1. © 1990-2005 J.Paul Robinson, Purdue University Lecture0005.ppt BMS 631 – Lecture 5 Properties and Applications of Light Sources J. Paul Robinson, PhD Professor of Immunopharmacology Professor of Biomedical Engineering Purdue University last modified February 2, 2005 At the conclusion of this lecture students will have an excellent understanding of the technical components and operation of flow cytometers with relation to the nature of light and its properties. Slides are designed w/o backgrounds to be printable on a B/W printer. Material relies heavily on Shapiro’s Practical Flow Cytometry, Wiley-Liss, 1994 or 2003 (4 th Ed) The WEB version of these slides can be found on http://www.cyto.purdue.edu/class

2. © 1990-2005 J.Paul Robinson, Purdue University Lecture0005.ppt Learning Objectives Identify the types of light sources used in flow cytometers Define the nature of each light source Understand the advantages and disadvantages of each system Understand the dangers involved with lasers

3. © 1990-2005 J.Paul Robinson, Purdue University Lecture0005.ppt Illumination Sources Lamps Xenon-Mercury Mercury Lasers Argon Ion (Ar) Krypton (Kr) Helium Neon (He-Ne) Helium Cadmium (He-Cd) YAG (solid State) Diodes Variety of wavelengths, cheap 3 rd Shapiro p 98 4 th Shapiro p 124 3 rd Shapiro p 98 4 th Shapiro p 124

4. © 1990-2005 J.Paul Robinson, Purdue University Lecture0005.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 Shapiro p 98 4 th Shapiro p 126 3 rd Shapiro p 98 4 th Shapiro p 126

5. © 1990-2005 J.Paul Robinson, Purdue University Lecture0005.ppt © J.Paul Robinson Mercury Arc Lamps Arc Lens

6. © 1990-2005 J.Paul Robinson, Purdue University Lecture0005.ppt Arc Lamp Excitation Spectra Irradiance at 0.5 m (mW m -2 nm -1 )         Xe Lamp Hg Lamp 3 rd Shapiro p 99 4 th Shapiro p 125 3 rd Shapiro p 99 4 th Shapiro p 125

7. © 1990-2005 J.Paul Robinson, Purdue University Lecture0005.ppt Lasers Coherent light Noncoherent light

8. © 1990-2005 J.Paul Robinson, Purdue University Lecture0005.ppt Lasers Hazards Laser light is very dangerous and should be treated as a significant hazard You should use laser protection goggles when using open lasers 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 Shapiro p 114 4 th Shapiro p 148 3 rd Shapiro p 114 4 th Shapiro p 148

9. © 1990-2005 J.Paul Robinson, Purdue University Lecture0005.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  m and 61  m at 458 nm 3 rd Ref: Shapiro p 103 4 th Ref: Shapiro p 130 3 rd Ref: Shapiro p 103 4 th Ref: Shapiro p 130

10. © 1990-2005 J.Paul Robinson, Purdue University Lecture0005.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 Ref: Shapiro p 106 4 th Shapiro p 136, 147 3 rd Ref: Shapiro p 106 4 th Shapiro p 136, 147

11. © 1990-2005 J.Paul Robinson, Purdue University Lecture0005.ppt © J.Paul Robinson 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 594nm or 611 nm 3 rd Shapiro p 110 4 th Shapiro 141 3 rd Shapiro p 110 4 th Shapiro 141

12. © 1990-2005 J.Paul Robinson, Purdue University Lecture0005.ppt © J.Paul Robinson Helium-Cadmium Lasers He-Cd laser 5-200mW power usually at 325 nm (UV) or 441 nm (blue) Wall power, air cooled Uses cadmium 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 3 rd Ref: Shapiro p 111 4 th Ref: Shapiro p 142 3 rd Ref: Shapiro p 111 4 th Ref: Shapiro p 142 He-Cd laser

13. © 1990-2005 J.Paul Robinson, Purdue University Lecture0005.ppt 405 nm & 375 nm Lasers These lasers are long lived and quite stable Can be fiber optically delivered but the fibers may not last long (1000 hours) Images from Point Source, www.point-source.com

14. © 1990-2005 J.Paul Robinson, Purdue University Lecture0005.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 this is the typical green laser pointer Noisy - and still reasonably expensive (particularly the double and tripled versions)

15. © 1990-2005 J.Paul Robinson, Purdue University Lecture0005.ppt About new diode lasers… The question: Which source of red >light is nowadays more suitable for flow cytometers in terms of power, stability (noise), life, maintenance and prize? Facscalibur has a red diode 635nm but I think that new LSR is provided with an He-Ne laser 633nm. The shortest answer is that whichever laser the manufacturer will sell you with some reasonable warranty should do the job. He-Ne lasers are larger, consume more power, and usually cost more per milliwatt than red diodes; they have nicer beam shapes (TEM00), and they don't have much (but do have some) long wavelength incoherent emission at wavelengths in the region of some of the fluorescence you're trying to excite with the primary beam. Noise on air-cooled He-Ne's with reasonable power is about 1% RMS. Diodes, while very small, more energy-efficient, and less expensive than He-Ne's, have ugly beams, which can be made reasonably smooth with appropriate optics, and can be made very quiet (a few hundredths of one per cent RMS noise), but they do emit long wavelength LED glow which usually requires that they be used with band pass excitation filters, and they can become unstable due to mode hopping. Diodes also vary over a range of a few nanometers in emission wavelength (635-640 nm); He-Ne's are really 633 nm, period. Both He-Ne and diode lasers should be good for over 10,000 hours of operation, but there seems to have been a higher failure rate among diodes, at least until recently. In general, the user isn't the one who puts the red laser into her or his instrument; the cytometer manufacturers do that, and they deal with the laser system manufacturers to get the specs they need. The FACSCalibur has extremely good red fluorescence sensitivity using a diode, and, if I'm not mistaken, it is a diode that is the standard red excitation source in the LSR, which also uses a He-Cd laser (*not* He-Ne) for UV - but if B-D is putting a red He-Ne into the LSR instead of the diode - possibly for more power - it should work just fine. Source: From: Howard Shapiro (hms@shapirolab.com) Date: Thu Feb 07 2002 - 19:38:14 ESThms@shapirolab.com http://www.cyto.purdue.edu/hmarchiv/current/1039.htm

16. © 1990-2005 J.Paul Robinson, Purdue University Lecture0005.ppt Argon and Krypton Ion Lasers

17. © 1990-2005 J.Paul Robinson, Purdue University Lecture0005.ppt 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 3 rd Shapiro p 82 4 th Shapiro p 135 3 rd Shapiro p 82 4 th Shapiro p 135 Ø r = tan -1 (n 2 /n 1 )

18. © 1990-2005 J.Paul Robinson, Purdue University Lecture0005.ppt Brewster’s Angle http://www.mic-d.com/java/brewster3d/ High reflector (back) Output coupler (front) © J.Paul Robinson

19. © 1990-2005 J.Paul Robinson, Purdue University Lecture0005.ppt Layout of Elite Cytometer with 4 Lasers (top view) Mirror 395 longPass He-Cd Laser 325/441 Argon Laser 353/488 nm (High speed sorting) He-Ne Laser 633 nm Argon Laser 488 nm 633 Beam Splitter UV\Beam Splitter 325 nm 353 nm 633 nm 488 nm Height Translators Optical bench computer

20. © 1990-2005 J.Paul Robinson, Purdue University Lecture0005.ppt Laser focusing There are several standards for creating a laser beam on a flow stream This has to do with the intensity of the focused beam There is also the issue of even cell illumination 60 microns 15 microns Want this as ‘flat” as possible stream

21. © 1990-2005 J.Paul Robinson, Purdue University Lecture0005.ppt Laser alignment A “translator’ can be used to move a beam in either the Vertical or horizontal direction without changing the alignment Beckman-Coutler’s Xl and MCL optical systemArgon laser He-Ne laser

22. © 1990-2005 J.Paul Robinson, Purdue University Lecture0005.ppt Use of Fiber Optics in Light delivery B-D Aria optical delivery via fiber optics

23. © 1990-2005 J.Paul Robinson, Purdue University Lecture0005.ppt Light Propagation & Vergence Considering a point source emission of light, rays emanate over 4pi steradians If the ray of light travels through a length L of a medium of RI n, the optical path length S=Ln (thus S represents the distance light would have traveled in a vacuum in the same time it took to travel the distance L in the medium (RI n). Rays diverge because the come from a point source Vergence is measured in diopters 3 rd Shapiro p 93 4 th Shapiro p 119 3 rd Shapiro p 93 4 th Shapiro p 119

24. © 1990-2005 J.Paul Robinson, Purdue University Lecture0005.ppt Summary and Learning Objectives covered Each instrument has a unique light path Some instruments use optical benches but they typically build their own bench The majority of instruments use “free space” optics and air cooled lasers Some are using fibers but there are problems in delivering lower wavelengths