BMS 631 - LECTURE 5 Flow Cytometry: Theory J.Paul Robinson Professor of Immunopharmacology & Biomedical Engineering Purdue University Light Sources & Optical systems Hansen Hall, B050 Purdue University Office:
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BMS 631 - LECTURE 5 Flow Cytometry: Theory J.Paul Robinson Professor of Immunopharmacology & Biomedical Engineering Purdue University Light Sources & Optical systems Hansen Hall, B050 Purdue University Office: 494 0757 Fax 494 0517 email\; [email protected] Shapiro 97-115 WEB http://www.cyto.purdue.edu © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT Page 1 Illumination Sources • Lamps • Xenon-Mercury • Mercury • Lasers • • • • • Argon Ion (Ar) Krypton (Kr) Helium Neon (He-Ne) Helium Cadmium (He-Cd) YAG 3rd Ed. Shapiro p 98 © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.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] 3rd Ed. Shapiro p 98 © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT Page 3 Mercury Arc Lamps Lens Arc Lens © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT Page 4 Arc Lamp Excitation Spectra Xe Lamp Irradiance at 0.5 m (mW m-2 nm-1) Hg Lamp 3rd Ed. Shapiro p 99 © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT Page 5 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 © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.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 3rd Ed. Shapiro p 103 © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT Page 7 Lasers • Coherent Enterprise laser - UV-visible • Air cooled laser (Argon) © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT Page 8 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 10KW power) 3rd Ed. Shapiro p 106 © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT Page 9 Lasers Images only available for in-house use Not for publication purposes © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT Page 10 Argon & Krypton Lasers Kr-Ar laser (488, 568, 647 nm lines) (Front) 3rd. Ed. Shapiro p 108 © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT Page 11 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 6G) 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 3rd. Ed.Shapiro p 110 © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.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 mW power • Lines available include green (543nm) and red 633 nm, 594nm or 611 nm. 3rd. Ed. Shapiro p 110 © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT Page 13 Helium-Cadmium Lasers • He-Cd laser • 5-200mW 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 kHz) • RMS noise mostly about 1.5% • Good for ratio measurements (pH or calcium) because power fluctuations don’t matter here – these lasers do have power fluctuation problems eventually. He-Cd laser 3rd. Ed. Shapiro p 111 © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.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 (GaAlAs) • 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 3rd Ed. Shapiro p113 © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.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 • 100s mWs 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) © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.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 3rd. Ed. Shapiro p 114 © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.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 © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.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 © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT Page 19 Optical Collection systems He-Cd Laser 2nd Argon Laser He-Ne Laser Argon Laser Optical layout of an Elite sorter at Purdue University © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT Page 20 Objectives • 1.3 NA objective Objective Harald Steen’s Bryte Cytometer © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT Page 21 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 © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.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. © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.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) © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT Page 24 Spectral Selection (Next lecture) • Monochromators Vs Filters • Filters are reasonably inexpensive © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT Page 25 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 © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT Page 26