Introduction To Flow Cytometry - A Learning Guide

Introduction to
Flow Cytometry:
A Learning Guide
11-11032-03 rev. A
December 2002
Introduction to Flow Cytometry: A Learning Guide
Copyright
© 2002 Becton, Dickinson and Company. All rights reserved. No part of this publication may be reproduced,
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Table of Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
For More Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Chapter 1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Review Questions: Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Chapter 2 Fluidics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Review Questions: Fluidics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Chapter 3 Generation of Scatter and Fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . 9
3.1 Light Scatter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Review Questions: Light Scatter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
3.2 Fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Review Questions: Fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Chapter 4 Optical System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
4.1 Optical Bench . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Review Questions: Optical Bench . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
4.2 Optical Filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Review Questions: Optical Filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
4.3 Signal Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Review Questions: Signal Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
4.4 Threshold . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Review Questions: Threshold . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Chapter 5 Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
5.1 Data Collection and Display . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Review Questions: Data Collection and Display . . . . . . . . . . . . . . . . . . . . . 28
5.2 Gating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Review Questions: Gating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
5.3 Data Analysis for Subsetting Applications . . . . . . . . . . . . . . . . . . . . . . . . . 30
5.4 Data Analysis for Other Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Review Questions: Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Introduction to Flow Cytometry: A Learning Guide iii

Chapter 6 Sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
6.1 Sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Review Questions: Sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Chapter 7 Lasers and Alignment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
7.1 How Lasers Work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
7.2 Laser Alignment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Review Questions: Lasers and Alignment . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Answer Key . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Review Questions: Overview on page 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Review Questions: Fluidics on page 8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Review Questions: Light Scatter on page 12 . . . . . . . . . . . . . . . . . . . . . . . . . 48
Review Questions: Fluorescence on page 15 . . . . . . . . . . . . . . . . . . . . . . . . . 49
Review Questions: Optical Bench on page 19 . . . . . . . . . . . . . . . . . . . . . . 49
Review Questions: Optical Filters on page 21 . . . . . . . . . . . . . . . . . . . . . . . . 49
Review Questions: Signal Detection on page 24 . . . . . . . . . . . . . . . . . . . . . . 50
Review Questions: Threshold on page 25 . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Review Questions: Data Collection and Display on page 28 . . . . . . . . . . . . 50
Review Questions: Gating on page 29 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Review Questions: Data Analysis on page 36 . . . . . . . . . . . . . . . . . . . . . . . . 51
Review Questions: Sorting on page 41 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Review Questions: Lasers and Alignment on page 45 . . . . . . . . . . . . . . . . . . 52
iv Introduction to Flow Cytometry: A Learning Guide

Preface
Learning to operate a flow cytometer is best achieved by using the instrument.

However, understanding the principles underlying this technology greatly facilitates
the process.
This document contains basic information on flow cytometry. Differences between

flow cell–based benchtop cytometers (BD FACScan™, BD FACSort™,
BD FACSCalibur™, and BD LSR cytometers) and stream-in-air cytometers
(BD FACS Vantage™ and BD FACSVantage™ SE cytometers) are described in
relevant sections. Reading this material and answering the questions at the end of each
section will enhance your hands-on training experience during Operator Training at
BD Biosciences.
This assignment will take approximately 2.5 hours to complete. Please review it
before you attend the training session. An answer key is provided.
If you have any questions or problems in the US, call 1.877.232.8995, prompts 2, 2.
In Europe or Canada, contact your local application specialist.
For More Information

For more information on general flow cytometry, review the following:
• Givan AL. Flow Cytometry: First Principles. New York, NY: Wiley-Liss; 2001
(ISBN 0-471-38224-8).
• Melamed MR. Flow Cytometry and Sorting. New York, NY: Wiley-Liss; 1990
(ISBN 0-471-56235-1).
• Shapiro H. Practical Flow Cytometry. 3rd ed. New York, NY: Alan R. Liss; 1995
(ISBN 0-471-30376-3).
v
vi
1
Overview
Flow cytometry is a technology that simultaneously measures and then analyzes

multiple physical characteristics of single particles, usually cells, as they flow in a fluid
stream through a beam of light. The properties measured include a particle’s relative
size, relative granularity or internal complexity, and relative fluorescence intensity.
These characteristics are determined using an optical-to-electronic coupling system
that records how the cell or particle scatters incident laser light and emits fluorescence.
A flow cytometer is made up of three main systems: fluidics, optics, and electronics.
• The fluidics system transports particles in a stream to the laser beam for
interrogation.
• The optics system consists of lasers to illuminate the particles in the sample stream
and optical filters to direct the resulting light signals to the appropriate detectors.
• The electronics system converts the detected light signals into electronic signals
that can be processed by the computer. For some instruments equipped with a
sorting feature, the electronics system is also capable of initiating sorting decisions
to charge and deflect particles.
In the flow cytometer, particles are carried to the laser intercept in a fluid stream. Any
suspended particle or cell from 0.2–150 micrometers in size is suitable for analysis.
Cells from solid tissue must be desegregated before analysis. The portion of the fluid
stream where particles are located is called the sample core. When particles pass
through the laser intercept, they scatter laser light. Any fluorescent molecules present
1
on the particle fluoresce. The scattered and fluorescent light is collected by

appropriately positioned lenses. A combination of beam splitters and filters steers the
scattered and fluorescent light to the appropriate detectors. The detectors produce
electronic signals proportional to the optical signals striking them.
List mode data are collected on each particle or event. The characteristics or
parameters of each event are based on its light scattering and fluorescent properties.
The data are collected and stored in the computer. This data can be analyzed to
provide information about subpopulations within the sample (Figure1-1).
sample core
laser
data displays
electronic pulses
Figure1-1 Scattered and emitted light signals converted to electronic pulses
2
Chapter : Overview
Review Questions: Overview
1 What properties of a cell or particle can be measured by a flow cytometer?
2 What light source is used in most flow cytometers?
3 What are the three main systems in a flow cytometer?
4 What type of biological sample is best suited for flow cytometric analysis?
5 What is the name given to the portion of the fluid stream where the cells are
located?
6 When cells labeled with fluorescent molecules pass through the focused laser
beam, what two types of light signals are generated?
7 Light emitted from a particle is collected by

_____________________________
8 The electronic signal produced by the detectors is proportional to:
9 All flow cytometric measurements can be made simultaneously on a single cell.
T F
10 Particles must be in single-cell suspension before flow cytometric analysis.
T F
3
4
2
Fluidics
The purpose of the fluidics system is to transport particles in a fluid stream to the laser
beam for interrogation. For optimal illumination, the stream transporting the
particles should be positioned in the center of the laser beam. In addition, only one
cell or particle should move through the laser beam at a given moment.
To accomplish this, the sample is injected into a stream of sheath fluid within the flow
chamber. The flow chamber in a benchtop cytometer is called a flow cell and the flow
chamber in a stream-in-air cytometer is called a nozzle tip. The design of the flow
chamber causes the sample core to be focused in the center of the sheath fluid where
the laser beam will then interact with the particles.
Based on principles relating to laminar flow, the sample core remains separate but
coaxial within the sheath fluid. The flow of sheath fluid accelerates the particles and
restricts them to the center of the sample core. This process is known as
hydrodynamic focusing. For an illustration of hydrodynamic focusing in each type of
flow cell, see Figure 2-1 and Figure 2-2.
5
low sample high sample

pressure pressure
(12 µL/min) laser beam (60 µL/min) laser beam
sheath sheath sheath sheath

fluid sample fluid fluid sample fluid
Figure 2-1 Hydrodynamic focusing of the sample core through a flow cell
sample
sheath sheath sheath sheath
laser laser
low differential pressure high differential pressure
Figure 2-2 Hydrodynamic focusing of the sample core through a nozzle tip
The sample pressure and the sheath fluid pressure are different from each other. The
sample pressure is always greater than the sheath fluid pressure. The sample pressure
regulator controls the sample flow rate by changing the sample pressure relative to the
sheath pressure.
• In BD benchtop cytometers, the sample stream is pressurized upward through an

optically clear region of the flow cell or cuvette; particles pass through the laser
beam while they are still within this flow cell (Figure 2-1). Most benchtop
cytometers have fixed sample pressure settings (LO, MED, and HI). The
6
Chapter : Fluidics
benchtop BD LSR cytometer also has a “fine” adjustment knob for intermediate
settings.
• In stream-in-air cytometers, the sample stream passes through a small orifice in a
nozzle tip before being intersected by the light beam in the open air (Figure 2-2).
Sample pressure settings can be adjusted within a dynamic range.
Increasing the sample pressure increases the flow rate by increasing the width of the
sample core. This, in turn, allows more cells to enter the stream within a given
moment. With a wider sample core, some cells could pass through the laser beam
off-center and intercept the laser beam at a less optimal angle. However, this might be
appropriate for your application.
• A higher flow rate is generally used for qualitative measurements such as

immunophenotyping. The data are less resolved, since the cells are less in line in
the wider core stream, but are acquired more quickly.
• A lower flow rate decreases the width of the sample core and restricts the position
of the cells to a smaller area. The majority of cells passes through the center of the
laser beam; thus the light illuminating the cells and emitted from the cells is more
uniform. A lower rate is generally used in applications where greater resolution is
critical, such as DNA analysis.
Proper operation of fluidic components is critical for particles to properly intercept

the laser beam. Therefore, the operator must always ensure that the fluidics system is
free of air bubbles and debris and is properly pressurized at all times.
7
Review Questions: Fluidics
1 The purpose of the fluidics system in a flow cytometer is:
2 What two factors can affect illumination of the particles within the laser beam?
3 How many cells or particles should pass through the laser beam at a given time?
_______________________
4 The particle suspension is injected into _________________ within the

_________________.
5 The process of centering the sample core within the sheath fluid is known as:
6 Which regulator controls the diameter of the sample core?
7 What are the three possible pressure settings for a benchtop flow cytometer?
8 Increasing sample pressure ______________ the sample flow rate and the
________________ of the sample core.
9 Good data resolution is required for DNA studies. What flow rate is
recommended?
10 A wider sample core decreases resolution.
T F
11 A high flow rate can be used when performing qualitative measurements.
T F
8
3
Generation of Scatter and
Fluorescence
In the last section, we learned how particles or cells are aligned to pass single file
through the sample core. Before describing how the flow cytometer detects and
processes signals, it is useful to understand what happens to the laser light as it strikes
the single-file particles.
9
3.1 Light Scatter

Light scattering occurs when a particle deflects incident laser light. The extent to
which this occurs depends on the physical properties of a particle, namely its size and
internal complexity. Factors that affect light scattering are the cell's membrane,
nucleus, and any granular material inside the cell. Cell shape and surface topography
also contribute to the total light scatter.
Forward-scattered light (FSC) is proportional to cell-surface area or size. FSC is a

measurement of mostly diffracted light and is detected just off the axis of the incident
laser beam in the forward direction by a photodiode (Figure 3-1). FSC provides a
suitable method of detecting particles greater than a given size independent of their
fluorescence and is therefore often used in immunophenotyping to trigger signal
processing.
Side-scattered light (SSC) is proportional to cell granularity or internal complexity.

SSC is a measurement of mostly refracted and reflected light that occurs at any
interface within the cell where there is a change in refractive index (Figure 3-1). SSC is
collected at approximately 90 degrees to the laser beam by a collection lens and then
redirected by a beam splitter to the appropriate detector.
side scatter detector
forward scatter detector

light source
Figure 3-1 Light-scattering properties of a cell
10
Chapter : Generation of Scatter and Fluorescence
Correlated measurements of FSC and SSC can allow for differentiation of cell types in
a heterogeneous cell population. Major leucocyte subpopulations can be differentiated
using FSC and SSC (Figure 3-2).
neutrophils
lysed whole blood
monocytes
lymphocytes
Figure 3-2 Cell subpopulations based on FSC vs SSC
11
Review Questions: Light Scatter
1 When does light scattering occur?
2 Which key cell components contribute to light scatter?
3 Light scattered in the same direction as the laser beam is called:
4 FSC is proportional to:

____________________________________________
5 Light scatter collected at 90 degrees to the laser beam is called:
6 SSC is proportional to the ___________ or _____________ of the cell.
7 Correlated measurements of both _______________ and _____________ can

allow differentiation of cells types in a heterogeneous cell population.
12
3.2 Fluorescence
A fluorescent compound absorbs light energy over a range of wavelengths that is
characteristic for that compound. This absorption of light causes an electron in the
fluorescent compound to be raised to a higher energy level. The excited electron
quickly decays to its ground state, emitting the excess energy as a photon of light. This
transition of energy is called fluorescence.
The range over which a fluorescent compound can be excited is termed its absorption
spectrum. As more energy is consumed in absorption transitions than is emitted in
fluorescent transitions, emitted wavelengths will be longer than those absorbed. The
range of emitted wavelengths for a particular compound is termed its emission
spectrum.
The argon ion laser is commonly used in flow cytometry because the 488-nm light
that it emits excites more than one fluorochrome. (See Chapter 7 for more
information about lasers.) One of these fluorochromes is fluorescein isothiocyanate
(FITC). In the absorption spectrum of FITC (Figure 3-3 on page 14), the 488-nm
line is close to the FITC absorption maximum. Excitation with this wavelength will
result in a high FITC emission. If the fluorochrome were excited by another
wavelength within its absorption spectrum, light emission of the same spectrum
would occur but it would not be of the same intensity.
More than one fluorochrome can be used simultaneously if each is excited at 488 nm
and if the peak emission wavelengths are not extremely close to each other. The
combination of FITC and phycoerythrin (PE) satisfies these criteria. The emission
spectrum of each of these fluorochromes is shown in Figure 3-4 on page 14. Although
the absorption maximum of PE is not at 488 nm, the fluorochrome is excited enough
at this wavelength to provide adequate fluorescence emission for detection. More
important, the peak emission wavelength is 520 nm for FITC and 575 nm for PE.
These peak emission wavelengths are far enough apart so that each signal can be
detected by a separate detector. The amount of fluorescent signal detected is
proportional to the number of fluorochrome molecules on the particle.
13
FITC = fluorescein isothiocyanate

PE = phycoerythrin
PerCP = peridinin chlorophyll protein
APC = allophycocyanin
Figure 3-3 Absorption spectra of four common fluorochromes
Figure 3-4 Emission spectra of the fluorochromes shown in Figure 3-3
When a fluorescent dye is conjugated to a monoclonal antibody, it can be used to

identify a particular cell type based on the individual antigenic markers of the cell
(Figure 3-5 on page 15). In a mixed population of cells, different fluorochromes can
be used to distinguish separate subpopulations. The staining pattern of each
subpopulation, combined with FSC and SSC data, can be used to identify which cells
are present in a sample and to count their relative percentages. The cells can also be
sorted if required.
14
fluorochrome-labeled antigenic
antibodies surface marker
Figure 3-5 Specific binding of fluorochrome-labeled antibodies to cell surface antigens
Review Questions: Fluorescence
1 When fluorescent compounds absorb light energy and then release excess energy,
they emit ____________________.
2 Characteristic wavelength ranges at which fluorescent compounds can be excited

are called _________________________________________.
3 The longer wavelengths of light emitted by a fluorochrome make up its

_______________________.
4 Which laser is most commonly used in flow cytometry? _________________
5 The FITC and PE fluorochromes are excited by this emission wavelength of an

argon-ion laser: _________________
6 Two fluorescent dyes commonly used in flow cytometry are ___________ and
____________.
7 Fluorochrome-labeled antibodies are used to detect

______________________.
15
16
4
Optical System
The optical system consists of excitation optics and collection optics. The excitation
optics consist of the laser and lenses that are used to shape and focus the laser beam.
The collections optics consist of a collection lens to collect light emitted from the
particle–laser beam interaction and a system of optical mirrors and filters to route
specified wavelengths of the collected light to designated optical detectors. The design
of the optical bench allows for these functions to occur.
4.1 Optical Bench

The optical bench in a flow cytometer provides a stable surface that holds the light
source and the excitation and collection optics in fixed positions. The alignment of a
benchtop analyzer is very stable because the flow cell is fixed in its alignment with the
laser beam. This ensures that the laser intercepts the sample stream consistently from
day to day. The optical bench for two benchtop cytometers is shown in Figure 4-1 and
Figure 4-2 on page 18.
In a stream-in-air cytometer, the laser beam is focused through an achromatic lens to

intercept the fluid stream at an optimal angle and position after the stream exits the
nozzle. Because of variations in focusing and in the position of the stream, the
alignment of a stream-in-air cytometer is less stable than that of a benchtop analyzer
and needs to be optimized on a daily basis. Misalignment of the laser beam with the
stream can result in uneven illumination of the particles and increased variability in
the signals emitted. This results in decreased precision. The optical bench for a
stream-in-air cytometer is shown in Figure 4-3 on page 19.
17
Figure 4-1 Optical bench diagram of the BD FACSCalibur™ benchtop flow cytometer
575/25
530/28
SSC
FL1 555 LP 488/25
FL5
FL6
510 LP
380 LP
620 SP 660/13
470 LP
670 LP
510/20 670 LP
FL4
488 nm
laser FL3
fluorescence
collection lens
633 nm 488 pass/
laser 633 reflect
325 nm FSC
laser flow cell
UV pass/
488 & 633 reflect focusing lens
Figure 4-2 Optical bench diagram of the BD LSR benchtop flow cytometer
18
Chapter : Optical System
detector
option 5 FL3 PMT
(laser #1) (laser #1)
FL1 PMT
(laser #1)
dichroic
filter mirror
filter
dichroic detector
mirror option 1
filter dichroic relay lens
mirror dichroic
mirror
lens filter intercept #2
filter 3 beam FL4, 5, 6 (option)
iris splitter
lens
FL2 PMT lens
(laser #1) iris filter
detector
dichroic option 2 FSC photodiode
mirror iris
lens
filter filter (488/10)
beam splitter lens
detector filter detector iris 45˚
option 3 option 4 mirror
lens FSC obscuration bar
intercept #3
FL4, 5, 6 (option)
lens
filter iris
SSC PMT FL objective lens lens
(laser #1) intercept
laser #3 FL obscuration bar point
prism 8
laser #2 achromatic
lens
prism 6
laser #1 prism 4 stream viewing
prism 7 video camera
prism 2
prism 5
prism 1
prism 3
Figure 4-3 Optical bench diagram of the BD FACSVantage SE™ stream-in-air flow
cytometer
Review Questions: Optical Bench
1 The optical bench provides a stable surface for the interaction of the laser light
with the ___________________________.
2 Which two components of the flow cytometer must be perfectly aligned to

illuminate cells uniformly? _____________________________________
3 The alignment of a stream-in-air cytometer requires daily optimization.
T F
4 For benchtop flow cytometers, the fixed _________________ ensures that the
laser intercepts the _________________ consistently from day to day.
19
4.2 Optical Filters

Once a cell or particle passes through the laser light, emitted SSC and fluorescence
signals are diverted to the photomultiplier tubes (PMTs) and a photodiode collects the
FSC signals. All of the signals are routed to their detectors via a system of mirrors and
optical filters. PMTs detect fluorescence signals, which are often weak. The specificity
of a detector for a particular fluorescent dye is optimized by placing a filter in front of
the PMT, which allows only a narrow range of wavelengths to reach the detector. This
spectral band of light is close to the emission peak of the fluorescent dye. Such filters
are called bandpass (BP) filters. For example, the filter used in front of the FITC
detector is labeled 530/30. This number gives the characteristics of the spectral band
transmitted: 530 ±15 nm, or wavelengths of light that are between 515 nm and
545 nm.
Other filters used in the flow cytometer are shortpass (SP) filters, which transmit
wavelengths of light equal to or shorter than a specified wavelength, and longpass (LP)
filters, which transmit wavelengths of light equal to or longer than a specified
wavelength (Figure 4-4 on page 21).
Beam splitters are devices that direct light of different wavelengths in different
directions. Dichroic mirrors are a type of beam splitter. The 560 SP dichroic mirror
shown in Figure 4-1 transmits wavelengths of light 560 nm or shorter. Wavelengths of
light longer than 560 nm are reflected at 45 degrees.
20
Longpass Shortpass Bandpass

480 520
480 500 520 480 500 520 460 500 540
LP500 SP500 500/50

% transmittance
% transmittance
% transmittance
400 500 600 400 500 600 450 500 550
wavelength (nm) wavelength (nm) wavelength (nm)
Figure 4-4 Light transmittance through longpass, shortpass, and bandpass filters
Review Questions: Optical Filters
1 Optical filters are placed in front of detectors to
2 A 530/30 bandpass filter transmits wavelengths of light between _________

and ________ nm.
3 A _________________ is used to split light signals by wavelength and redirect

the light signals to the appropriate detector.
4 _______________ filters transmit wavelengths equal to or shorter than a

specified wavelength, whereas ________________ filters transmit wavelengths
equal to or longer than a specified wavelength.
21
4.3 Signal Detection

Light signals are generated as particles pass through the laser beam in a fluid stream.
These light signals are converted to electronic signals (voltages) by photodetectors and
then assigned a channel number on a data plot. There are two types of photodetectors
in BD flow cytometers: photodiodes and photomultiplier tubes (PMTs). The
photodiode is less sensitive to light signals than the PMTs and thus is used to detect
the stronger FSC signal. PMTs are used to detect the weaker signals generated by SSC
and fluorescence.
A voltage pulse is created when a particle enters the laser beam and starts to scatter
light or fluoresce. Once the light signals, or photons, strike one side of the PMT or
the photodiode, they are converted into a proportional number of electrons that are
multiplied, creating a greater electrical current. The electrical current travels to the
amplifier and is converted to a voltage pulse. The highest point of the pulse occurs
when the particle is in the center of the beam and the maximum amount of scatter or
fluorescence is achieved. As the particle leaves the beam, the pulse comes back down
to the baseline (Figure 4-5).
Voltage
Laser
Time
Voltage
Laser
Time
Voltage
Laser
Time
Figure 4-5 Creation of a voltage pulse
The size of the voltage pulse depends on the number of photons detected, the PMT
voltage or pre-amplifier gain, and the amplifier gain. Signals can be amplified by
applying a voltage to the PMTs, thus creating a greater electrical current, or by
increasing the amplification gain. Amplifier settings can be linear or logarithmic (Lin
22
or Log). Log amplification is often used to separate negative from dim positive signals,
whereas Lin amplification is often used to amplify scatter and fluorescent parameters.
The Analog-to-Digital Converter (ADC) converts a 0–10 V pulse to a digital number.

In analog systems, such as the BD FACSCalibur, BD LSR, and BD FACSVantage SE
cytometers, the voltage pulse is assigned a digital value representing 0–1000 channels.
The channel number is transferred to the computer via the General Purpose In/Out
(GPIO) cable (Figure 4-6). The light signal is then displayed in an appropriate
position on the data plot.
Figure 4-6 Voltage pulses converted to channel values by ADC converter
In digital systems, such as the BD FACSVantage SE cytometer equipped with the

BD FACSVDiVa™ option, the voltage corresponding to each signal is digitized into
one of 16,384 possible levels 10 million times per second by ADCs. Signals are
continuously digitized during normal operation, whether a pulse is present or not,
and all digitized signals are represented as numbers in memory. See Figure 4-7 on
page 24 (numbers of lines for illustration only; 16,384 levels and 1 sample every
0.1 µsec). The signals are transferred from the cytometer to the computer via Ethernet
cables. Log amplifiers are not used in digital systems. Instead, the logarithm of each
digitized data point is computed and displayed via the software. As with analog
systems, light signal is then displayed in an appropriate position on the data plot.
23
digitization sampling
Figure 4-7 Signal generation and processing
Regardless of whether the system is digital or analog, when multiple lasers are used,
the data derived from the earlier laser are delayed to coincide with data from the last
laser on a per event basis. This ensures that data from a single event are processed
together.
Review Questions: Signal Detection
1 Forward-scattered light is detected by a _____________________.
2 Side-scattered light and fluorescence are detected by highly sensitive

_____________________________________________________________
24
3 The light detectors generate current, which travels to an amplifier. It converts

the current to voltages which are proportional to the intensity of light striking
them.
T F
4 What does the ADC do?
4.4 Threshold
An electronic threshold can be used to limit the number of events acquired by the
flow cytometer. A threshold is set on one parameter. When a threshold value is
defined, only signals with an intensity greater than or equal to the threshold channel
value will be processed and sent to the computer. For example, when running
immunophenotyping samples, the threshold can be set on FSC to eliminate events
such as debris that are smaller than the threshold channel number. Other parameters
can be used to set the threshold depending on the application.
A second threshold parameter is available on some benchtop analyzers equipped with

the second-laser option. If two threshold parameters are chosen, then the particle
must meet the values of both thresholds to be processed as an event.
Review Questions: Threshold
1 FSC threshold can be used to eliminate signals generated from:
2 If two threshold parameters are chosen, the event must meet the values of one or
the other parameter to be processed.
T F
25
26
5
Data Analysis
5.1 Data Collection and Display

Once light signals have been converted to electronic pulses and then converted to
channel numbers by the ADC, the data must be stored by the computer system.
Flow cytometric data is stored according to a standard format, the flow cytometry
standard (FCS) format, developed by the Society for Analytical Cytology.* According
to the FCS standard, a data storage file includes a description of the sample acquired,
the instrument on which the data was collected, the data set, and the results of data
analysis.
A single cell analyzed for four parameters (FSC, SSC, FITC, and PE fluorescence)
generates 8 bytes of data. When multiplied by the approximately 10,000 events
collected for a single sample, an FCS data file typically contains 80 kB of data.
Once a data file has been saved, cell populations can be displayed in several different
formats. A single parameter such as FSC or FITC (FL1) can be displayed as a single-
parameter histogram, where the horizontal axis represents the parameter’s signal value
in channel numbers and the vertical axis represents the number of events per channel
number (Figure 5-1). Each event is placed in the channel that corresponds to its signal
* Data file standard for flow cytometry. Data File Standards Committee of the Society for Analytical Cytology.
Cytometry. 1990;11(3):323-332.
27
value. Signals with identical intensities accumulate in the same channel. Brighter
signals are displayed in channels to the right of the dimmer signals.
Two parameters can be displayed simultaneously in a plot. One parameter is displayed

on the x-axis and the other parameter is displayed on the y-axis. In BD CellQuest™
and BD Attractors software, three-dimensional data can also be viewed where the x-
and y-axes represent parameters and the z-axis displays the number of events per
channel (Figure 5-1).
Histogram 2-D plot 3-D plot 3-D plot

BD CellQuest software BD Attractors software
Figure 5-1 Graphic representations of flow cytometric data
Review Questions: Data Collection and Display
1 What does the horizontal axis in a histogram represent?
2 A dot plot can be used to display __________ parameters.
3 What does the z-axis on a 3-D plot represent in BD CellQuest software?
28
Chapter : Data Analysis
5.2 Gating
A subset of data can be defined through a gate. A gate is a numerical or graphical
boundary that can be used to define the characteristics of particles to include for
further analysis. For example, in a blood sample containing a mixed population of
cells, you might want to restrict your analysis to only the lymphocytes. Based on FSC
or cell size, a gate can be set on the FSC vs SSC plot to allow analysis only of cells the
size of lymphocytes. The resulting display would reflect the fluorescence properties of
only the lymphocytes (Figure 5-2).
ungated data
gated data
Figure 5-2 Use of gating to restrict analysis to one population
Review Questions: Gating
1 A gate can be used to restrict the analysis to a specific population within the
sample.
T F
29
5.3 Data Analysis for Subsetting Applications

Data analysis consists of displaying the data from a list-mode file in a plot, then
measuring the distribution of the events within the plots. As mentioned earlier, several
types of plots can be used to present the data. Data can also be subdivided by gating
upon specific populations.
For example, in the dot plot shown in Figure 5-3, a gate is drawn around the
population of interest, which in this case is the lymphocytes. A gate or a region is a
boundary drawn around a subpopulation to isolate events for analysis or sorting.
Figure 5-3 Dot plot with a gate encompassing the lymphocyte population
Data for events within this gate can then be displayed in subsequent plots. In the
examples that follow, you will see different ways to analyze fluorescence data from
events in this gate to determine the percentages of various subpopulations (subsets)
present.
You can make a single-parameter histogram plot with histogram markers, a two-
parameter dot plot with a quadrant marker, a two-parameter dot plot with regions,
and three-dimensional plots. You can also create statistics and export the results that
are associated with these plots to a spreadsheet.
30
A histogram allows you to view a single parameter against the number of events. A
subclass control is used to determine where the markers will be placed. Histogram
markers are used to specify a range of events for a single parameter (Figure 5-4). In the
first histogram, marker M1 is placed around the negative peak of the subclass control.
Marker M2 is placed to the right of M1 to designate positive events. The second
histogram shows events from a CD3 FITC sample.
Figure 5-4 Histograms of subclass control (NORM001) and CD3 FITC

(NORM002) with histogram markers M1 and M2
Figure 5-5 shows 619 events in M1 and 2272 in M2. To find out statistical
percentages of the negatives and the positives, compare the event count with the gated
events. There are 6000 events in the data file, but 2891 events found inside the
lymphocyte gate. We want the percentage of lymphocytes that are CD3 positive, so
we would look at the %Gated for M2: 2272/2891 = 78.59%.
Figure 5-5 Histogram statistics
31
A dot plot provides a two-parameter display of data. Each dot represents one or more
events. The first dot plot in Figure 5-6 is the isotype or subclass control. A subclass
control is used to determine where the quadrant markers will be placed. A quadrant
marker divides two-parameter plots into four sections to distinguish populations that
are considered negative, single positive, or double positive. The lower-left quadrant
displays events that are negative for both parameters. The upper-left quadrant
contains events that are positive for the y-axis parameter (CD19 PE) but negative for
the x-axis (CD3 FITC) parameter. The lower-right quadrant contains events that are
positive for the x-axis parameter (CD3 FITC) but negative for the y-axis (CD19 PE)
parameter. The upper-right quadrant contains events that are positive for both
parameters (CD19+/CD3+), or double positive.
Figure 5-6 Dot plots of subclass control (NORM001) and CD3 FITC/CD19 PE
(NORM002) with quadrant markers
To find out the percentages of CD19+/CD3- lymphocytes, look at the %Gated of the
upper left (UL) quadrant divided by gated events (Figure 5-7): 296/2839 = 10.43%.
Figure 5-7 Quadrant statistics
32
An alternative way to get statistics is to create regions around the populations instead
of using a quadrant marker. You can create differently shaped regions (Figure 5-8);
then use region statistics to find out the percentages of specific populations. In
Figure 5-9, the %Gated of R4 is the CD3–/CD4+ lymphocytes: 40/2866 = 1.40%.
Figure 5-8 Dot plot of CD3 FITC/CD4 PE with four regions
Figure 5-9 Region statistics
There is a disadvantage in using both of these methods of analysis when you have
several files to analyze from different donor samples. If you draw the regions around
populations or create quadrant markers from one data file and then read in another
file, it is possible that the populations will fall outside the regions or markers due to
sample variability. In this case, you will have to readjust the regions or markers for
every file.
There is a new analysis method available to avoid this situation. This innovative
technology is called cluster analysis. BD MultiSETTM and BD AttractorsTM software
use cluster analysis to analyze data. In these software programs, regions shift their
positions to encompass clusters from one data file to the next (Figure 5-10).
33
Figure 5-10 Two-parameter plot in BD Attractors software with ellipsoid regions before
and after analysis
5.4 Data Analysis for Other Applications

The analysis methods illustrated so far are used for estimating percentages of discrete
populations. This is why you only used the %Gated and %Total statistics.
If you were analyzing a cloned cell line to determine if it were positive for a particular
molecule, you would most likely not use percentages. Since a cloned cell line consists
of a single population, in most cases it would either be 100% negative or 100%
positive. It’s also possible that it would express low amounts of the molecule in
question. In that case, it would still be positive but dim.
For these cases where you want to compare fluorescence intensities and measure the
degree of positivity, you would compare the geometric means or medians of the
subclass control data versus the sample data. If the sample data statistic is greater than
that of the subclass control data by some limit set by the user, it would be considered
positive. The greater the difference between the two, the more molecules are expressed
per cell and the more positive, or brighter the population.
The dot plot in Figure 5-11 shows a single population by scatter. The histogram
overlays show data from a subclass control and staining with two different antibodies.
The geometric mean for each histogram is respectively 2, 5, and 20 (left to right). In
this example, the user would decide if sample data in the middle histogram was
positive.
34
Figure 5-11 Analysis of a cell line
Besides being used to measure positivity, geometric means or medians can be used to
estimate the quantity of molecules (ligands) expressed per cell. Specialized software
programs such as QuantiCALC™ use the median in conjunction with data from a
standard curve to calculate the number of antibodies bound per cell. An example is
shown in Figure 5-12; circles on the y-axis indicate information from a standard
curve. This information can be used to estimate the number of ligands per cell.
information from a standard curve
Figure 5-12 QuantiCALC estimate of number of antibodies bound per cell*
* Data provided courtesy of Dr. David Miller, Community Blood Centers of South Florida.
35
For DNA content analysis, another specialized software program such as

ModFit LT™ can be used. Because the populations that represent a DNA histogram
(G0/G1, S, and G2+M) are not discrete (Figure 5-13), special algorithms are used. The
area under the curve is integrated; then the percentages of each population present are
calculated.
Figure 5-13 DNA histogram
Review Questions: Data Analysis
1 Why would you use a two-dimensional dot plot rather than a histogram?
2 Referring to Figure 5-4 and Figure 5-5,
What percentage of the lymphocytes is not CD3+? __________________
What percentage of the total events is CD3+? ________________________
3 The population in the LL quadrant is the double-positive population.
T F
4 Referring to Figure 5-6 and Figure 5-7, what is the percentage of lymphocytes
that are CD3+/CD19–? _____________________________
36
5 Regions can only be drawn as rectangles.
T F
6 What is the disadvantage of using regions to analyze several files?
7 What kind of data analysis avoids the problem of shifting groups of cell
populations?
8 What statistics are used to measure degree of positivity and for quantitation
studies?
37
38
6
Sorting
6.1 Sorting
In most applications, after a particle exits the laser beam, it is sent to waste. Sorting
allows us to capture and collect cells of interest for further analysis. Once collected,
the cells can be analyzed microscopically, biochemically, or functionally. Not all
benchtop flow cytometers are equipped with a sorting feature; however, they can be
upgraded to perform this function.
To sort particles or cells, the cytometer first needs to identify the cells of interest, then
separate out the individual cells. Once the population of interest has been identified
on a data acquisition plot, a region is drawn around that population. A logical gate is
created from the regions. This gate is then loaded into the cytometer’s software as the
sort gate. The sort gate identifies cells of interest to be sorted out of the stream.
Different cytometers have different methods of capturing a particle of interest. The

BD FACSCalibur system, a benchtop analyzer, uses a mechanical device called a
catcher tube to sort cells. This catcher tube is located in the upper portion of the flow
cell. It moves in and out of the sample stream to collect a population of desired cells at
a rate of up to 300 cells per second.
As a cell passes through the laser beam, the BD FACSCalibur system electronics,
using the sort gate characteristics, quickly determines if the cell is a target. The target
cell is captured according to the preselected sort mode. A sort mode is the criteria of
capturing the target cells relative to accurate cell count or purity. Because laser
39
alignment and stream velocity are fixed, the time it takes for desired cells to travel
from the laser intercept to the catcher tube is constant.
When the decision is made to capture the target cell, the electronics system waits for a
fixed period of time to allow the cell to reach the catcher tube. It then triggers the
catcher tube to swing into the sample stream to capture the cell. Figure 6-1(left) shows
the catcher tube in its resting position in the sheath stream. Figure 6-1 (right) shows
the catcher tube positioned in the sample core stream ready to capture a target cell.
sorted
cell
collection
waste waste
catcher
s heath sheath
Figure 6-1 (Left) catcher tube in sheath stream; (right) catcher tube in sample stream
The BD FACSVantage SE system, a stream-in-air flow cytometer, isolates a cell of

interest by vibrating the entire stream. The sample stream vibrates along its axis and
breaks up into drops. The distance between drops is fixed. When the sheath velocity
and the vibration speed of the nozzle tip are constant, the pattern of drop formation is
fixed. With the fixed drop formation, the BD FACSVantage SE instrument is able to
calculate the distance between the drops precisely, which allows for the isolation of
individual cells.
The BD FACSVantage SE cytometer applies a voltage charge to drops containing a

cell that meets the predefined sorting criteria. Positively and negatively charged plates
are present on either side of the vibrating stream. As the charged drops pass by the
40
Chapter : Sorting
charged plates, the droplets are deflected to the collection tubes, depending on the
droplet’s charge polarity (Figure 6-2).
charging electrode
sample injection tube

sheath tube
vent tube
deflection plates
waste collection
collection tubes
Figure 6-2 Sorting components on the BD FACSVantage SE flow cytometer
Review Questions: Sorting
1 To separate individual cells, the liquid stream of the BD FACSVantage SE

cytometer _________________________ along its axis.
2 How many cells are contained in each sorted drop on the BD FACSVantage SE
instrument?
3 What is the name of the device used by the BD FACSCalibur instrument to

capture sorted cells?
4 What does the BD FACSVantage SE system use to attract or repel charged

particles?
41
42
7
Lasers and Alignment
7.1 How Lasers Work

LASER is an acronym for light amplification by stimulated emission of radiation.
Light is generated in the following manner. Gas lasers consist of cylinders, or plasma
tubes, filled with an inert gas such as argon. The inert gas is ionized by a high-voltage
electrical pulse, so that the electrons in the ionized gas atoms absorb energy and move
to a higher energy state. As the excited electrons return to the ground state, all at once
or in several rapid steps, they give off photons with wavelengths specific to each
transition level.
Optics at each end of the plasma tube reflect photons back and forth through the
tube. These photons interact with other exited electrons, resulting in the release of
more photons that are identical in wavelength, phase, and direction. As each photon is
capable of stimulating more photons, light amplification or lasing occurs. Brewster
windows seal each end of the plasma tube and transmit the light in one plane of
polarization.
An electromagnet placed around the plasma tube creates a magnetic field that
compresses the electrons towards the center of the tube. This prevents the electrons
from hitting the sides of the tube, increasing the current density and causing greater
stimulated emission of light.
43
A small percentage of this light is transmitted through the optic at the front (output
coupler) of the tube. This transmitted light is the laser beam. The wavelength of the
primary laser beam in benchtop flow cytometers is fixed at 488 nm by placing a filter
in front of the output coupler (Figure 7-1). A secondary laser, the red-diode laser, is
available for the benchtop analyzer. This is a solid-state laser that emits a 635-nm
wavelength. The wavelength of the laser beam in stream-in-air cytometers is selected
by adjusting the high reflector optic position to reflect just one of the refracted
wavelengths of light traveling through the prism. Only the selected wavelength will be
reflected between the output coupler and the high reflector. The output of the laser is
monitored and maintained at a constant level through the use of feedback circuitry.
Figure 7-1 Generation of laser light in a gas laser
7.2 Laser Alignment

In the flow cytometer, the laser beam is focused on the sample core. This point of
intercept must remain constant. To accomplish this, the laser head is held in a fixed
position. The beam passes through an optic that causes the beam shape to be elliptical.
The beam next passes through a focusing lens that focuses the beam at the point of
intercept between the laser beam and the sample core. In benchtop flow cytometers,
the laser is fixed in proper alignment within the cytometer and no operator
adjustment is needed. Stream-in-air flow cytometers use removable lasers and laser
optics; therefore, operator adjustment is necessary.
44
Chapter : Lasers and Alignment
Review Questions: Lasers and Alignment
1 A(n) _______________ laser emits light that has a wavelength of 488 nm.
2 The laser beam shape is elliptical after it travels through the focusing optics.
T F
3 What is the wavelength of the red-diode laser in the bench top analyzer?
4 The wavelength of the laser used in the stream-in-air flow cytometer can be
selected by adjusting the ______________ optic position at the rear of the laser.
45
46
Answer Key
Review Questions: Overview on page 3
1 Relative size, relative granularity or internal complexity, and relative fluorescence

intensity
2 A laser
3 The fluidics, the optics, and the electronics
4 A single cell suspension
5 Sample core
6 Scattered light and fluorescence
7 Lenses
8 The amount of light striking them
9 True
10 True
47
Review Questions: Fluidics on page 8
1 To position the sample core in the center of the laser beam
2 The width and the positioning of the stream
3 One
4 Sheath fluid; flow chamber
5 Hydrodynamic focusing
6 The sample pressure regulator
7 LO, MED, and HI
8 Increases, width
9 A lower flow rate
10 True
11 True
Review Questions: Light Scatter on page 12
1 When a particle deflects incident laser light
2 The cell membrane, nucleus, and internal granular components
3 Forward-scattered light (FSC)
4 Cell surface area or size
5 Side-scattered light (SSC)
6 Granularity or internal complexity
7 FSC and SSC
48
Answer Key
Review Questions: Fluorescence on page 15
1 Photons of light
2 Absorption spectra
3 Emission spectrum
4 The argon-ion laser
5 488 nm
6 FITC and PE
7 Antigenic markers
Review Questions: Optical Bench on page 19
1 The excitation and collection optics
2 The laser beam and the sample stream
3 True
4 Flow cell, sample stream
Review Questions: Optical Filters on page 21
1 Limit the range of wavelengths reaching the detector
2 515 and 545
3 Beam splitter
4 Shortpass, longpass
49
Review Questions: Signal Detection on page 24
1 Photodiode
2 Photomultiplier tubes
3 True
4 Converts voltage pulses to channel numbers
Review Questions: Threshold on page 25
1 Dust, debris, or electronic noise
2 False
Review Questions: Data Collection and Display on page 28
1 The parameter’s signal value in channel numbers
2 Two
3 The number of events per channel
Review Questions: Gating on page 29
1 True
50
Answer Key
Review Questions: Data Analysis on page 36
1 A dot plot allows you to view a two-parameter display of data. Each event has a
position in the plot according to its channel values for both parameters.
Histograms can only display data of one parameter in relationship to the
number of events.
2 21.41%
37.87%
3 False
4 79.57%
5 False
6 As you read in one data file to the next in the plot, if the regions are created
tightly around the population, the subsequent populations could fall outside of
the regions because of sample variability.
7 Cluster analysis
8 Geometric mean or median
Review Questions: Sorting on page 41
1 Vibrates
2 One
3 Catcher tube
4 Deflection plates
51
Review Questions: Lasers and Alignment on page 45
1 Argon
2 False
3 635 nm
4 High reflector
52

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Introduction To Flow Cytometry - A Learning Guide

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Introduction to

Introduction to Flow Cytometry: A Learning Guide iii

iv Introduction to Flow Cytometry: A Learning Guide

Learning to operate a flow cytometer is best achieved by using the instrument.

This document contains basic information on flow cytometry. Differences between

For More Information

Flow cytometry is a technology that simultaneously measures and then analyzes

on the particle fluoresce. The scattered and fluorescent light is collected by

Figure1-1 Scattered and emitted light signals converted to electronic pulses

Review Questions: Overview

1 What properties of a cell or particle can be measured by a flow cytometer?

2 What light source is used in most flow cytometers?

3 What are the three main systems in a flow cytometer?

7 Light emitted from a particle is collected by

8 The electronic signal produced by the detectors is proportional to:

9 All flow cytometric measurements can be made simultaneously on a single cell.

10 Particles must be in single-cell suspension before flow cytometric analysis.

low sample high sample

sheath sheath sheath sheath

sheath sheath sheath sheath

low differential pressure high differential pressure

• In BD benchtop cytometers, the sample stream is pressurized upward through an

• A higher flow rate is generally used for qualitative measurements such as

Proper operation of fluidic components is critical for particles to properly intercept

Review Questions: Fluidics

1 The purpose of the fluidics system in a flow cytometer is:

4 The particle suspension is injected into _________________ within the

6 Which regulator controls the diameter of the sample core?

10 A wider sample core decreases resolution.

11 A high flow rate can be used when performing qualitative measurements.

3.1 Light Scatter

Forward-scattered light (FSC) is proportional to cell-surface area or size. FSC is a

Side-scattered light (SSC) is proportional to cell granularity or internal complexity.

side scatter detector

forward scatter detector

Figure 3-1 Light-scattering properties of a cell

lysed whole blood

Figure 3-2 Cell subpopulations based on FSC vs SSC

Review Questions: Light Scatter

1 When does light scattering occur?

2 Which key cell components contribute to light scatter?

3 Light scattered in the same direction as the laser beam is called:

4 FSC is proportional to:

5 Light scatter collected at 90 degrees to the laser beam is called:

6 SSC is proportional to the ___________ or _____________ of the cell.

7 Correlated measurements of both _______________ and _____________ can

FITC = fluorescein isothiocyanate

Figure 3-3 Absorption spectra of four common fluorochromes

Figure 3-4 Emission spectra of the fluorochromes shown in Figure 3-3

When a fluorescent dye is conjugated to a monoclonal antibody, it can be used to

Figure 3-5 Specific binding of fluorochrome-labeled antibodies to cell surface antigens

Review Questions: Fluorescence

2 Characteristic wavelength ranges at which fluorescent compounds can be excited

3 The longer wavelengths of light emitted by a fluorochrome make up its

4 Which laser is most commonly used in flow cytometry? _________________

5 The FITC and PE fluorochromes are excited by this emission wavelength of an

7 Fluorochrome-labeled antibodies are used to detect

4.1 Optical Bench

In a stream-in-air cytometer, the laser beam is focused through an achromatic lens to

6 SSC is proportional to the _ or ___ of the cell.

7 Correlated measurements of both ___ and _ can