© J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT Page 1 BMS 633A-BME 695Y - Week 1 Flow Cytometry Module for Engineering Students Contact Information: Hansen Hall, B050 Purdue University Office: Fax \; WEB Last modified January 9, 2004 J. Paul Robinson Professor of Biomedical Engineering Professor of Immunopharmacology School of Veterinary Medicine, Purdue University 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

© J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT Page 2 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

© J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT Page 3 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%

© J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT Page 4 Sources of information Flow Cytometry and Sorting, 2nd ed. (M.R. Melamed, T. Lindmo, M.L. Mendelsohn, eds.), Wiley-Liss, New York, 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 Practical Flow Cytometry 3nd 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 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! More information on flow cytometry books can be found on our website at: 4 th Edn Some slides are based on slides taken from Dr. Robert Murphy [RFM]

© J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT Page 5 Primary Text Practical Flow Cytometry 3nd edition (2003),H. Shapiro: Alan R. Liss, New York - referred to as PFC Amazon Order page for Shapiro 2 nd Ed You can buy 2 nd hand copies for around $80 on Amazon and its related sites

© J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT Page 6 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

© J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT Page 7 Additional Sources Powerpoint presentations references as J.Paul Robinson ( JPR ); Robert Murphry ( RFM ), Carleton Stewart ( CS ) Web sources of these presentation are: Additional Sources include the Purdue Cytometry CD-ROM series Vol. 5 Vol. 6 Vol. 7 Vol. 1 Vol. 2 Vol. 3 Vol. 4 Free copies of any of these Are available to you just For the asking!

© J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT Page 8 Key Reference Text The course will use Shapiro: Practical Flow Cytometry, 3nd 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.

© J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT Page 9 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 1-60; Watson pp 1-4; Givan pp 1-9)

© J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT Page 10 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

© J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT Page 11 What can Flow Cytometry Do? Enumerate particles in suspension Determine “biologicals” from “non-biologicals” Separate “live” from “dead” particles Evaluate 10 5 to 10 6 particles in less than 1 min Measure particle-scatter as well as innate fluorescence or 2 o fluorescence Sort single particles for subsequent analysis

© J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT Page 12 The growth of flow cytometry based on publications 52,196 references 1 st use of keyword Publications using the keyword “ flow cytometry ”

© J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT Page 13 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: 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

© J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT Page 14 Fluorescence Labeling Technique Coons et al 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 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: , 1941 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: , 1941 “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) 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

© J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT Page 15 Basics of Flow Cytometry...cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence that is collected, filtered and converted to digital values that are stored on a computer... Fluidics Optics Electronics

© J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT Page 16 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

© J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT Page 17 Commercial Instruments

© J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT Page 18 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

© J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT Page 19 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

© J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT Page 20 Technical Components Detection Systems Photomultiplier Tubes (PMTs) Historically 1-2 Current Instruments 3-15 Diodes Light scatter detectors (plus PMTs) Illumination Systems Lasers ( , 420, 457, 488, 514, 532, 600, 633 nm) Argon ion, Krypton ion, HeNe, HeCd, YAG Arc Lamps Mercury, Mercury-Xenon (most lines)

© J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT Page 21 Data are collected as histograms Parameter 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.

© J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT Page 22 Data Analysis Concepts 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 As part of that comparison of histograms, it is necessary to create complex multivariate data sets. Many variables are compared simultaneously with Boolean logic.

© J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT Page 23 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 FITC Fluorescence Mo1 CD4 CD8 CD45 leu11a CD20 Tube ID - TIP Tube Identifier Parameter: Reference - Cytometry 12:82-90, TIG – Time Interval Gating: Refgerence: Cytometry, 12: , 1991

© J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT Page 24 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

© J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT Page 25 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 ( 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]

© J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT Page 26 Whether flow will be laminar can be determined from the Reynolds number When R e < 2300, flow should be laminar When R e > 2300, flow can be turbulent Fluidics - Laminar Flow [RFM]

Figure from V. Kachel, H. Fellner-Feldegg & E. Menke - MLM Chapt. 3 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] Fluidics

© J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT Page 28 Fluidics How do we accomplish sample injection and regulate sample flow rate? –Differential pressure –Volumetric injection [RFM]

© J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT Page 29 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]

© J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT Page 30 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]

© J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT Page 31 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 Positive Displacement Syringe Systems 1-2 ms -1 flow rate Fixed volume (50  l or 100  l) Absolute number calculations possible Usually fully enclosed flow cells 100  l Sample loop Sample Waste Flowcell 3-way valve Syringe

© J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT Page 32 Sample tube Sample station of the Coulter XL analyzer Sample is delivered to flow Cell from here Waste tanks accept waste Sheath tanks deliver sheath

© J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT Page 33 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]

© J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT Page 34 Syringe systems Bryte HS Cytometer 3 way valve Syringe Sample line Sheath fluid

Fluidics - Volumetric Injection System H.B. Steen - MLM Chapt. 2 nozzle

© J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT Page 36 Hydrodynamic Systems MicroscopeObjective Waste FlowCell CoverslipSignals Microscope Objective Waste Flow Cell Coverslip Signals

© J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT Page 37 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]

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.” Figure from V. Kachel, et al. - MLM Chapt. 3 [RFM]

© J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT Page 39 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]

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

© J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT Page 41 Coulter XL Analyzer Sample tube Sheath and waste system

© J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT Page 42 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 best optical properties, can’t sort [RFM]

Fluidics - Flow Chambers Modified Figure from H.B. Steen - MLM Chapt. 2 Flow through cuvette (sense in quartz) [RFM] droplets Sheath flow

Fluidics - Flow Chambers Modified figure from H.B. Steen - MLM Chapt. 2 Closed cross flow chamber [RFM]

© J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT Page 45 Flow Cell Injector Tip Fluorescence signals Focused laser beam Sheath fluid Forward scatter

© J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT Page 46 Hydrodynamic Systems Sample in Sheath Sheath in Laser beam Piezoelectric crystal oscillator Fluorescence Sensors Scatter Sensor Core Sheath

© J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT Page 47 Flow chamber blockage 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. hair

© J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT Page 48 From laser Fluorescence collection lens, optical filters, dichroic filter, band pass filter Beam shaping lens reflector Flow cell body dichroics

© J.Paul Robinson, Purdue University BMS 633A –BME 695Y LECTURE 1.PPT Page 49 Lecture 1 Summary History – how, when, where and why Technical highlights – operational principles Mechanics of flow Fluidics