1. Page 1 © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT BMS 631 - LECTURE 5 Flow Cytometry: Theory J.Paul Robinson Professor of Immunopharmacology & Biomedical Engineering Purdue University Hansen Hall, B050 Purdue University Office: 494 0757 Fax 494 0517 email\; robinson@flowcyt.cyto.purdue.edu WEB http://www.cyto.purdue.edu Light Sources & Optical systems Shapiro 97-115

2. Page 2 © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT 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

3. Page 3 © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT 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

4. Page 4 © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT Mercury Arc Lamps Arc Lens

5. Page 5 © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT Arc Lamp Excitation Spectra Irradiance at 0.5 m (mW m -2 nm -1 ) Xe Lamp Hg Lamp 3 rd Ed. Shapiro p 99

6. Page 6 © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT Optics - Optical Channels An optical channel is a path that light can follow from the illuminated volume to a detector separation wavelength selectionOptical elements provide separation of channels and wavelength selection

7. Page 7 © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT 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

8. Page 8 © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT Lasers Coherent Enterprise laser - UV-visible Air cooled laser (Argon)

9. Page 9 © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT 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) 3 rd Ed. Shapiro p 106

10. Page 10 © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT Lasers Images only available for in-house use Not for publication purposes

11. Page 11 © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT Argon & Krypton Lasers 3 rd. Ed. Shapiro p 108 Kr-Ar laser (488, 568, 647 nm lines) (Front)

12. Page 12 © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT 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 3 rd. Ed.Shapiro p 110

13. Page 13 © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT 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. 3 rd. Ed. Shapiro p 110

14. Page 14 © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT 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 dont matter here – these lasers do have power fluctuation problems eventually. 3 rd. Ed. Shapiro p 111 He-Cd laser

15. Page 15 © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT 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 3 rd Ed. Shapiro p113

16. Page 16 © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT 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)

17. Page 17 © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT 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

18. Page 18 © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT 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

19. Page 19 © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT Goals of Light Collection Maximum signal, minimum noise Maximum area of collection Inexpensive system if possible Easy alignment Reduced heat generation Reduced power requirement

20. Page 20 © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT Optical Collection systems He-Cd Laser Argon Laser He-Ne Laser 2 nd Argon Laser Optical layout of an Elite sorter at Purdue University

21. Page 21 © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT Objectives 1.3 NA objective Objective Harald Steens Bryte Cytometer

22. Page 22 © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT 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

23. Page 23 © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT Optical translators The laser beam remains parallel, but horizontally translated. This reduces the difficulty in aligning the laser. No cytometer should be without one!!!

24. Page 24 © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT 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)

25. Page 25 © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT Spectral Selection (Next lecture) Monochromators Vs Filters Filters are reasonably inexpensive

26. Page 26 © 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT 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