Some Basic Physics & Optics 2:34 PM

Photons and Quantum Theory 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 10-34 joule-seconds c = speed of light (3x108 m/s) 3rd Ed Shapiro p 76 2:34 PM

Electromagnetic Spectrum We can see from about 370 nm to about 700 nm This is known as the visible spectrum Images from: http://www.lbl.gov/MicroWorlds/ALSTool/EMSpec/EMSpec2.html 2:34 PM

Interference A+B A B C+D C D Wavelength Amplitude Constructive The frequency does not change, but the amplitude is doubled A Amplitude B Constructive Interference Here we have a phase difference of 180o (2 radians) so the waves cancel each other out C+D C D Destructive Interference Figure modified from Shapiro 3rd Ed “Practical Flow Cytometry” Wiley-Liss, p79 4th Ed. Shapiro p 109 Shapiro 4th Ed P105 2:34 PM

Reflection and Refraction Snell’s Law: The angle of reflection (Ør) is equal to the angle of incidence (Øi) regardless of the surface material The angle of the transmitted beam (Øt) is dependent upon the composition of the material Transmitted (refracted)Beam Reflected Beam r t i Incident Beam n1 sin Øi = n2 sin Øt The velocity of light in a material of refractive index n is c/n 3rd Ed. Shapiro p 81 4th Ed. Shapiro p 106 2:34 PM

Refraction & Dispersion Short wavelengths are “bent” more than long wavelengths dispersion Light is “bent” and the resultant colors separate (dispersion). Red is least refracted, violet most refracted. 2:34 PM

Wavelength = Frequency The light spectrum Wavelength = Frequency Blue light 488 nm short wavelength high frequency high energy (2 times the red) Photon as a wave packet of energy Red light 650 nm long wavelength low frequency low energy 2:34 PM

Introduction to light scatter in flow Light scattering is a complex phenomenon. If the refractive index is not different from the surrounding medium (which may be water) there will be a negligible difference between RI of cell and medium Light scattering can be measured based on light scattered by a particle suspension light scattered by an individual particle The scattered fields; scattering angle and/or wavelength are important Particle parameters can be: Size, morphology, viability, structure, effective refractive index, etc. 2:34 PM

Light scattering Scattering Absorption Incident Beams Emission (Mie, Rayleigh) Absorption Emission (Fluorescence) Particles Incident Beams The interaction of light with a particle in terms of scattering, absorption, and emission. 2:34 PM 4

Forward & Side Scatter Forward Scatter (FSC): Diffracted light FSC is related to cell surface area (at narrow angles) FSC is detected along axis of incident light in the forward direction BUT – the accuracy for cell sizing is not good – it is very dependent upon the exact angle of scatter which can vary even between similar instruments Side Scatter (SSC): Reflected and Refracted light SSC is related to cell granularity and complexity SSC is detected (mostly) at 90° to the laser beam Factors that affect light scatter Shape, surface, size, granularity, internal complexity, refractive index. 2:34 PM

Why look at FSC vs SSC Since FSC ~ size and SSC ~ internal structure, a correlated measurement between them provides a basis for a simple differentiation between the major populations Sometimes scatter is presented this way….. FSC SSC Granulocytes Lymphocytes Monocytes RBCs, Debris, Dead Cells 2:34 PM

White blood cells Light Scatter Gating And sometimes scatter is presented this way….. 200 400 600 800 1000 Side Scatter Projection Forward Scatter Projection 90 Degree Scatter Neutrophils Lymphocytes Monocytes Light Scatter Gating 2:34 PM

© 1990-2012 J. Paul Robinson, Purdue University Lecture0004.ppt Fluorescence Photon emission as an electron returns from an excited state to ground state © 1990-2012 J. Paul Robinson, Purdue University Lecture0004.ppt 2:34 PM

© 1990-2012 J. Paul Robinson, Purdue University Lecture0004.ppt Fluorescence Excitation Spectrum Intensity of emission as a function of exciting wavelength (this is the absorbance component) Chromophores are components of molecules which absorb light They are frequently aromatic rings © 1990-2012 J. Paul Robinson, Purdue University Lecture0004.ppt 2:34 PM

Electromagnetic Spectrum Arc lamps and lasers for flow cytometry restricted to this region © Microsoft Corp, 1995 Only a very small region within the ES is used for flow cytometry applications © 1990-2012 J. Paul Robinson, Purdue University Lecture0004.ppt 2:34 PM

© 1990-2012 J. Paul Robinson, Purdue University Lecture0004.ppt Fluorescence Stokes Shift is the energy difference between the lowest energy peak of absorbance and the highest energy of emission Stokes Shift is 20 + nm Fluorescein molecule 495 nm 518 nm Fluorescnece Intensity © 1990-2012 J. Paul Robinson, Purdue University Lecture0004.ppt Wavelength 2:34 PM

Some Common Laser lines 600 nm 300 nm 500 nm 700 nm 400 nm 457 350 514 610 632 488 PE-TR Conj. Texas Red PI Ethidium PE FITC 325nm cis-Parinaric acid © 1990-2012 J. Paul Robinson, Purdue University Lecture0004.ppt 2:34 PM

© 1990-2012 J. Paul Robinson, Purdue University Lecture0004.ppt Photobleaching Defined as the irreversible destruction of an excited fluorophore Photobleaching is not a big problem for flow cytometry as long as the time window for excitation is very short (a few hundred microsconds) © 1990-2012 J. Paul Robinson, Purdue University Lecture0004.ppt 2:34 PM

Excitation - Emission Peaks % Max Excitation at 488 568 647 nm Fluorophore EXpeak EM peak FITC 496 518 87 0 0 Bodipy 503 511 58 1 1 Tetra-M-Rho 554 576 10 61 0 L-Rhodamine 572 590 5 92 0 Texas Red 592 610 3 45 1 CY5 649 666 1 11 98 Note: You will not be able to see CY5 fluorescence under the regular fluorescent microscope because the wavelength is too high. 2:34 PM © 1990-2012 J. Paul Robinson, Purdue University Lecture0004.ppt

© 1990-2012 J. Paul Robinson, Purdue University Lecture0004.ppt Probes for Proteins Probe Excitation Emission FITC 488 525 PE 488 575 APC 630 650 PerCP™ 488 680 Cascade Blue 360 450 Coumerin-phalloidin 350 450 Texas Red™ 610 630 Tetramethylrhodamine-amines 550 575 CY3 (indotrimethinecyanines) 540 575 CY5 (indopentamethinecyanines) 640 670 © 1990-2012 J. Paul Robinson, Purdue University Lecture0004.ppt 2:34 PM

Specific Organelle Probes Probe Site Excitation Emission BODIPY Golgi 505 511 NBD Golgi 488 525 DPH Lipid 350 420 TMA-DPH Lipid 350 420 Rhodamine 123 Mitochondria 488 525 DiO Lipid 488 500 diI-Cn-(5) Lipid 550 565 diO-Cn-(3) Lipid 488 500 BODIPY - borate-dipyrromethene complexes NBD - nitrobenzoxadiazole DPH - diphenylhexatriene TMA - trimethylammonium © 1990-2012 J. Paul Robinson, Purdue University Lecture0004.ppt 2:34 PM

© 1990-2012 J. Paul Robinson, Purdue University Lecture0004.ppt Fluorescence Overlap © 1990-2012 J. Paul Robinson, Purdue University Lecture0004.ppt 2:34 PM

© 1990-2012 J. Paul Robinson, Purdue University Lecture0004.ppt Fluorescence Overlap © 1990-2012 J. Paul Robinson, Purdue University Lecture0004.ppt 2:34 PM

© 1990-2012 J. Paul Robinson, Purdue University Lecture0004.ppt Fluorescence Overlap This is your bandpass filter Overlap of FITC fluorescence in PE PMT a © 1990-2012 J. Paul Robinson, Purdue University Lecture0004.ppt Overlap of PE fluorescence in FITC PMT b 2:34 PM

© 1990-2012 J. Paul Robinson, Purdue University Lecture0004.ppt Mixing fluorochromes When there are two molecules with different absorption spectra, it is important to consider where a fixed wavelength excitation should be placed. It is possible to increase or decrease the sensitivity of one molecule or another. © 1990-2012 J. Paul Robinson, Purdue University Lecture0004.ppt 2:34 PM

© 1990-2012 J. Paul Robinson, Purdue University Lecture0004.ppt Excitation of 3 Dyes with emission spectra J. Paul Robinson, Class lecture notes, BMS 631 © 1990-2012 J. Paul Robinson, Purdue University Lecture0004.ppt 2:34 PM

© 1990-2012 J. Paul Robinson, Purdue University Lecture0004.ppt Change of Excitation J. Paul Robinson, Class lecture notes, BMS 631 © 1990-2012 J. Paul Robinson, Purdue University Lecture0004.ppt 2:34 PM

Hydrodynamic Systems – Increase Sample Pressure Injector Tip Flow Chamber Sheath fluid Fluorescence signals Focused laser beam Increase sample pressure: Widen Core Increase turbulence 2:34 PM

Closed flow chambers – e.g. Beckman Elite, Altra, XL Forward Scatter detector Laser direction Fluorescence signals Photo: J. P Robinson 2:34 PM

Data Acquisition Each measurement from each detector is referred to as a “parameter” or “variable” Data are acquired as a “list” of the values for each “parameter” (variable) for each “event” (cell) .Data Collection Most flow cytometers collect between 3 and 10 "parameters" or "variables". Each variable can be used to discriminate some component of the cell populations. The standard for data collection has been known as the "Flow Cytometry Standard" or FCS data file structure. Data can be collected in either histogram mode or listmode. It is now routine to collect flow cytometry data in listmode because there are few constraints in the size of data collection files. Originally, flow cytometers did not have good network capability, small hard disks (5-20 Mb, and floppy drive that could save only about 1 Mb of data). With high capacity hard disks and Ethernet access now routine, listmode is the most useful collection modality. Data can be collected in a gated or ungated mode. If the data are ungated, all events registered by the instruments electronic circuits will be saved in listmode files. If gated collection is chosen, it is possible to collect a listmode file of only those events that satisfy certain criteria established by the operator. [RFM]

Data Acquisition - Listmode Structure of a list mode file .List Mode Data Essentially however, a listmode file is a series of numbers which sequentially list data for each variable collected on each cell. This is shown in the associated table provided.

Using Gates R1 log PE Region 1 established Gated on Region 1

Quadrant Analysis ( - +) (+ +) log PE (- -) (+ -) 2:34 PM