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

3. Background Information on Flow Cytometry
Section I
Background Information on Flow Cytometry

4. 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

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

7. 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.

8. 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

9. 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

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

11. 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

12. What Happens in a Flow Cytometer (Simplified)

13. 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)

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

15. 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.

16. 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

17. 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

18. 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

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

20. 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!

21. Low pressure
High pressure

22. 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.

23. 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

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

25. 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

26. 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.

27. 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

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

29. 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

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

31. 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

32. 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.

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

34. 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

35. 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

36. 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

37. 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

38. 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 .

39. 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

40. 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

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

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

44. 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

49. .

54. Compensation-Practical Eg.

55. 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.

56. 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.

57. 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

58. 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

59. 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

60. 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

61. Photoelectric Effect
Einstein- Nobel Prize 1921

63. 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

64. 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

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

66. 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

67. 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.

68. The Pulse
Time
 Photons/Detector (V)

69. 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

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

71. 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.

72. 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 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 ; 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 and linear values ; the third log decade channels and linear values ; and the fourth log decade channels and linear values 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

73. Analog to Digital Conversion
8-bit binary code
ADC
Time
Voltage Intensity
ADC
204
Relative
bin number
10-bit binary code
228
Relative
bin number

74. 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

75. 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

76. 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

77. 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

78. 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

79. 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

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

81. 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

82. Gating Example

83. Red Detector
Blue Detector

84. PE detector

85. Red Detector
Blue Detector

86. PE detector

87. 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

88. 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.

89. 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

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

91. Flow Lab Contact Info…
James Marvin, Managing Director
Jeff Nelson, Sr. Research Technologist
Paul Mehl, Research Tech
Location:
Main Lab: Olson Bldg 8505