Downloaded 2,215 times
Uploaded byRobert (Rob) Salomon
Introduction to Flow Cytometry
This document provides an introduction to flow cytometry. It defines flow cytometry as the measurement of physical and chemical characteristics of cells as they flow in a fluid stream through a beam of light. It describes the key components of a flow cytometer including fluidics to deliver cells to the laser, optics to excite and collect light, and electronics to amplify and process signals. It explains the different types of signals detected including light scatter and fluorescence, and how these can be used to characterize cells. The document provides guidance on choosing fluorochromes and considerations for multi-color panels such as spectral overlap. It outlines some common applications of flow cytometry and contact details.
More Related Content
Similar to Introduction to Flow Cytometry
Introduction to Flow Cytometry
- 1. MLC Flow CytometryFacility Introduction To Flow Cytometry Rob Salomon Garvan Institute of Medical Research Darlinghurst NSW Flow Cytometry
- 2. What Is FlowCytometry ?
- 3. What Is FlowCytometry ? Measurement METRY
- 4. What Is FlowCytometry ? Cells Measurement CYTO METRY
- 5. What Is FlowCytometry ? Flow Cells Measurement Flow CYTO METRY
- 6. What Is FlowCytometry ? Flow Cells Measurement Flow CYTO METRY Flow Cytometry
- 7. Prerequisites for Flow Cytometry 1. Cells in single cell suspension 2. Fluorescent probes 3. Cytometer The key to good result is good sample Preparation http://www.photobiology.info/Zimmer.html - from Roger Y.Tsien)
- 8. What does aFlow Cytometer do? Analyses light signals to determine: Phenotype and Function Cd3
- 9. What’s inside aFlow Cytometer ? • Flow cytometers have 3 key systems – Fluidics – Optics – Electronics
- 10. Fluidics Delivery of Low sample to laser intercept (interrogation Medium point) Legend Laser intercept High Core Stream
- 11. Optics • Allow theexcitation and the collection of the emitted light Steering LASER mirrors emission Flow Cell - Steering interrogation mirrors point
- 12. Optics cont.. Fluorescent and SSC Detectors Signal Detection FSC is achieved by detector collecting emitted or scattered light
- 13. Optics cont.. B530 Detector – FITC GFP 530/30 488/10 SSC Fluorescent and Detector 506 LP SSC signals are collected at right Emission angles to the from blue 575/26 excitation laser laser are progressively B575 Detector picked off to – PE, PI 556 LP facilitate multiple fluorochrome use
- 14. Electronics Detector or PMT Electron Cascade Digitisation and processing Amplification Voltage http://sales.hamamatsu.com /assets/applications/ETD/p mt_handbook_complete.pdf
- 15. What type ofsignals do we see with Flow ? • Scatter – Forward Scatter (FSC) • parallel or Perpendicular FSC – Side scatter (SSC) • Fluorescence – FITC , PE, APC, GFP, DAPI (plus lots lots more)
- 16. Understanding Scatter Signals •WBC discrimination FSC has some similarities to size SSC has some similarities to granularity and complexity
- 17. Fluorescent Signals • Fluorescencemay be used in the detection of : – Protein, RNA and DNA – DNA synthesis – Dye efflux – Organelle Activity A cytometer can – Change in pH detected light from – Protein interactions any system you can design that – Cell movement and division utilises – etc fluorescence
- 18. Examples of fluorescentprobe use 10 5 Fluorescence 2 (CD4) <B670L_B-A>: CD8-PerCP55 104 3 10 0 0 103 104 105 <R780_A-A>: bTCR-APCAF750 Fluorescence 1 (βTCR)
- 19. Understanding Fluoroscence The fluorescent Excited molecule is excited e- by the excitation state e- source (laser). This imparts energy to e- electrons in the e- molecule which in Resting e- then released as Mechanism of the molecule relaxes. The Fluorscence energy is released as light.
- 20. How do Ichoose my Fluorochromes ? • Antibody availability • Function – i.e. Mcherry Vs GFP • Fluorochrome brightness • Excitation source • Emission filters • Other fluorochromes/ Signals present in my sample (spectral overlap)
- 21. Fluorochrome Brightness Probe QY AF488 0.92 R-Pe 0.82 AF546 0.79 AF594 0.66 Quantum yield : APC 0.68 Is a measure of the A647 0.33 relative brightness of eGFP 0.6 the fluorochrome. IT is measured as: Azumi Green 0.74 ZS Green 1 0.91 http://en.wikipedia.org/wiki/Fluorophore
- 22.
- 23. Choosing your Fluorochromes spectral viewers http://www.bdbioscience s.com/research/multicolo r/spectrum_viewer/index. jsp http://www.invitrogen.co m/site/us/en/home/supp ort/Research- Tools/Fluorescence- SpectraViewer.htmlUse the
- 24. Choosing your Fluorochromes spectral viewers http://www.bdbioscience s.com/research/multicolo r/spectrum_viewer/index. jsp http://www.invitrogen.co m/site/us/en/home/supp ort/Research- Tools/Fluorescence- SpectraViewer.htmlUse the
- 25. Choosing your Fluorochromes spectral viewers http://www.bdbioscience s.com/research/multicolo r/spectrum_viewer/index. jsp http://www.invitrogen.co m/site/us/en/home/supp ort/Research- Tools/Fluorescence- SpectraViewer.htmlUse the
- 26. Choosing your Fluorochromes spectral viewers http://www.bdbioscience s.com/research/multicolo r/spectrum_viewer/index. jsp http://www.invitrogen.co m/site/us/en/home/supp ort/Research- Tools/Fluorescence- SpectraViewer.htmlUse the
- 27. Understanding Spectral Overlap Effect of spectral overlap - Instrument View 120% 100% Percentage of Signal 80% in Detector 60% 40% 20% Spectral overlap 0% B 530 B 585 occurs when PE 5% 87% fluorochromes FITC 95% 13% excited by the same lasers emit in similar ranges.
- 28. Compensation Signal from FITC bright Compensation Controls 120 Signal Strength 100 120% 80 60 100% 40 20 80% Axis Title 0 overlap B 530 B 585 60% FITC bright 100 13 40% overlap 20% FITC dull 0% 120 Signal Strength B 530 B 585 100 FITC 100% 13% 80 Compensation is PE 5% 100% 60 40 applied at the 20 0 single event B 530 B 585 FITC dull 50 6 level
- 29. Effect of Compensation Digital compensation doesn’t change the underlying data it just allows us to Uncompensated Data interpret it
- 30. Effect of Compensation Digital compensation doesn’t change the underlying data it just allows us to Compensated Data interpret it
- 31. How many Fluorochromescan I use ? • Most flow = 1- 3 fluorochromes • Basic phenotyping panel = 6-8 fluorochromes • Complicated panels = 11-12 flourochromes • High end = 17 fluorochromes Seventeen-colour flow cytometry: unravelling the immune system Stephen P. Perfetto, Pratip K. Chattopadhyay & Mario Roederer
- 32. Impact of increasingFlourochromes • Data get dramatically more complex Parameters 2 3 4 8 12 18 22 Populations 22 23 24 28 212 218 222 Populations 4 8 16 256 4,096 262,144 4,194,304 With 3 12 24 48 768 12,288 786,432 12,582,912 scatter populations Number of populations – assuming each fluorochromes gives rise to only a positive and negative population
- 33. Visualising Signal Data Dot Density plot plot Statistical measures are also used to Contour plot identify Histogram changes
- 34. Basics uses ofFlow Cytometry ? • Phenotyping • Apoptosis and cell death • Cell cycle, cell divising and DNA synthesis • Transduction/transfection confirmation • Cell tracking • Small particle analysis • Functional analysis – calcium flux, gene expression, dye efflux, mitochondrial activity • Marine and microorganism identification
- 35. Why use FlowCytometry ? • Rapid analysis ( 3k- 200k events/second) • Individual event analysis • Quantifiable results • Multiple parameter analysis • Statistical relevance
- 36. Flow and ImagingComparison Imaging Flow Cytometry Cells per field/sec) Approx 100 20, 000 No. of parameter <6 <24 Quantifiable Maybe (using complex Yes analysis tool) 18 bit resolution 12- 16 bit (< 65,536 (262,144 channels) channels) Ave number of 1,000 - >10, 000 analysed cells 10 field of 100 Anatomical localisation Yes no
- 37. How do Iget more ? Analysis Cell Sorting Sorting See It Sort It
- 38. Contact Details • RobSalomon – r.salomon@garvan.org.au – (02) 9295 8431 • Bookings (Nikki and David) – Flow@garvan.org.au – (02) 9295 8432 • http://linkage.garvan.unsw.edu.au/Flow/index.html