Presentation on theme: "Introduction to Flow Cytometry IGC Workshop General Programme TimeTutorialRoomSpeaker Day 1 9h30-10h45Fundamentals of Flow CytometryIoniansRui Gardner."— Presentation transcript:
Introduction to Flow Cytometry IGC Workshop General Programme TimeTutorialRoomSpeaker Day 1 9h30-10h45Fundamentals of Flow CytometryIoniansRui Gardner 11h00-13h00 Module 1 – Group 1 Module 2 – Group 2 Analyzer room(Hands-on) Day 2 9h30-10h45Multicolor AnalysisIoniansRui Gardner 11h00-13h00 Module 1 – Group 3 Module 2 – Group 1 Analyzer room(Hands-on) Day 3 9h30-10h45Applications in Flow CytometryIoniansClaudia Bispo 11h00-13h00 Module 1 – Group 2 Module 2 – Group 3 Analyzer room(Hands-on) Day 4 9h30-11h00Cell SortingIoniansRui Gardner 11h15-13h00FlowJo Basic TrainingGiordano Bruno Module 1 – Cell Cycle Analysis using FACScan (Cláudia Bispo) Module 2 – Multicolor Analysis using CyAn ADP (Rui Gardner)
What is Flow Cytometry? Flow Cytometry uic Fundamentals of Flow Cytometry IGC – April 2, 2013 Introduction to Flow Cytometry IGC Workshop
Overview 3 Definitions: What is flow cytometry and what does it measure? Fluidics: Hydrodynamic focusing, Flow Rate... Optics: Light, fluorescence, emission and detection... Electronics: pulse, data acquisition... Analysis: Tips on Data handling
Resources 4 Books: Shapiro, H. Practical Flow Cytometry, 4ed, Online version. http://PracticalFlowCytometry.com Purdue Univ. Cytometry Labs: all you need to know about Cytometry! http://www.cyto.purdue.edu History of Flow Cytometry: Shapiro, H. (2007) Cytometry and Cytometers: Development and Growth, in Flow Cytometry with Plant Cells (Eds, J. Dolezel, J. Greilhuber, J. Suda), WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Tutorials: http://www.invitrogen.com/site/us/en/home/support/Tutorials.html
What is Flow Cytometry? 9 Cytometry: Measurement of physical and chemical properties of cells. Flow Cytometry: Characterization of cells flowing in a stream of fluid Flow Cytometry is not equivalent to FACS: Fluorescence Activated Cell Sorting What is Flow Cytometry used for? Flow cytometry can be used to count large numbers of cells or particles based on size, internal compexity, phenotype, cellular state, cell function, DNA content, gene expression, and to quantify these same cellular properties at a single-cell level.
Key Advantages of Flow Cytometry Population data Measure thousands of cells/particles per second Measure multiple parameters simultaneously. Detection of extremely rare populations Cell sorting High purity, speed, and yield. 0.02% Flow Cytometry does not... Locate where a certain component is within a cell Measure distribution of cellular components Measure cell morphology
Flow Cytometer in a nutshell 11 Cells in suspension flow in single file through… Fluidics an illuminated volume where they scatter light and emit fluorescence that is filtered, collected and… Optics converted to digital values that are processed and analyzed in a computer. Electronics
Simplified Schematics Sheath Flow Sheath Flow Sample Injected Laser Flow Chamber Hydrodynamic Focusing Flow Chamber in a BD FACSAria Cell SorterFlow Chamber in a BD LSR Fortessa Sheath Flow Sheath Flow Sample Injected Laser Flow Chamber
Flow Rate 16 More cells pass per unit time More cells pass per unit time Sheath Flow Sheath Flow Sample Flow Laser High Flow Rate Less cells pass per unit time Less cells pass per unit time Sheath Flow Sheath Flow Sample Flow Laser Low Flow Rate CyAn ADP LSR Fortessa FACSCalibur FACScan
Flow Rate 17 Low Flow Rate Laser Sheath Sample High Flow Rate Laser Sheath Sample High Power Lower Power High Power Lower Power Samples should be run at the lowest flow rate (differential pressure) as possible. If increasing flow rate has: No impact on population distribution: Use high flow rate. Changes population distribution: Use low flow rate and increase cellular concentration.
Typical jet-in air cytometer Exposure time 18 v ~ 5-30m/s Therefore exposure time ~ 10 -7 -10 -5 s Cell velocity is proportional to Sheath Pressure Laser beam height ~ 5-20 µm Typically, in sorters, sheath pressure can be reduced to increase exposure time, and therefore sensitivity and resolution of acquisition. Sheath pressure in Analyzers is usually fixed.
Excitation - Lasers 20 488 nm Most common laser wavelengths available 442
Multiple Laser Systems 21 Laboratory of Molecular Immunology Institute Molecular Genetics, Academy of Sciences, Czech Republic http://mi.img.cas.cz/images/intercepts.jpg Laser Intercepts in Flow Cell Spatially Separated Laser Beams FACSAria III (BD Biosciences) Time delay between lasers must be determined for each instrument for a given sheath pressure. Changes in Sheath Pressure will impact measurements.
Emission - Light Scatter 22 Light is reflected towards all directions Low angle: Forward Scatter (FSC) High angle: Side Scatter (SSC)
Forward Scatter (FSC) 23 The magnitude of Forward Scatter is roughly proportional to the size of the cell.
Side Scatter (SSC) 24 Side Scatter is caused by granulosity and structural complexity inside the cell.
(2D) Scatter Plot 25
Fluorescence (1) 27 Low Energy High Energy Ground State Absorbed Light Excited State Emitted Light
Fluorescence (2) Low Energy High Energy 1 2 3 1 2 3
Excitation-Emission Spectrum (2) Emission maximaExcitation maxima Each Fluorochrome has a specific Excitation and Emission Spectra
Reactive and Conjugated Probes Cascade Blue Pacific Blue Pacific Orange Alexa Dyes Lucifer yellow NBD R-Phycoerythrin (PE) PE-Cy5 conjugates PE-Cy7 conjugates PE-Texas Red PerCP TruRed PerCP-Cy5.5 conjugate Fluorescein (FITC) BODIPY-FL TRITC X-Rhodamine XRITC Lissamine Rhodamine B Texas Red Allophycocyanin (APC) APC-Cy7 conjugates Etc… Immunohistochemistry First dye-coupled antibody using fluorescein isothiocyanate (FITC) patented by Joseph Burckhalter and Robert Seiwald, in 1960 Direct methodIndirect method Pictures adapted from wikipedia
DNA and Cell Function Probes CalciumMembranepHCell TrackerDNAMitocondria Fura 2FM 1-43SNAFLECMRADAPINonyl Acridine O Indo 1FM 4-64SNARFCMTPXHOECHSTMitoSox Bapta/dyeDiOBCECFBMQCTOPRO3Mitotracker Calcium GreenANNINE 6pHRodoDMFDAPIRhodamine 123 Fluo 4Di4ANNEPS hqlysosensorCMTMRDRAQ5MitoProbe
Fluorescent Proteins (1) GFP was first noted by Shimomura et al., in the jelly fish Aequoria Victoria, in 1962 and cloned 30 years later
Fluorescent Proteins (2) San Diego beach scene drawn with living bacteria expressing 8 different colors of fluorescent proteins http://www.tsienlab.ucsd.edu/HTML/Images/IMAGE - PLATE - Beach.jpg Roger Tsien, Nobel Lectures, 2009 Tsien Lab
What is Flow Cytometry? Flow Cytometry uic Introduction to Flow Cytometry IGC Workshop Coffee Break…!