BMS 631 — LECTURE 5 Flow Cytometry Theory

BMS 631 - LECTURE 5 Flow Cytometry: Theory J. Paul Robinson Professor of Immunopharmacology & Biomedical Engineering Purdue University Light Sources & Optical systems Hansen Hall, B 050 Purdue University Office: 494 0757 Fax 494 0517 email; robinson@flowcyt. cyto. purdue. edu Shapiro 97 -115 WEB http: //www. cyto. purdue. edu © 1990 -2002 J. Paul Robinson, Purdue University BMS 631 – LECTURE 005. PPT Page 1

Illumination Sources • Lamps • Xenon-Mercury • Mercury • Lasers • • • Argon Ion (Ar) Krypton (Kr) Helium Neon (He-Ne) Helium Cadmium (He-Cd) YAG 3 rd Ed. Shapiro p 98 © 1990 -2002 J. Paul Robinson, Purdue University BMS 631 – LECTURE 005. PPT Page 2

Optics - Light Sources Epilumination in Flow Cytometers • Arc-lamps – provide mixture of wavelengths that must be filtered to select desired wavelengths – provide milliwatts of light – inexpensive, air-cooled units – provide incoherent light [RFM] 3 rd Ed. Shapiro p 98 © 1990 -2002 J. Paul Robinson, Purdue University BMS 631 – LECTURE 005. PPT Page 3

Mercury Arc Lamps Lens Arc Lens © 1990 -2002 J. Paul Robinson, Purdue University

Arc Lamp Excitation Spectra Xe Lamp Irradiance at 0. 5 m (m. W m-2

Optics - Optical Channels • An optical channel is a path that light can follow from the illuminated volume to a detector • Optical elements provide separation of channels and wavelength selection © 1990 -2002 J. Paul Robinson, Purdue University BMS 631 – LECTURE 005. PPT Page 6

Spot Illumination - Lasers • Advantages are that the pathway is easier to define (you know where the light is going !!) • It is usually monochromatic light so excitation filters are not needed • Brighter source of light than arc lamps (higher radiance) • Spot size (d) can be calculated by formula – d=1. 27( F/D) where D is the beam diameter in mm and F is the focal distance from the lens • For a 125 mm focal length spherical lens at 515 nm is 55 um and 61 um at 458 nm 3 rd Ed. Shapiro p 103 © 1990 -2002 J. Paul Robinson, Purdue University BMS 631 – LECTURE 005. PPT Page 7

Lasers • Coherent Enterprise laser - UV-visible • Air cooled laser (Argon) © 1990

Laser Power & Noise Light Amplification by Stimulated Emission of Radiation • Laser light is coherent and monochromatic (same frequency and wavelength) • this means the emitted radiation is in phase with and propagating in the same direction as the stimulating radiation • ION lasers use electromagnetic energy to produce and confine the ionized gas plasma which serves as the lasing medium. • Lasers can be continuous wave (CW) or pulsed (where flashlamps provide the pulse) • Laser efficiency is variable - argon ion lasers are about 0. 01% efficient (1 W needs 10 KW power) 3 rd Ed. Shapiro p 106 © 1990 -2002 J. Paul Robinson, Purdue University BMS 631 – LECTURE 005. PPT Page 9

Lasers Images only available for in-house Not for publication purposes © 1990 -2002 J.

Argon & Krypton Lasers Kr-Ar laser (488, 568, 647 nm lines) (Front) 3 rd.

Dye Lasers • Dye lasers use a source laser known as the pump laser to excite another laser known as the dye laser. • The dye laser consists of a flowing dye which exhibits desirable properties such as excitation and emission. • The lasing medium is a fluorescent dye (e. g. Rhodamine 6 G) which is dissolved in an organic solvent such as ethanol or ethylene glycol • The laser can be tuned, usually by a rotatable filter or prism • The dye must be circulated and cooled to prevent it being bleached or over-heated 3 rd. Ed. Shapiro p 110 © 1990 -2002 J. Paul Robinson, Purdue University BMS 631 – LECTURE 005. PPT Page 12

Helium-Neon Lasers • He-Ne - low power, no cooling needed • Cheap, mostly emit red light at 633 nm • Generally 0. 1 W to 50 m. W power • Lines available include green (543 nm) and red 633 nm, 594 nm or 611 nm. 3 rd. Ed. Shapiro p 110 © 1990 -2002 J. Paul Robinson, Purdue University BMS 631 – LECTURE 005. PPT Page 13

Helium-Cadmium Lasers • He-Cd laser • 5 -200 m. W power usually at 325 nm (UV) or 441 nm (blue) • Wall power, air cooled • Uses cadium vapor as the lasing medium • Major problem is noise (plasma noise between 300 -400 k. Hz) • RMS noise mostly about 1. 5% • Good for ratio measurements (p. H or calcium) because power fluctuations don’t matter here – these lasers do have power fluctuation problems eventually. He-Cd laser 3 rd. Ed. Shapiro p 111 © 1990 -2002 J. Paul Robinson, Purdue University BMS 631 – LECTURE 005. PPT Page 14

Diode Lasers • Small, efficient, cheap • Only red wavelengths available at reasonable prices (blue works, but still problems) • Mostly made of Gallium aluminum arsenide (Ga. Al. As) • Emission ratio is varied by changing the ration of gallium to aluminum in the semiconductor • Main use is CD players (now 2 in every household!! One in the stereo and one in the computer! And maybe one in the laser printer!) • Biggest problem is not power - but lack of fluorescent probes to be excited at 650 -900 nm • Problem is poor beam profiles for diode lasers • Noise levels are generally 0. 05% or less compared to 1% for air cooled argon and. 02% with water cooled argon lasers 3 rd Ed. Shapiro p 113 © 1990 -2002 J. Paul Robinson, Purdue University BMS 631 – LECTURE 005. PPT Page 15

Solid State Lasers • Neodynymium-YAG (Yttrium aluminum garnet) lasers • Lasing medium is a solid rod of crystalline material pumped by a flashlamp or a diode laser • 100 s m. Ws at 1064 nm • can be doubled or tripled to produce 532 nm or 355 nm • Noisy - and still reasonably expensive (particularly the double and tripled versions) © 1990 -2002 J. Paul Robinson, Purdue University BMS 631 – LECTURE 005. PPT Page 16

Lasers Hazards • Laser light is very dangerous and should be treated as a significant hazard • Water cooled lasers have additional hazards in that they require high current and voltage in addition to the water hazard • Dye lasers use dyes that can be potentially carcinogenic 3 rd. Ed. Shapiro p 114 © 1990 -2002 J. Paul Robinson, Purdue University BMS 631 – LECTURE 005. PPT Page 17

Summary so far…. • Arc lamps are useful for flow cytometry because of low cost and wide spectral characteristics • Arc lamps require more complex optical trains • Lasers provide light at high radiance • Lasers are essentially monochromatic, coherent • Lasers represent a significant hazard © 1990 -2002 J. Paul Robinson, Purdue University BMS 631 – LECTURE 005. PPT Page 18

Goals of Light Collection • • • Maximum signal, minimum noise Maximum area of collection Inexpensive system if possible Easy alignment Reduced heat generation Reduced power requirement © 1990 -2002 J. Paul Robinson, Purdue University BMS 631 – LECTURE 005. PPT Page 19

Optical Collection systems He-Cd Laser 2 nd Argon Laser He-Ne Laser Argon Laser Optical layout of an Elite sorter at Purdue University © 1990 -2002 J. Paul Robinson, Purdue University BMS 631 – LECTURE 005. PPT Page 20

Objectives • 1. 3 NA objective Objective Harald Steen’s Bryte Cytometer © 1990 -2002

Field stops & obscuration bars • Obscuration bar is placed along the path of the illuminating beam • It blocks the direct light but allows the fluorescence signal (which is going in all directions) • In a capillary or cuvet system, a field stop which is placed in the image plane achieves the same result © 1990 -2002 J. Paul Robinson, Purdue University BMS 631 – LECTURE 005. PPT Page 22

Optical translators No cytometer should be without one!!! The laser beam remains parallel, but horizontally translated. This reduces the difficulty in aligning the laser. © 1990 -2002 J. Paul Robinson, Purdue University BMS 631 – LECTURE 005. PPT Page 23

The point of a good optical system is to obtain a good Signal Vs Noise • Good optical filters • Remove as much excitation signal as possible • Collect as much fluorescence as possible (use concave spherical mirrors) © 1990 -2002 J. Paul Robinson, Purdue University BMS 631 – LECTURE 005. PPT Page 24

Spectral Selection (Next lecture) • Monochromators Vs Filters • Filters are reasonably inexpensive ©

Lecture Summary • After completing this lecture you should understand: • Excitation light sources and their properties • Each light source has unique utility • Optical components together with light source creates an optical system • The general nature of optical systems in typical cytometers © 1990 -2002 J. Paul Robinson, Purdue University BMS 631 – LECTURE 005. PPT Page 26