Flow Cytometry Basic Training

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Flow Cytometry Basic Training

• A Look Inside the Box

Ryan DugganSr. Research TechnologistFlow
Cytometry Facility University of Chicago
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Section I

• Background Information on Flow Cytometry

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Purpose of the Courses(Who Should Attend?)

• Introduce the research community to the
  progressively useful tool of flow cytometry.
• Provide the tools necessary to use flow cytometry
  properly.
• To get people to think creatively about new
  applications using flow in their research.
• Everyone who uses flow should attend these
  courses.

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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
Sorting Basics
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What Is Flow Cytometry?

• Everyone has done some form of cytometry
• Cyto cell
• Metry measure
• Add some flow cells in motion
• Measuring properties of cells in flow

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Cytometry vs. Flow Cytometry

• Cytometry
• 1st look at cells under a microscope-late 1600s
• Localization of antigen is possible
• Poor enumeration of cell subtypes
• Limiting number of simultaneous measurements
• Cost for basic fluorescence microscope 25,000

• Flow Cytometry.
• 1st proposal to count cells while in flow-1934.
• Cannot tell you where antigen is.
• Can analyze many cells in a short time frame.
• Can look at numerous parameters at once.
• Cost for basic flow cytometer 100,000.00

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

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Background Info Summary

• Flow Cytometry is a relatively new technology
• Has continually increased in popularity since the
  mid 1980s.
• Gives us the ability to analyze many properties
  of many cells in little time
• Instrumentation can be expensive

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Section II

• The 4 Main Components of a Flow Cytometer

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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.

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What Happens in a Flow Cytometer (Simplified)
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The Fluidics SystemCells 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)

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Fluidics Schematic
Sample Tube
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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.

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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
Original from Purdue University Cytometry
Laboratories, Modified by R. Duggan
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Sample Differential

• 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 CVs and increase in multiple
  cells passing through the laser at once.
  No more single cell analysis!

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Common Types of Flow Cell

• Quartz cuvette
• Good optical properties can be used for sorting
• Quartz cuvette with optical gel interface
• Excellent optical properties
• Jet-in-air
• Best for sorting, inferior optical properties

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Quartz Cuvette

• Laser interrogates cells inside the quartz flow
  cell
• Light emitted from the cells passes through the
  quartz and then to the collection lens
• Minimal light is lost due to air impedance.
• Laser light is better focused

Original from Purdue University Cytometry
Laboratories, Modified by R. Duggan
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Quartz Flow Cell with Optical Gel

• Laser interrogates cell inside the quartz.
• Emitted light passes through quartz and optical
  gel interface
• Less light is lost due to air
• Laser light is better focused

Original from Purdue University Cytometry
Laboratories, Modified by R. Duggan
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Jet-in-air Flow Cell

• Laser interrogates cell outside the flow cell
• Emitted light passes through air towards the
  collection lens
• Some fluorescence is lost
• Flow cell is free to vibrate and make droplets,
  sorting is optimal

Original from Purdue University Cytometry
Laboratories, Modified by R. Duggan
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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.
• Some flow cells allow for greater optical
  sensitivity, while others are best for sorting

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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.

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Interrogation

• Light source needs to be focused on the same
  point where cells were focused.
• Two types of light sources
• Lasers
• Arc-lamps

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LasersLight 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 having waves with similar direction,
amplitude, and phase that are capable of
exhibiting interference
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Arc Lamps

• Arc lamps provide a mixture of wavelengths that
  must be filtered to select the desired excitation
  wavelength
• Provide milliwatts of light
• Gives non-uniform, incoherent light

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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 called a channel
  to a detector.

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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 is proportional to
  cell size and membrane integrity
• Forward ScatterFSCFALSLALS

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Forward Scatter
Original from Purdue University Cytometry
Laboratories, Modified by R. Duggan
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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. granulated nuclei, cell inclusions, etc.)
• Side ScatterSSCRALS90 degree Scatter

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Side Scatter
Original from Purdue University Cytometry
Laboratories, Modified by R. Duggan
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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

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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
• Those photons pass through the collection lens
  and are split and steered down specific channels
  with the use of filters.

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Fluorescence Detectors
Original from Purdue University Cytometry
Laboratories, Modified by R. Duggan
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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

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Long Pass Filters

• Transmit all wavelengths greater than specified
  wavelength
• Example 500LP will transmit all wavelengths
  greater than 500nm

Original from Cytomation Training Manual,
Modified by R. Duggan
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Short Pass Filter

• Transmits all wavelengths less than specified
  wavelength
• Example 600SP will transmit all wavelengths
  less than 600nm.

Original from Cytomation Training Manual,
Modified by R. Duggan
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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)

Original from Cytomation Training Manual,
Modified by R. Duggan
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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.

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Optical Bench Layout

• To observe scatter and multiple fluorescence
  simultaneously from each cell, you need multiple
  channels
• The design of a multi-channel layout must
  consider
• Spectral Properties of the fluorochromes used
• The appropriate positioning of filters

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Spectra of Common Fluorochromes
Laser Lines (nm)
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 R. Duggan
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Example Channel Layout
Detector4
Dichroic Mirrors
Detector3
Bandpass Filters
Detector2
Detector1
Original from Purdue University Cytometry
Laboratories, Modified by R. Duggan
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Channel Layout Model
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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 registers a signal from only 1
  fluorochrome

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Compensation-Practical Eg.
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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.

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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.

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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

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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.

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Electronics

• Detectors basically collect photons of light
• The electronics must process that light signal
• The job of the electronics is to convert an
  analog light signal detected by the photo
  detector into a digitized value that can be used
  by a computer

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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
• The detector can be made more or less sensitive
  depending on how much voltage is applied.

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PMT Sensitivity
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Electronics Schematic
Detector Photons converted to ? no. of electrons
Linear Amplification
Voltage
Current out ? Photons in
Voltage
or
Log Amplification
Time
PMT Voltage Input 150V-999V
Original from Becton Dickinson Training manual,
Modified by R. Duggan
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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
• The threshold can be set on any parameter, but is
  usually set on FSC

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

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The Pulse
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Measurements of the Pulse
Voltage Intensity
Time
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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.

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Analog to Digital Converters

• An ADCs takes the voltage pulse and converts it
  to discrete binary numbers depending on total
  resolution
• ADCs are either 8-bit (having 256 bin resolution)
  or 10-bit (having 1024 bin 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.

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Analog to Digital Conversion
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List Mode File
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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

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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.

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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

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Creation of a Histogram
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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

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Plots
Contour Plot
Density Plot
Dot Plot
Greyscale Density
www.treestar.com
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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

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Gating Example
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PE detector
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PE detector
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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

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Everythings Relative

• The relative bin numbers are just thatrelative.
• 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.

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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.

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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/

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Flow Lab Contact Info

• Ryan Duggan, Sr. Research Technologist
• rcduggan_at_midway.uchicago.edu
• 702-9212
• James Marvin, Research Technologist
• jmarvin_at_flowcity.bsd.uchicago.edu
• 702-9212
• Location
• Main Lab Kovler Room 037
• Satellite Facility Knapp Center Room 107, BSLC
• Satellite Facility Billings Hospital Room S-301
• Website http//iacf.bsd.uchicago.edu/index.htm