Principles of Flow Cytometry: 1 Dr. Prabesh Kumar Choudhary NAMS, Bir Hospital Principles of Flow Cytometry Quartz nozzle Fluorescence signals Focalized laser beam Injection of cells History: 2 History Flow cytometry (FC) is the measurement of numerous cell properties (cytometry) as the cells move in the single file (flow) in a fluid column and interupt a beam of laser light. 1930; Casperson & Schultz: nucleic acid measurement of the cell Coons & Kaplan: conjugation of fluorescein to antibody PowerPoint Presentation: 3 Flow cytometry integrates fluidics, laser technologies, optics, electronics, computer , and software in a single platform. Cellular Parameters Measured by Flow Cytometry: 4 Cellular Parameters Measured by Flow Cytometry No reagents or probes required ( Structural ) Cell size (Forward Light Scatter) Cytoplasmic granularity (Side Scatter) Photosynthetic pigments Reagents are required. Structural DNA content DNA base ratios RNA content Functional Immunophenotyping . DNA synthesis DNA degradation (apoptosis) Cytoplasmic Ca++ Gene expression Intrinsic Extrinsic Flow Cytometry Applications: 5 Flow Cytometry Applications Immunophenotyping Cell Cycle Kinetics Cell Kinetics Genetics Molecular Biology Animal Husbandry (and Human as well) Microbiology Biological Oceanography Parasitology Bioterrorism PowerPoint Presentation: 6 PowerPoint Presentation: 7 The 4 Main Components of a Flow Cytometer What Happens in a Flow Cytometer?: 8 What Happens in a Flow Cytometer? Cells in suspension flow in single file Focused laser where the cell scatter light and emit fluorescence that is filtered and collected then converted to digitized values that are stored in a file Which can then be read by specialized software. Interrogation Fluidics Electronics Interpretation Fluidic System: 9 Fluidic System In order to do analyses in Flow Cytometers, the machine needs to analyze cells individually To accomplish this, cells are run through the machine in fast-moving stream of fluid (hence the name flow cytometry): Single file This process is called “Hydrodynamic Focusing” http://www.bdbiosciences.com Fluidics Schematic: 10 Fluidics Schematic Sheath Tank Waste Tank Line Pressure Vacuum Sample Pressure (Variable) Sheath Pressure (Constant) Sample Tube Fluidic System: 11 Fluidic System Velocity of the core stream: sheath pressure Diameter of the core stream: sample delivery rate Resolution: CV> Relative SD of signal Sensitivity: product of intensity of excitation> time spent by the cell> flow velocity ; Shape of the beam Resolution: function of diameter Sensitivity: velocity>sheath pressure PowerPoint Presentation: 12 The sample fluid pressure is always higher than the sheath fluid The relative pressure in the sample fluid controls the velocity of the stream as it flows through the laser beam, or interrogation point High sheath pressure > increased velocity> decreased sensitivity High sample delivery rate >increase diameter > poor resolution http://www.bdbiosciences.com PowerPoint Presentation: 13 Incoming Laser Sample Sheath Sheath Sheath Sample Sample Core Stream Low Differential High Differential Laser Focal Point Sample Differential: 14 Sample Differential 10 psi 10.2 psi 10 psi 10.4 psi 10 psi 10.8 psi Difference in pressure between sample and sheath This will control sample volume flow rate The greater the differential, the wider the sample core. If differential is too large, cells will no longer line up single file Results in wider CV’s and increase in multiple cells passing through the laser at once. No more single cell analysis! PowerPoint Presentation: 15 Low pressure High pressure Fluidics Recap: 16 Fluidics Recap Purpose is to have cells flow one-by-one past a light source. Cells move out of tube because there is slightly greater pressure on the sample than on the sheath Cells are “focused” due to hydrodynamic focusing and laminar flow. What Happens in a Flow Cytometer?: 17 What Happens in a Flow Cytometer? Cells in suspension flow in single file Focused laser where the cell scatter light and emit fluorescence that is filtered and collected then converted to digitized values that are stored in a file Which can then be read by specialized software. Interrogation Fluidics Electronics Interpretation Light Source and Light Beam: 18 Light Source and Light Beam Light Amplification by Stimulated Emission of Radiation Argon laser (488 nm) Krypton laser (407 nm) Dye lasers (595 nm) Diameter of the beam: 650um> narrowed by two cylindrical lenses Vertical lens> longer vertical dimension> increases the sensitivity; Immunophenotyping Horizontal lens> longer horizontal dimension> increases the resolution; DNA quantification Light Scatter: 19 Light Scatter When light from a laser interrogates a cell, that cell scatters light in all directions. The scattered light can travel from the interrogation point down a path to a detector. Forward scatter (FS) Side scatter (SS) Forward Scatter: 20 Forward Scatter Light scattered in the forward direction (along the same axis the laser is traveling) is detected in the Forward Scatter Channel (FSC) . The intensity of this signal has been attributed to cell size , refractive index (membrane permeability) Forward Scatter: 21 Forward Scatter FSC Detector Laser Beam Original from Purdue University Cytometry Laboratories Side Scatter: 22 Side Scatter Laser light that is scattered at 90 degrees to the axis of the laser path is detected in the Side Scatter Channel (SSC) . The intensity of this signal is proportional to the amount of cytosolic structure in the cell (eg. granules , cell inclusions, etc.) Side Scatter: 23 Side Scatter FSC Detector Collection Lens SSC Detector Laser Beam Original from Purdue University Cytometry Laboratories Why Look at FSC v. SSC: 24 Why Look at FSC v. SSC Since FSC ~ size and SSC ~ internal structure, a correlated measurement between them can allow for differentiation of cell types in a heterogenous cell population FSC SSC Lymphocytes Monocytes Granulocytes RBCs, Debris, Dead Cells Fluorescence Channels: 25 Fluorescence Channels As the laser interrogates the cell, fluorochromes on/in the cell ( intrinsic or extrinsic ) may absorb some of the light and become excited As these fluorochromes leave their excited state, they release energy in the form of a photon with a specific wavelength, longer than the excitation wavelength>> stoke shift The photons pass through the collection lens and are split and steered down specific channels with the use of filters. Fluorescence Detectors: 26 Fluorescence Detectors FSC Detector Collection Lens Laser Beam Fluorescence Detector A, B, C, etc… Original from Purdue University Cytometry Laboratories, Modified by James Marvin Filters: 27 Filters Many wavelengths of light will be scattered from a cell, we need a way to split the light into its specific wavelengths in order to detect them independently. This is done with filters Optical filters are designed such that they absorb or reflect some wavelengths of light, while transmitting other. 3 types of filters Long Pass filter Short Pass filter Band Pass filter Long Pass Filters: 28 Long Pass Filters Transmit all wavelengths greater than specified wavelength Example: 500LP will transmit all wavelengths greater than 500nm 400nm 500nm 600nm 700nm Transmittance Original from Cytomation Training Manual, Modified by James Marvin Short Pass Filter: 29 Short Pass Filter Transmits all wavelengths less than specified wavelength Example: 600SP will transmit all wavelengths less than 600nm. 400nm 500nm 600nm 700nm Transmittance Original from Cytomation Training Manual, Modified by James Marvin Band Pass Filter: 30 Band Pass Filter Transmits a specific band of wavelengths Example: 550/20BP Filter will transmit wavelengths of light between 540nm and 560nm (550/20 = 550+/-10, not 550+/-20) 400nm 500nm 600nm 700nm Transmittance Original from Cytomation Training Manual, Modified by James Marvin Dichroic Filters: 31 Dichroic Filters Can be a long pass or short pass filter Filter is placed at a 45 º angle to the incident light Part of the light is reflected at 90º to the incident light, and part of the light is transmitted and continues on. Dichroic Filter Detector 1 Detector 2 PowerPoint Presentation: 32 Abdcerotec.com Optical Bench Layout: 33 Optical Bench Layout To separate scatter and multiple fluorescence wavelengths simultaneously from each cell The design of a multi-channel layout must consider Spectral Properties of the fluorochromes used The appropriate positioning of filters Spectra of Common Fluorochromes: 34 Spectra of Common Fluorochromes Laser Lines (nm) 350 457 488 514 610 632 300 400 500 600 700 PE-Texas Red Texas Red PI Ethidium PE FITC cis-Paranaric Acid Original from Purdue University Cytometry Laboratories, Modified by James Marvin Detectors: 35 Detectors There are two main types of photo detectors used in flow cytometry Photo-diodes Used for strong signals, when saturation is a potential problem (eg. FSC detector ) Photomultiplier tubes (PMT) More sensitive than a Photodiode, a PMT is used for detecting small amounts of fluorescence emitted from fluorochromes. Photodiodes and PMTs: 36 Photodiodes and PMTs Photo Detectors usually have a band pass filter in front of them to only allow a specific band width of light to reach it Therefore, each detector has a range of light it can detect, once a filter has been placed in front of it. Interrogation Recap: 37 Interrogation Recap A focused light source (laser) interrogates a cell and scatters light That scattered light travels down a channel to a detector FSC ~ size and cell membrane shape SSC ~ internal cytosolic structure Fluorochromes on/in the cell will become excited by the laser and emit photons These photons travel down channels and are steered and split by dichroic (LP/SP) filters Specific wavelengths are then detected by PMTs that have a BP filter in front of them What Happens in a Flow Cytometer?: 38 What Happens in a Flow Cytometer? Cells in suspension flow single file past a focused laser where they scatter light and emit fluorescence that is collected, filtered and converted to digitized values that are stored in a file Which can then be read by specialized software. Interrogation Fluidics Electronics Interpretation Electronics: 39 Electronics Detectors basically collect photons of light and convert them to current The electronics must process that light signal and convert the current to a digitized value that the computer can graph What Happens in the PMT: 40 What Happens in the PMT A voltage is applied to the detector which makes electrons available for the photons to “pick up” As the number of photons increase, more and more electrons are “picked up” yielding a greater current output from the detector Also, as the voltage applied to the detector increases the same amount of photons will have a greater current output Electronics Schematic; Photons in> Voltage out: 41 Electronics Schematic; Photons in> Voltage out Detector PMT Voltage Input 150V-999V Current out Photons in Linear Amplification Log Amplification or Voltage Time Voltage Original from Becton Dickinson Training manual, Modified by James Marvin Photons Threshold: 42 Threshold When the laser interrogates an object, light is scattered. If the amount of light scattered surpasses a threshold, then the electronics opens a set window of time for signal detection The threshold can be set on any parameter, but is usually set on FSC Threshold: 43 Threshold FSC Detector FSC Detector Time Time Threshold (eg. 52) Threshold (eg. 52) The Voltage Pulse: 44 The Voltage Pulse As the cell passes through the laser, more and more light is scattered until the cell is in the center of the laser (maxima) As the cell leaves the laser, less and less light is scattered After a set amount of time, the window closes until another object scatters enough light to be triggered. The Pulse: 45 The Pulse Time Photons/Detector (V) Measurements of the Pulse: 46 Measurements of the Pulse Pulse Height Pulse Width Pulse Area Time Voltage Intensity PowerPoint Presentation: 47 (Volts) 0 10 (Volts) Relative Brightness Channel Number 6.21 volts 1.23 volts 3.54 volts 10 1 .1 .01 .001 10 100 1000 10,000 1 256 196 64 0 128 (1mV) Pg 255 Linear and Log Amplifiers: 48 Linear and Log Amplifiers The current exiting the detector passes through either a linear or log amplifier where it is converted into a voltage pulse. The use of a log amp is beneficial when there is a broad range of fluorescence as this can then be compressed; this is generally true of most biological distributions. Linear amplification is used when there is not such a broad range of signals e.g. in DNA analysis and calcium flux measurement. Analog to Digital Converters: 49 Analog to Digital Converters An ADCs takes the voltage pulse and converts it to discrete binary numbers depending on total resolution The binary signal generated is converted to a relative bin number Those relative bin numbers are acquired as a list of values from each detector for each event (cell) and are eventually plotted on a graph. Analog to Digital Conversion: 50 228 Relative bin number Analog to Digital Conversion Time Voltage Intensity ADC 11001100 8-bit binary code 204 Relative bin number 2 7 + 2 6 + 2 5 + 2 4 + 2 3 + 2 2 + 2 1 + 2 0 ADC 0011100100 10-bit binary code 2 9 + 2 8 + 2 7 + 2 6 + 2 5 + 2 4 + 2 3 + 2 2 + 2 1 + 2 0 List Mode File: 51 List Mode File Event # Param 1 FS Param2 SSC Param 3 FITC Param 4 PE Param 5 APC 1 100 500 10 650 4 2 110 505 700 700 6 3 90 480 720 670 10 4 95 490 15 720 15 Electronics Recap: 52 Electronics Recap The varying number of photons reaching the detector are converted to a proportional number of electrons The number of electrons exiting a PMT can be multiplied by making more electrons available to the detector (increase Voltage input) The current generated goes to a log or linear amplifier where it is amplified (if desired) and is converted to a voltage pulse The voltage pulse goes to the ADC to be digitized The values are placed into a List Mode File What Happens in a Flow Cytometer?: 53 What Happens in a Flow Cytometer? Cells in suspension flow single file past a focused laser where they scatter light and emit fluorescence that is collected, filtered and converted to digitized values that are stored in a file Which can then be read by specialized software. Interrogation Fluidics Electronics Interpretation Interpretation: 54 Interpretation Once the values for each parameter are in a list mode file, specialized software can graphically represent it. The data can be displayed in 1, 2, or 3 dimensional format Common programs include… Cell Quest Flowjo WinMDI FCS Express Creation of a Histogram: 55 Creation of a Histogram Event # Param 1 FSC Param2 SSC Param 3 FITC Param 4 PE Param 5 APC 1 100 500 10 650 4 2 110 505 700 700 6 3 90 480 720 670 10 4 95 490 15 720 15 0………10………100………1000…….10000 Types of Plots: 56 Types of Plots Single Color Histogram Fluorescence intensity (FI) versus count Two Color Dot Plot FI of parameter 1 versus FI of Parameter 2 Two Color Contour Plot FI of P1 versus FI of P2. Concentric rings form around populations. The more dense the population, the closer the rings are to each other Two Color Density Plot FI of P1 versus FI of P2. Areas of higher density will have a different color than other areas Plots: 57 Plots Contour Plot Density Plot Greyscale Density Dot Plot www.treestar.com Gating: 58 Gating Is used to isolate a subset of cells on a plot Allows the ability to look at parameters specific to only that subset Can use multiple gates Important Points on Analysis: 59 Important Points on Analysis 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 param1 and param2 What kind of statistics are available CV Median Anything you can do with a list of numbers What Happens in a Flow Cytometer?: 60 What Happens in a Flow Cytometer? Cells in suspension flow single file past a focused laser where they scatter light and emit fluorescence that is collected, filtered and converted to digitized values that are stored in a file Which can then be read by specialized software. Interrogation Fluidics Electronics Interpretation PowerPoint Presentation: 61 Thanks for your Attention!!