Flow Cytometry Basic Training A Look Inside the Box James Marvin Flow Cytometry Facility Northwestern University

Background Information on Flow Cytometry Section I Background Information on Flow Cytometry

The Many Parts of Flow Experimental design Sample preparation Choosing the proper instrument Setting up the instrument Collecting the proper data Interpreting the data Graphics presentation and publication Sorting Specific Applications Courses Flow Basics Data Analysis

What Is Flow Cytometry? Flow ~ cells in motion Cyto ~ cell Metry ~ measure Measuring properties of cells while in a fluid stream

Cytometry vs. Flow Cytometry Localization of antigen is possible Poor enumeration of cell subtypes Limiting number of simultaneous measurements Flow Cytometry. Cannot tell you where antigen is. Can analyze many cells in a short time frame. Can look at numerous parameters at once.

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

Background Info Summary Flow Cytometry is a quickly expanding technology Has continually increased in popularity since the mid 1980s. Gives us the ability to analyze many properties of many cells in very little time

The 4 Main Components of a Flow Cytometer Section II The 4 Main Components of a Flow Cytometer

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 filtered and collected then converted to digitized values that are stored in a file Which can then be read by specialized software. Fluidics Interrogation Electronics Interpretation

What Happens in a Flow Cytometer (Simplified)

The Fluidics System “Cells in suspension flow single file” You need to have the cells flow one-by-one into the cytometer to do single cell analysis Accomplished through a pressurized laminar flow system. The sample is injected into a sheath fluid as it passes through a small orifice (50um-300um)

Fluidics Schematic Sheath Tank Waste Line Pressure Vacuum Sample (Variable) Sheath Pressure (Constant) Sample Tube

How The Flow Cell Works The cells from the sample tube are injected into the sheath stream Flow in a flow cell is laminar. Hydrodynamic focusing pushes the cells to line up single file along their long axis. The shape of the flow cell provides the means for hydrodynamic focusing.

Fluidics Notice how the ink is focused into a tight stream as it is drawn into the tube under laminar flow conditions. Notice also how the position of the inner ink stream is influenced by the position of the ink source. V. Kachel, H. Fellner-Feldegg & E. Menke - MLM Chapt. 3

Particle Orientation and Deformation a: Native human erythrocytes near the margin of the core stream of a short tube (orifice). The cells are uniformly oriented and elongated by the hydrodynamic forces of the inlet flow. b: In the turbulent flow near the tube wall, the cells are deformed and disoriented in a very individual way. v>3 m/s. V. Kachel, et al. - MLM Chapt. 3

The Flow Cell 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 Cell Sample Stream Original from Purdue University Cytometry Laboratories, Modified by James Marvin

Low Differential High Differential Sample Sample Sheath Sheath Sheath Core Stream Laser Focal Point Incoming Laser Low Differential High Differential

Sample Differential Difference in pressure between sample and sheath 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!

Low pressure High pressure

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? Cells in suspension flow single file past a focused laser where they scatter light and emit fluorescence that is filtered, collected and converted to digitized values that are stored in a file Which can then be read by specialized software. Fluidics Interrogation Electronics Interpretation

Interrogation Light source needs to be focused on the same point where cells are focused. Light source On all flow lab instruments-Lasers

Lasers Light amplification by stimulated emission of radiation Lasers can provide a single wavelength of light (monochromatic) They can provide milliwatts to watts of power Also provide coherent light All help to create a stable and reliable signal . Coherent: all emmiting photons have same wavelength, phase and direction as stimulation photons

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 Light that is scattered in the forward direction (along the same axis the laser is traveling) is detected in the Forward Scatter Channel. The intensity of this signal has been attributed to cell size, refractive index (membrane permeability) Forward Scatter=FSC=FALS=LALS

Forward Scatter Laser Beam FSC Detector Original from Purdue University Cytometry Laboratories

Side Scatter Laser light that is scattered at 90 degrees to the axis of the laser path is detected in the Side Scatter Channel The intensity of this signal is proportional to the amount of cytosolic structure in the cell (eg. granules, cell inclusions, etc.) Side Scatter=SSC=RALS=90 degree Scatter

Side Scatter Laser Beam FSC Detector Collection Lens SSC Detector Original from Purdue University Cytometry Laboratories

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 Granulocytes Monocytes RBCs, Debris, Dead Cells

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 those fluorochromes leave their excited state, they release energy in the form of a photon with a specific wavelength, longer than the excitation wavelength Those photons pass through the collection lens and are split and steered down specific channels with the use of filters.

Fluorescence Detectors Laser Beam FSC Detector Collection Lens Fluorescence Detector A, B, C, etc… Original from Purdue University Cytometry Laboratories, Modified by James Marvin

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 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 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 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 Can be a long pass or short pass filter Filter is placed at a 45º 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 .

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 Laser Lines (nm) 350 457 488 514 610 632 PE-Texas Red Texas Red PI Ethidium PE FITC cis-Paranaric Acid 300 400 500 600 700 Original from Purdue University Cytometry Laboratories, Modified by James Marvin

Example Channel Layout Detector#4 Dichroic Mirrors Detector#3 Bandpass Filters Detector#2 Detector#1 Original from Purdue University Cytometry Laboratories

Channel Layout Model SSC PE-Cy5 FITC <505nm >605nm 505nm-555nm

Compensation Fluorochromes typically fluoresce over a large part of the spectrum (100nm or more) Depending on filter arrangement, a detector may see some fluorescence from more than 1 fluorochrome. (referred to as bleed over) You need to “compensate” for this bleed over so that 1 detector reports signal from only 1 fluorochrome

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Compensation-Practical Eg.

Detectors There are two main types of photo detectors used in flow cytometry Photodiodes 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 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 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 filter in front of them

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. Fluidics Interrogation Electronics Interpretation

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 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

Photoelectric Effect Einstein- Nobel Prize 1921

Electronics Schematic Detector Linear Amplification Current out  Photons in Voltage Voltage or Log Amplification Photons Time PMT Voltage Input 150V-999V Original from Becton Dickinson Training manual, Modified by James Marvin

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 FSC Detector Threshold (eg. 52) Time FSC Detector Threshold

Photons In ~ Voltage Out Detector Photons converted to  no. of electrons Linear Amplification Current out  Photons in Voltage Voltage or Log Amplification Time PMT Voltage Input 150V-999V Original from Becton Dickinson Training manual, Modified by James Marvin

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 Time  Photons/Detector (V)

10 256 10 10,000 1 196 1000 .1 128 (Volts) (Volts) Relative Brightness Channel Number 3.54 volts .01 64 10 1.23 volts .001 1 (1mV) Pg 255

Measurements of the Pulse Pulse Area Pulse Width Pulse Height Voltage Intensity Time

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. You can adjust the intensity of the voltage by amplifying it on a linear scale or converting it to a logarithmic scale 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 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. As we have seen, each PMT/ADC circuit divides the continuous data distribution that is acquired from cells into a discrete distribution. To do this it forms a histogram where the x axis is divided into a certain number of channels. This is either 256 or 1024 channels depending on the type of ADC used (an 8-bit ADC gives 2^8 i.e. 256 channels, a 10-bit ADC gives 2^10 i.e. 1024 channels - 10 bit ADCs are more common and will be considered in the following examples). So each data point will fall into a particular channel depending on its level of fluorescence. To get that value we need to look at the scale of the x axis. Regardless of whether data have been acquired using a linear or logarithmic amplifier, histograms can be displayed on a linear or a logarithmic scale. The scale itself does not affect the raw data - the histogram depends on the amplifier that collected the data - but will affect any derived data e.g. mode, mean, median etc. If the scale is linear the display is as channel numbers i.e. 0-1023; if it is logarithmic, it is as linear values (just to make things crystal clear!). For data that have been acquired by linear amplification, channel numbers and linear values are equivalent, but for log amplified data we can choose either channel numbers or linear values. We can relate the two: the log amps used in the flow cytometer are generally 4 decade logs so the range is 10^0-10^4 and each log decade takes up a quarter of the available channels. Therefore, the first log decade takes up channels 0 to 255 and linear values 1 to 10; the second log decade channels 256-511 and linear values 10-100; the third log decade channels 512-767 and linear values 100-1000; and the fourth log decade channels 768-1023 and linear values 1000-10000. The linear value for a particular channel number can be calculated by using: 10 channel no/256 or vice versa, a linear value can be converted to a channel number by: log(linear value) x 256

Analog to Digital Conversion 11001100 8-bit binary code ADC 27+26+25+24+23+22+21+20 Time Voltage Intensity ADC 204 Relative bin number 0011100100 10-bit binary code 29+28+27+26+25+24+23+22+21+20 228 Relative bin number

List Mode File Event # Param 1 FSC Param2 SSC Param 3 FITC Param 4 PE APC 1 100 500 10 650 4 2 110 505 700 6 3 90 480 720 670 95 490 15

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? 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. Fluidics Interrogation Electronics Interpretation

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… CellQuest Flowjo WinMDI FCS Express

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 6 3 90 480 720 670 95 490 15 0………10………100………1000…….10000

Types of Plots Single Color Histogram Two Color Dot Plot 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 www.treestar.com Contour Plot Density Plot Greyscale Density Dot Plot www.treestar.com

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 boolean logic to include or exclude multiple gates

Gating Example

Red Detector Blue Detector

PE detector

Red Detector Blue Detector

PE detector

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 MFI (geometric or arithmetic) %-ages CV Median Anything you can do with a list of numbers

Everything’s Relative The relative bin numbers are just that…relative. Saying your cells have a mean fluorescence intensity of 100 means absolutely nothing until you compare it to a negative. The fact that everything is relative allows you to compare 2, 3, or 20 samples using the same instrument settings.

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. Fluidics Interrogation Electronics Interpretation

References Numerous References available in the Flow Lab Cytometry Current Protocols in Flow Cytometry Many more reference books available Purdue University Cytometry Laboratories website: http://www.cyto.purdue.edu/ Dr. Robert Murphy, Carnegie Mellon University- Basic Theory 1 and 2 powerpoint slides The Scripps Research Institute Flow Cytometry Core Facility: http://facs.scripps.edu/

Flow Lab Contact Info… James Marvin, Managing Director J-marvin@northwestern.edu 908-1294 Jeff Nelson, Sr. Research Technologist J-nelson@northwestern.edu Paul Mehl, Research Tech P-mehl@northwestern.edu Location: Main Lab: Olson Bldg 8505