BMS 633 A-BME 695 Y

BMS 633 A-BME 695 Y - 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, B 050 Purdue University Office: 494 0757 Fax 494 0517 email; robinson@flowcyt. cyto. purdue. edu WEB http: //www. cyto. purdue. edu http: //tinyurl. com/385 ss © 1988 -2004 J. Paul Robinson, Purdue University BMS 633 A –BME 695 Y LECTURE 1. PPT J. Paul Robinson Professor of Biomedical Engineering Professor of Immunopharmacology School of Veterinary Medicine, Purdue University Last modified January 9, 2004

Learning Goals of this Course • • Become familiar with the terms and features of flow cytometry Understand each of the technology components of flow cytometers – – 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 633 A –BME 695 Y LECTURE 1. PPT 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 633 A –BME 695 Y LECTURE 1. PPT 3

Sources of information • • Flow Cytometry and Sorting, 2 nd 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 4 th Edn Practical Flow Cytometry 3 nd edition (1994), 4 th 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. (2 nd 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 B 50 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 633 A –BME 695 Y LECTURE 1. PPT 4

Primary Text • Practical Flow Cytometry 3 nd edition (2003), H. Shapiro: Alan R. Liss, New York - referred to as PFC Amazon Order page for Shapiro 2 nd Ed http: //tinyurl. com/2 ldlf You can buy 2 nd hand copies for around $80 on Amazon and its related sites © 1988 -2004 J. Paul Robinson, Purdue University BMS 633 A –BME 695 Y LECTURE 1. PPT 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 633 A –BME 695 Y LECTURE 1. PPT 6

Additional Sources • • 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/educate 1. 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 633 A –BME 695 Y LECTURE 1. PPT Vol. 7 7

Key Reference Text The course will use Shapiro: Practical Flow Cytometry, 3 nd edition (1994) and 4 th 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 633 A –BME 695 Y LECTURE 1. PPT

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: (3 rd Ed Shapiro pp 1 -5; 4 th Ed Shapiro p -4; Givan pp 1 -9) © 1988 -2004 J. Paul Robinson, Purdue University BMS 633 A –BME 695 Y LECTURE 1. PPT 1 -60; Watson pp 1

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 633 A –BME 695 Y LECTURE 1. PPT

What can Flow Cytometry Do? • • • 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 2 o fluorescence • Sort single particles for subsequent analysis © 1988 -2004 J. Paul Robinson, Purdue University BMS 633 A –BME 695 Y LECTURE 1. PPT 11

Publications using the keyword “flow cytometry” 52, 196 references 1 st use of keyword The growth of flow cytometry based on publications © 1988 -2004 J. Paul Robinson, Purdue University BMS 633 A –BME 695 Y LECTURE 1. PPT

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 633 A –BME 695 Y LECTURE 1. PPT

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 633 A –BME 695 Y LECTURE 1. PPT 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 633 A –BME 695 Y LECTURE 1. PPT 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 633 A –BME 695 Y LECTURE 1. PPT

Commercial Instruments © 1988 -2004 J. Paul Robinson, Purdue University BMS 633 A –BME

What are the principles? • • • 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 633 A –BME 695 Y LECTURE 1. PPT 18

Technical Components • • • 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 633 A –BME 695 Y LECTURE 1. PPT 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, He. Ne, He. Cd, YAG Arc Lamps Mercury, Mercury-Xenon (most lines) © 1988 -2004 J. Paul Robinson, Purdue University BMS 633 A –BME 695 Y LECTURE 1. PPT 20

Data are collected as histograms Number of events 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 633 A –BME 695 Y LECTURE 1. PPT

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) • • 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 633 A –BME 695 Y LECTURE 1. PPT

Data Presentation Formats What might flow cytometry data look like? • Histogram • Dot plot • Contour plot • 3 D plots/isometric • Dot plot with projection • Overviews (multiple histograms) • TIP* or TIG+ position formats CD 45 CD 20 FITC Fluorescence • + 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 633 A –BME 695 Y LECTURE 1. PPT be Tu leu 11 a ID CD 8 Mo 1 • - CD 8 CD 4

Hydrodynamics and Fluid Systems • • • 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 633 A –BME 695 Y LECTURE 1. PPT 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 633 A –BME 695 Y LECTURE 1. PPT 25

Fluidics - Laminar Flow • Whether flow will be laminar can be determined from the Reynolds number • When Re < 2300, flow should be laminar • When Re > 2300, flow can be turbulent [RFM] © 1988 -2004 J. Paul Robinson, Purdue University BMS 633 A –BME 695 Y LECTURE 1. PPT 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 © 1988 -2004 J. Paul Robinson, Purdue University BMS 633 A –BME 695 Y LECTURE 1. PPT V. Kachel, H. Fellner-Feldegg & E. Menke - MLM Chapt. 3 27

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 633 A –BME 695 Y LECTURE 1. PPT 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 633 A –BME 695 Y LECTURE 1. PPT 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 633 A –BME 695 Y LECTURE 1. PPT 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 © 1988 -2004 J. Paul Robinson, Purdue University BMS 633 A –BME 695 Y LECTURE 1. PPT Flowcell 100 l • • 3 -way valve Sample Waste Sample loop 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 633 A –BME 695 Y LECTURE 1. PPT

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 633 A –BME 695 Y LECTURE 1. PPT 33

Syringe systems • Bryte HS Cytometer Syringe Sample line Sheath fluid 3 way valve

Fluidics - Volumetric Injection System nozzle © 1988 -2004 J. Paul Robinson, Purdue University

Hydrodynamic Systems Signals Flow Cell Coverslip Signals Flow Cell Microscope Objective Waste Coverslip Microscope Objective Waste © 1988 -2004 J. Paul Robinson, Purdue University BMS 633 A –BME 695 Y LECTURE 1. PPT 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 633 A –BME 695 Y LECTURE 1. PPT 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] © 1988 -2004 J. Paul Robinson, Purdue University BMS 633 A –BME 695 Y LECTURE 1. PPT Figure from V. Kachel, et al. - MLM Chapt. 3 38

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 633 A –BME 695 Y LECTURE 1. PPT 39

Closed flow cell Forward scatter signal direction fluorescence signal direction © 1988 -2004 J. Paul Robinson, Purdue University BMS 633 A –BME 695 Y LECTURE 1. PPT flow direction (it can go “up” or “down” depending on the orientation of the flow cell) Laser direction 40

Coulter XL Analyzer Sample tube © 1988 -2004 J. Paul Robinson, Purdue University BMS

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 633 A –BME 695 Y LECTURE 1. PPT 42

Fluidics - Flow Chambers Sheath flow Flow through cuvette (sense in quartz) droplets [RFM] © 1988 -2004 J. Paul Robinson, Purdue University BMS 633 A –BME 695 Y LECTURE 1. PPT Modified Figure from H. B. Steen - MLM Chapt. 2 43

Fluidics - Flow Chambers Closed cross flow chamber Modified figure from H. B. Steen

Flow Cell Injector Tip Sheath fluid Fluorescence signals Focused laser beam Forward scatter ©

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 633 A –BME 695 Y LECTURE 1. PPT 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 633 A –BME 695 Y LECTURE 1. PPT 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 633 A –BME 695 Y LECTURE 1. PPT

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 633 A –BME 695 Y LECTURE 1. PPT 49

BMS 633 A-BME 695 Y — Week 1