© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt Page 1 BMS Lecture 2 Who’s and Why’s of Flow Cytometry The History of Flow Cytometry: An introduction to the early beginnings of flow cytometers; the rationale for early investigations; a summary of the state-of-the-art; the events that led to modern cytometry; early fluorescent dyes; image analysis; DNA cytology J.Paul Robinson, PhD Professor of Immunopharmacology and Bioengineering References: (Shapiro 3rd ed. pp43-71) Note: these slides were converted to web slides by Microsoft PowerPoint directly. Microsoft made such a bad job of this process that all text boxes had to be eliminated because they did not translate at all – so forgive the problems – they are mostly Bad Microsoft programming thanks Bill!

© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt Page 2 Herzenberg Lou Herzenberg sorter based on fluorescence (arc lamp) built after working with one of Kamentsky’s RCS systems where they built an instrument they called the Fluorescence Activated Cell Sorter (FACS) Photos ©2000 – J.P. Robinson

© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt Page 3 Dittrich & Göhde Dittrich & Gohde Impulscytophotometer (ICP)- used ethidium bromide for a DNA stain and a high NA objective used as a condenser and collection lens Laerum, Göhde, Darzynkiewicz (1998) Photos ©2000 – J.P. Robinson Göhde and Laerum (1998)

© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt Page 4 Kamentsky Kamentsky - Bio/Physics Systems commercial cytometer - the “Cytograph” He-Ne laser system at 633 nm for scatter (and extinction) - supposedly the first commercial instrument incorporating a laser. It could separate live and dead cells by uptake of Trypan blue. A fluorescence version called the “Cytofluorograph” followed using an air cooled argon laser at 488 nm excitation 1970 Cytograph presently at the Purdue University Cytometry Laboratories Photo ©2000 – J.P. Robinson

© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt Page 5 History Phywe AG of Gottingen (1970) - produced a commercial version of the ICP built around a Zeiss fluorescent microscope Don’t have photo….

© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt Page 6 Herzenberg & Becton Dickinson Herzenberg Argon laser flow sorter - placed an argon laser onto their sorter and successfully did high speed sorting - Coined the term Fluorescence Activated Cell Sorting (FACS) This instrument could detect weak fluorescence with rhodamine and fluorescein tagged antibodies. A commercial version was distributed by B-D in 1974 and could collect forward scatter and fluorescence above 530 nm. Photo ©2000 – J.P. Robinson

© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt Page 7 Mack Fulwyler Coulter Electronics manufactured the TPS-1 (Two parameter sorter) in 1975 which could measure forward scatter and fluorescence using a 35mW argon laser. This photo (©2000 – J.P. Robinson) is one of only one or two surviving TPS Instruments. It is very similar to the Coulter Counter of the day. Photo ©2000 – J.P. Robinson

© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt Page 8 Shapiro Shapiro and the Block instruments ( ) - a series of multibeam flow cytometers that did differentials and multiple fluorescence excitation and emission Photos ©2000 – J.P. Robinson

© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt Page 9 Hemalog D Technicon - Hemalog D first commercial differential flow cytometer - light scatter and absorption at different wavelengths - chromogenic enzyme substrates were used to identify neutrophils and eosinophils by peroxidase and monocytes by esterase, basophils were identified by the presence of glycosaminoglycans using Alcian Blue - the excitation for all measurements was a tungsten-halogen lamp Insert photos on page 60 Image from Shapiro “Practical Flow Cytometry”, Wiley-Liss, 1995

© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt Page 10 Coulter Electronics developed the Epics series of instruments which were essentially 5 watt argon ion laser instruments, complete with a multiparameter data analysis system, floppy drive and graphics printer. Epics V front end (left) and MDADS (right)currently at Purdue University Photo ©2000 – J.P. Robinson

© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt Page 11 Biophysics -Ortho Ortho Diagnostics (Johnson and Johnson) purchased Biophysics in 1976 and in 1977 the System 50 Cytofluorograph was developed - this was a droplet sorter, with a flat sided flow cell, forward and orthogonal scatter, extinction, 2 fluorescence parameters, multibeam excitation, computer analysis option NIH scientists had added a krypton laser at 568 nm to excite Texas Red fluorescence at 568 nm and emit at nm. Argon (488 nm FITC was measured simultaneously without signal cross-talk - thus the FACS IV was developed (B-D). Photo ©2000 – J.P. Robinson

© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt Page 12 Stuart Schlossman Schlossman at the Farber Institute in Boston, began to make monoclonal antibodies to white blood cell antigens in Eventually he collaborated with Ortho Diagnostics who distributed the famous “OK T4” etc., Mabs Coulter Immunology also distributed his antibodies

© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt Page 13 Introductory Terms and Concepts Parameter/Variable Light Scatter- Forward (FALS), narrow (FS) - Side, Wide, 90 deg, orthogonal Fluorescence - Spectral range Absorption Time Count

© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt Page 14 Concepts Scatter: Size, shape, granularity, polarized scatter (birefringence) Fluorescence: Intrinsic: Endogenous pyridines and flavins Extrinsic: All other fluorescence profiles Absorption: Loss of light (blocked) Time: Useful for kinetics, QC Count: Always part of any collection

© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt Page 15 Instrument Components Electronics: Control, pulse collection, pulse analysis, triggering, time delay, data display, gating, sort control, light and detector control Optics: Light source(s), detectors, spectral separation Fluidics: Specimen, sorting, rate of data collection Data Analysis: Data display & analysis, multivariate/simultaneous solutions, identification of sort populations, quantitation

© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt Page 16 Fundamentals of a Flow Cytometer

© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt Page 17 Data Analysis Concepts Gating Single parameter Dual parameter Multiple parameter Back Gating Note: these terms are introduced here, but will be discussed in more detail in later lectures

© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt Page 18 Data Presentation Formats Histogram Dot plot Contour plot 3D plots Dot plot with projection Overviews (multiple histograms)

© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt Page 19 Light and Matter J.Paul Robinson Professor of Immunopharmacology School of Veterinary Medicine, Purdue University Hansen Hall, B050 Purdue University Office: Fax \; WEB Shapiro p 75-93

© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt Page 20 Light and Matter Energy –joules, radiant flux (energy/unit time) –watts (1 watt=1 joule/second) Angles –steradians - sphere radius r - circumference is 2  r 2 ; the angle that intercepts an arc r along the circumference is defined as 1 radian. (57.3 degrees) a sphere of radius r has a surface area of 4  r 2. One steradian is defined as the solid angle which intercepts as area equal; to r 2 on the sphere surface 3 rd Ed - Shapiro p 75

© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt Page 21 Terms Side scatter, forward angle scatter, cell volume, coulter volume: Understand light scattering concepts; intrinsic and extrinsic parameters Photometry: Light - what is it - wavelengths we can see nm, most sensitive around 550 nm. Below 400 nm essentially measuring radiant energy. Joules (energy) radiant flux (energy per unit time) is measured in watts (1 watt=1 joule/second). Steradian (sphere radius r has surface area of 4  r 2 ; one steradian is defined as that solid angle which intercepts an area equal to r 2 on the surface. Mole - contains Avogadro's number of molecules (6.02 x ) and contains a mass in grams = molecular weight. Photons - light particles - waves - Photons are particles which have no rest mass - pure electromagnetic energy - these are absorbed and emitted by atoms and molecules as they gain or release energy. This process is quantized, is a discrete process involving photons of the same energy for a given molecule or atom. The sum total of this energy gain or loss is electromagnetic radiation propagating at the speed of light (3 x 10 8 m/s). The energy (joules) of a photon is E=h and E=h /l [ -frequency, l-wavelength, h-Planck's constant 6.63 x joule- seconds] Energy - higher at short wavelengths - lower at longer wavelengths.

© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt Page 22 Photons and Quantum Theory Photons –particles have no rest mass - composed of pure electromagnetic energy - the absorption and emission of photons by atoms and molecules is the only mechanism for atoms and molecules can gain or lose energy Quantum mechanics –absorption and emission are quantized - i.e. discrete process of gaining or losing energy in strict units of energy - i.e. photons of the same energy (multiple units are referred to as electromagnetic radiation) Energy of a photon –can be computed from its frequency ( ) in hertz (Hz) or its wavelength (l) in meters from E=h and E=hc/ = wavelength h = Planck’s constant (6.63 x joule-seconds c = speed of light (3x10 8 m/s) 3 rd Ed Shapiro p 76

© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt Page x Laser power One photon from a 488 nm argon laser has an energy of E= 6.63x joule-seconds x 3x10 8 To get 1 joule out of a 488 nm laser you need 2.45 x photons 1 watt (W) = 1 joule/second a 10 mW laser at 488 nm is putting out 2.45x10 16 photons/sec E=h and E=hc/ = 4.08x J 3 rd Ed. Shapiro p 77

© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt Page x What about a UV laser? E= 6.63x joule-seconds x 3x10 8 = 6.12 x J so 1 Joule at 325 nm = 1.63x10 18 photons What about a He-Ne laser? 633 x E= 6.63x joule-seconds x 3x10 8 = 3.14 x J so 1 Joule at 633 nm = 3.18x10 18 photons 3 rd Ed. Shapiro p 77

© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt Page 25 Polarization and Phase: Interference Electric and magnetic fields are vectors - i.e. they have both magnitude and direction The inverse of the period (wavelength) is the frequency in Hz 3 rd Ed. Shapiro p 78 Wavelength (period T) Axis of Magnetic Field Axis of Propagation Axis of Electric Field

© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt Page 26 Interference Constructive Interference Destructive Interference A B C D A+B C+D Amplitude 0o0o 90 o 180 o 270 o 360 o Wavelength Figure modified from Shapiro “Practical Flow Cytometry” Wiley-Liss, p79 Here we have a phase difference of 180 o (2  radians) so the waves cancel each other out The frequency does not change, but the amplitude is doubled

© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt Page 27 Light Scatter Materials scatter light at wavelengths at which they do not absorb If we consider the visible spectrum to be nm then small particles (< 1/10 ) scatter rather than absorb light For small particles (molecular up to sub micron) the Rayleigh scatter intensity at 0 o and 180 o are about the same For larger particles (i.e. size from 1/4 to tens of wavelengths) larger amounts of scatter occur in the forward not the side scatter direction - this is called Mie Scatter (after Gustav Mie) - this is how we come up with forward scatter be related to size Shapiro p 79

© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt Page 28 Rayleigh Scatter Molecules and very small particles do not absorb, but scatter light in the visible region (same freq as excitation) Rayleigh scattering is directly proportional to the electric dipole and inversely proportional to the 4th power of the wavelength of the incident light the sky looks blue because the gas molecules scatter more light at shorter (blue) rather than longer wavelengths (red)

© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt Page 29 Reflection and Refraction Snell’s Law: The angle of reflection (Ø r ) is equal to the angle of incidence (Ø i ) regardless of the surface material The angle of the transmitted beam (Ø t ) is dependent upon the composition of the material Shapiro p 81 tt ii rr Incident Beam Reflected Beam Transmitted (refracted)Beam n 1 sin Ø i = n 2 sin Ø t The velocity of light in a material of refractive index n is c/n

© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt Page 30 Refraction & Dispersion Light is “bent” and the resultant colors separate (dispersion). Red is least refracted, violet most refracted. dispersion Short wavelengths are “bent” more than long wavelengths ref rac tion

© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt Page 31 Brewster’s Angle Brewster’s angle is the angle at which the reflected light is linearly polarized normal to the plane incidence At the end of the plasma tube, light can leave through a particular angle (Brewster’s angle) and essentially be highly polarized Maximum polarization occurs when the angle between reflected and transmitted light is 90 o thus Ø r + Ø t = 90 o since sin (90-x) = cos x Snell’s provides (sin Ø i / cos Ø i ) = n 2 /n 1 Ø r is Brewster’s angle Shapiro p 82 Ø r = tan -1 (n 2 /n 1 )

© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt Page 32 Brewster’s Angle Photo ©2000 – J.P. Robinson

© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt Page 33 Lecture Summary History of Flow Principles of light and matter Basic Optics Essentials of lasers