BMS 633A-BME 695Y - Week 1
Flow Cytometry Module for Engineering
Students
A course designed for engineering students who may not have strong biology backgrounds.
This course consists of a series of lectures and practicals that allow engineering students to
understand biotechnology and its application. At the conclusion of this 2 credit hour course
students will have an excellent understanding of the technical components and operation of
flow cytometers, understand the biological principals of operation, be familiar with the
applications of the technology and be able to intelligently discuss the technological
implications of flow cytometry with a person who is well versed in the field.
Slides are mostly designed w/o backgrounds to be printable on a B/W printer
The WEB version of these slides can be found on
Contact Information:
Hansen Hall, B050
Purdue University
Office: 494 0757
Fax 494 0517
email\; robinson@flowcyt.cyto.purdue.edu
WEB http://www.cyto.purdue.edu
http://tinyurl.com/385ss
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
J. Paul Robinson
Professor of Biomedical Engineering
Professor of Immunopharmacology
School of Veterinary Medicine, Purdue University
Last modified January 9, 2004
Page 1
Learning Goals of this Course
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Become familiar with the terms and features of flow cytometry
Understand each of the technology components of flow cytometers
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Electronics
Fluidics
Optics
Data Collection and Analysis
Understand the background and biological principles of the field
Know the applications and uses of the technology
Students will become familiar with sample preparation, pipetting,
spectroscopy, buffers, blood collection, phenotyping, DNA analysis,
kinetic analysis, and observe cell sorting.
Develop sufficient laboratory skills to collect, prepare and run
specimens on a flow cytometer
Be able to converse intelligently with an expert in the field on the
general uses, applications and operating principles of the technology
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
Page 2
Structure of this course
• Lectures: There are five lectures, one for each week of the
course. Each lecture will be given in two parts, each part
approximately 1 hour.
• Practicals: There are 5 practicals that will be performed over
the 5 weeks, each practical will be divided into 2 sessions of
approximately 4 hours each, a total of 8 hours each week.
• The 5 week module is designed to give engineering students a
highly condensed, rapid learning opportunity in both theory
and practice. They will also familiarize you with the PUCL
environment in which you will find most of the CYTOMIC tools
you will need to study cellular systems
• Grading:
End of course theory exam: 25%
End of course Practical Exam: 45%
Attendance at 10 sessions: 2%/session (10%)
Presentation of Class manual: 20%
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
Page 3
Sources of information
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Flow Cytometry and Sorting, 2nd ed. (M.R. Melamed, T. Lindmo, M.L. Mendelsohn, eds.), Wiley-Liss, New York, 1990
- referred to here as MLM
Flow Cytometry: Instrumentation and Data Analysis (M.A. Van Dilla, P.N. Dean, O.D. Laerum, M.R. Melamed, eds.),
Academic Press, London, 1985 – referred to as VDLM
4th
Edn
Practical Flow Cytometry 3nd edition (1994),4th Ed (2003) H. Shapiro: Alan R. Liss, New York - referred to as PFC
Introduction to Flow Cytometry. J. Watson, Cambridge Press, 1991 referred to as IFC
Methods in Cell Biology: v.40,41, 63, 64 Darzynkiewicz, Robinson & Crissman, Academic Press, 1994, 2000 MCB
Data Analysis in Flow Cytometry:A Dynamic Approach-Book on CDROM M. Ormerod referred to as DAFC
Flow Cytometry: First Principles. (2nd Ed) Alice Longobardi Givan, Wiley-Liss, 2001 referred to as AFCFP
More information on flow cytometry books can be found on our website at:
http://www.cyto.purdue.edu/flowcyt/books/bookindx.htm
Note: All of these books are in Prof. Robinson’s library in Hansen Hall, Room B50 and may be
checked out for 24 hour periods with permission and by signing off on the sign-out chart. At
least one finger must be left in the shelf as hostage for return of my books!
Some slides are based on slides taken from Dr. Robert Murphy [RFM]
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
Page 4
Primary Text
• Practical Flow Cytometry 3nd edition (2003),H.
Shapiro: Alan R. Liss, New York - referred to as
PFC
Amazon Order page for Shapiro 2nd Ed
http://tinyurl.com/2ldlf
You can buy 2nd hand copies for around
$80 on Amazon and its related sites
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
Page 5
Methods and Practical Assistance
• For help with protocols there are several choices including
the MCB references on the previous slide (Methods in Cell
Biology)
• The Handbook of Flow Cytometry Methods
• Current Protocols in Cytometry
Both of these are in the lab
You may used them at any time
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
Page 6
Additional Sources
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Powerpoint presentations references as J.Paul Robinson (JPR); Robert Murphry
(RFM), Carleton Stewart (CS)
Web sources of these presentation are:
http://www.cyto.purdue.edu/flowcyt/educate/pptslide.htm
http://www.cyto.purdue.edu/flowcyt/educate1.htm
Additional Sources include the Purdue Cytometry CD-ROM series
Vol. 1
Vol. 2
Vol. 3
Vol. 4
Free copies of any of these
Are available to you just
For the asking!
Vol. 5
Vol. 6
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
Vol. 7
Page 7
Key Reference Text
The course will use Shapiro:
Practical Flow Cytometry, 3nd edition (1994) and 4th Ed
(2003), Alan R. Liss, New York, as the main reference
text.
Supplementary texts have been referenced on the
previous slides
There are several copies of this text in the
Cytometry Laboratories available for students to
use. They may not be checked out.
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
Page 8
Week 1
• Introduction to the course.
• Discussion of texts and associated reading
materials.
• Discussion of expectations of students and special
concerns. Evaluation criteria
• Overview of flow cytometry. Each system
presented
•Types of data to expect, fluidics and hydrodynamics
including flow cells and liquid handling systems
•This is a whirlwind course! 5 lectures, 9 x 4 hour
pracs. It is a superfast introduction to a technology
•You must read the material and attend all the
sessions to keep up
References:(3rd Ed Shapiro pp 1-5; 4th Ed Shapiro p
1-4; Givan pp 1-9)
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
1-60; Watson pp
Page 9
General introduction to flow
cytometry
Introduction to the terminology, types of
measurements, capabilities of flow cytometry, uses &
applications
• Comparison between flow cytometry and fluorescence
microscopy
• Transmitted light
• Scatter
• Sensitivity, precision of measurements, statistics,
populations
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
Page 10
What can Flow Cytometry
Do?
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Enumerate particles in suspension
Determine “biologicals” from “non-biologicals”
Separate “live” from “dead” particles
Evaluate 105 to 106 particles in less than 1 min
Measure particle-scatter as well as innate
fluorescence or 2o fluorescence
• Sort single particles for subsequent analysis
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
Page 11
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
•2000-2001
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10000
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•1976-1977
•1975-1976
Publications using the keyword
“flow cytometry”
52,196 references
1st use of keyword
The growth of flow cytometry based on publications
Page 12
Historical Overview
Historical approach to cytometry…….
Early nucleic acid measurements
Aerosols... Lomonosov, Moldavan, Gucker
Moldavan (1934) demonstrates use of a suspending fluid in which were blood cells - the
measurements were made in a capillary tube using a photoelectric sensor to make extinction
measurements
FT Gucker: 1947 - used a system of suspending bacteria in aerosols then enumerating them thus the organisms were counted in air not water as we do now. The system used a dark field
illumination illuminated by a Ford headlight and the detector was a PMT
Applications to cancer
Mid nucleic acid measurements,
•Casperson
•Avery
•Papanicolaou & Traut
•Friedman
•Mellors & Silver
Antibodies - fluorescence advances, Coons & Kaplan
Automated counters - sheath flow principle - Gucker, Crosland-Taylor blood cell
counter, Coulter orifice
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
Page 13
Fluorescence Labeling Technique
Coons et al 1941 - developed the
fluorescence antibody technique they labeled antipneumococcal
antibodies with anthracine allowing
them to detect both the organism and
the antibody in tissue using UV
excited blue fluorescence
“Moreover, when Type II and III
organisms were dried on different parts of
the same slide, exposed to the conjugate
for 30 minutes, washed in saline and
distilled water, and mounted in glycerol,
individual Type III organisms could be
seen with the fluorescence
microscope……” (ref see below)
Key Publication
Immunological Properties of an Antibody Containing a
Fluorescent Group
Albert H. Coons, Hugh J. Creech and R. Norman Jones
Department of Bacteriology and Immunology, Harvard Medical School, and the
Chemical Laboratory, Harvard University
Proc. Soc. Exp.Biol.Med. 47:200-202, 1941
Coons and Kaplan (1950) - conjugated fluorescein with
isocyanate - better blue green fluorescent signal - further
away from tissue autofluorescence. This method used a
very dangerous preparative step using phosgene gas
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
Page 14
Basics of Flow Cytometry
Fluidics ...cells in suspension
flow in single-file through
an illuminated volume where they
Optics
scatter light and emit fluorescence
that is collected, filtered and
Electronics converted to digital values
that are stored on a computer...
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
Page 15
Instrument Components
Fluidics: Specimen manipulation, sorting, rate of data
collection
Optics: Light source(s), detectors, spectral separation
Electronics: Control, pulse collection, pulse analysis,
triggering, time delay, data display, gating, sort control,
light and detector control
Computation-Data Analysis: Data display & analysis,
multivariate/simultaneous solutions, identification of sort
populations, quantitation, standards
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
Page 16
Commercial Instruments
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
Page 17
What are the principles?
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Hydrodynamically focused stream of particles
Light scattered by a laser or arc lamp
Specific fluorescence detection
Electrostatic particle separation for sorting
Multivariate data analysis capability
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
Page 18
Technical Components
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Illumination Sources - Electrical Engineering /Optics
Detection Systems - Electrical Engineering /Optics
Fluidics - Mechanical Engineering
Sorting - Mechanical Engineering
Data Acquisition - Electrical Engineering /Signal Processing
Data Analysis - Electrical Engineering /Signal Processing.
Computer science
• Biological Systems - Biomedical Engineering
– Stains - Chemistry, biological systems- immunology &
biochemistry, microbiology
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
Page 19
Technical Components
• Detection Systems
Photomultiplier Tubes (PMTs)
Historically 1-2
Current Instruments 3-15
Diodes
Light scatter detectors (plus PMTs)
• Illumination Systems
Lasers
(350-363, 420, 457, 488, 514, 532, 600, 633 nm)
Argon ion, Krypton ion, HeNe, HeCd, YAG
Arc Lamps
Mercury, Mercury-Xenon (most lines)
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
Page 20
Data are collected as histograms
As cells or particles pass the observation point, scattered light is collected at various
angles and sent to detectors which convert the light into a voltage and record the result
as a histogram. Comparison of histograms is essentially what happens when we evaluate
flow cytometry data.
Parameter
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
Page 21
Data Analysis Concepts
As part of that comparison of histograms, it is necessary to create
complex multivariate data sets. Many variables are compared
simultaneously with Boolean logic.
Gating (multivariate analysis)
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Single parameter
Dual parameter
Multiple parameter
Back-gating
Note: these terms are introduced here, but will be discussed in more detail in later
lectures
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
Page 22
Data Presentation Formats
What might flow cytometry data look like?
• Histogram
• Dot plot
• Contour plot
• 3D plots/isometric
• Dot plot with projection
• Overviews (multiple histograms)
•TIP* or TIG+ position formats
CD45
CD8
CD4
CD8
leu11a
Mo1
CD20
FITC Fluorescence
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TIP Tube Identifier Parameter: Reference - Cytometry 12:82-90,1991
•+ TIG – Time Interval Gating: Refgerence: Cytometry, 12:701-706, 1991
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
Page 23
Hydrodynamics and Fluid
Systems
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Cells are always in suspension
The usual fluid for cells is saline
The sheath fluid can be saline or water
The sheath must be saline for sorting
Samples are driven either by syringes or
by pressure systems
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
Page 24
Fluidics
• Need to have cells in suspension flow in single file
through an illuminated volume
• In most instruments, accomplished by injecting
sample into a sheath fluid as it passes through a
small (50-300 µm) orifice
• When conditions are right, sample fluid flows in a
central core that does not mix with the sheath fluid
• This is termed Laminar flow
• 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
[RFM]
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
Page 25
Fluidics - Laminar Flow
• Whether flow will be laminar can be
determined from the Reynolds number
Re

d v

where
d  tube diameter
  density of fluid
v  mean velocity of fluid
  viscosity of fluid
• When Re < 2300, flow should be laminar
• When Re > 2300, flow can be turbulent
[RFM]
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
Page 26
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.
[RFM]
Figure from V. Kachel, H. Fellner-Feldegg & E. Menke - MLM Chapt. 3
Fluidics
• How do we accomplish sample injection
and regulate sample flow rate?
– Differential pressure
– Volumetric injection
[RFM]
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
Page 28
Fluidics - Differential Pressure
System
• Use air (or other gas) to pressurize
sample and sheath containers
• Use pressure regulators to control
pressure on each container separately
[RFM]
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
Page 29
Fluidics - Differential
Pressure System
• Sheath pressure will set the sheath volume
flow rate (assuming sample flow is
negligible)
• Difference in pressure between sample and
sheath will control sample volume flow
rate
• Control is not absolute - changes in friction
cause changes in sample volume flow rate
[RFM]
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
Page 30
Fluidics Systems
Positive Pressure Systems
• Based upon differential pressure
between sample and sheath fluid.
• Require balanced positive pressure
via either air or nitrogen
• Flow rate varies between 6-10 ms-1
+++
+++
+++
Positive Displacement Syringe Systems
1-2 ms-1 flow rate
Syringe
Fixed volume (50 l or 100 l)
Absolute number calculations possible
Usually fully enclosed flow cells
Flowcell
100 l
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3-way valve
Sample
Waste
Sample loop
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
Page 31
Sample tube
Sheath tanks
deliver sheath
Sample station of the
Coulter XL analyzer
Sample is delivered to flow
Cell from here
Waste tanks
accept waste
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
Page 32
Fluidics - Volumetric Injection
System
• Use air (or other gas) pressure to set
sheath volume flow rate
• Use syringe pump (motor connected to
piston of syringe) to inject sample
• Sample volume flow rate can be changed
by changing speed of motor
• Control is absolute (under normal
conditions)
[RFM]
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
Page 33
Syringe systems
• Bryte HS
Cytometer
Syringe
Sample
line
Sheath
fluid
3 way valve
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
Page 34
Fluidics - Volumetric Injection System
nozzle
H.B. Steen - MLM Chapt. 2
Hydrodynamic
Systems
Signals
Flow
Cell
Coverslip
Signals
Flow
Cell
Microscope
Objective
Waste
Coverslip
Microscope
Objective
Waste
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
Page 36
Fluidics - Particle Orientation
and Deformation
• As cells (or other particles) are
hydrodynamically focused, they experience
different shear stresses on different points
on their surfaces (an in different locations in
the stream)
• These cause cells to orient with their long
axis (if any) along the axis of flow
• The shear stresses can also cause cells to
deform (e.g., become more cigar-shaped)
[RFM]
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
Page 37
Fluidics - 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.”
[RFM]
Figure from V. Kachel, et al. - MLM Chapt. 3
Fluidics - Flow Chambers
• The flow chamber
– defines the axis and dimensions of
sheath and sample flow
– defines the point of optimal
hydrodynamic focusing
– can also serve as the interrogation point
(the illumination volume)
[RFM]
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
Page 39
Closed flow cell
fluorescence
signal
direction
Forward
scatter
signal
direction
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
flow direction
(it can go “up”
or “down”
depending on
the orientation
of the flow cell)
Laser
direction
Page 40
Coulter XL Analyzer
Sample tube
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
Sheath and waste system
Page 41
Fluidics - Flow Chambers
• Four basic flow chamber types
– Jet-in-air
• best for sorting, inferior optical properties
– Flow-through cuvette
• excellent optical properties, can be used for
sorting
– Closed cross flow
• best optical properties, can’t sort
– Open flow across surface
[RFM]
• best optical properties, can’t sort
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
Page 42
Fluidics - Flow Chambers
Sheath flow
Flow through
cuvette (sense
in quartz)
droplets
[RFM]
Modified Figure from H.B. Steen - MLM Chapt. 2
Fluidics - Flow Chambers
Closed cross
flow chamber
Modified figure from H.B. Steen - MLM Chapt. 2
[RFM]
Flow Cell
Injector
Tip
Sheath
fluid
Fluorescence
signals
Focused laser
beam
Forward scatter
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
Page 45
Hydrodynamic Systems
Sample in
Sheath
Piezoelectric
crystal oscillator
Sheath in
Fluorescence
Sensors
Laser beam
Scatter Sensor
Sheath
Core
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
Page 46
Flow chamber blockage
hair
A human hair blocks the flow cell channel.
Complete disruption of the flow results.
Frequently the only way to remove these
objects is to use a very fine wire to force the
object out.
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
Page 47
Fluorescence collection lens, optical filters,
dichroic filter, band pass filter
dichroics
Flow cell body
From
laser
reflector
Beam shaping lens
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
Page 48
Lecture 1 Summary
• History – how, when, where and why
• Technical highlights – operational
principles
• Mechanics of flow
• Fluidics
© 1988-2004 J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT
Page 49