introduction to flow cytometry - … cells in suspension flow in single-file through an illuminated...
TRANSCRIPT
![Page 1: INTRODUCTION TO FLOW CYTOMETRY - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/1.jpg)
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 - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/2.jpg)
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 - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/3.jpg)
Cellular Parameters
Lymphocytes Basophils
Monocytes
Eosinophils
Neutrophils
Granulocytes
![Page 4: INTRODUCTION TO FLOW CYTOMETRY - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/4.jpg)
Cellular Parameters
![Page 5: INTRODUCTION TO FLOW CYTOMETRY - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/5.jpg)
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 - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/6.jpg)
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 - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/7.jpg)
Flow Cytometer
![Page 8: INTRODUCTION TO FLOW CYTOMETRY - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/8.jpg)
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 - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/9.jpg)
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 - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/10.jpg)
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 - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/11.jpg)
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 - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/12.jpg)
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 - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/13.jpg)
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 - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/14.jpg)
Histogram
Forward Scatter FSC
0 10 100 1000 10000
Coun
t
smaller bigger
![Page 15: INTRODUCTION TO FLOW CYTOMETRY - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/15.jpg)
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 - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/16.jpg)
Light Scattering in Whole Blood
![Page 17: INTRODUCTION TO FLOW CYTOMETRY - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/17.jpg)
Colours
• Antibodies • Fluorescent dyes • Fluorescent proteins
NEW HAT? NO FACS
ANTIBODY!
![Page 18: INTRODUCTION TO FLOW CYTOMETRY - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/18.jpg)
Fluorescence
absorbed excitation
light
emitted fluorescence
light
![Page 19: INTRODUCTION TO FLOW CYTOMETRY - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/19.jpg)
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 - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/20.jpg)
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 - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/21.jpg)
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 - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/22.jpg)
More Fluorescent Parameters…
![Page 23: INTRODUCTION TO FLOW CYTOMETRY - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/23.jpg)
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 - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/24.jpg)
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 - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/25.jpg)
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 - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/26.jpg)
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 - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/27.jpg)
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 - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/28.jpg)
N-Dimensional Plots
![Page 29: INTRODUCTION TO FLOW CYTOMETRY - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/29.jpg)
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 - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/30.jpg)
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 - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/31.jpg)
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 - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/32.jpg)
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 - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/33.jpg)
Application: Clinical phenotyping
Blood from Patient
Normal blood sample
www.med4you.at
![Page 34: INTRODUCTION TO FLOW CYTOMETRY - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/34.jpg)
Application: Clinical phenotyping
www.med4you.at
Blood from Patient
Normal blood sample
![Page 35: INTRODUCTION TO FLOW CYTOMETRY - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/35.jpg)
Application: Cell function
• Intracellular staining of cytokines, cytoskeleton, enzymes, transcription factors, signaling molecules Fixation
Permeabilization
![Page 36: INTRODUCTION TO FLOW CYTOMETRY - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/36.jpg)
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 - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/37.jpg)
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 - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/38.jpg)
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 - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/39.jpg)
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 - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/40.jpg)
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 - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/41.jpg)
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 - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/42.jpg)
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 - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/43.jpg)
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:
![Page 44: INTRODUCTION TO FLOW CYTOMETRY - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/44.jpg)
Electronics – Voltage Pulse
Laser Beam
Transmitted Light
Detector
Volta
ge
Time
![Page 45: INTRODUCTION TO FLOW CYTOMETRY - … Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence to digital values that are](https://reader033.vdocument.in/reader033/viewer/2022051802/5af624ee7f8b9a154c90b71b/html5/thumbnails/45.jpg)
Measurements of the Electronic Signal
Volta
ge
Time
Area A
High H
Width W