector

Volta

ge

Time

Page 45: INTRODUCTION TO FLOW CYTOMETRY - Biozentrum:  · PDF fileDEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY Janine Zankl FACS Core Facility . 17. October 2012

Measurements of the Electronic Signal

Volta

ge

Time

Area A

High H

Width W