1	BMS 631 - LECTURE 5 Flow Cytometry: Theory J.Paul Robinson Professor of Immunopharmacology & Biomedical Engineering Purdue University
2	Illumination Sources Lamps Xenon-Mercury Mercury Lasers Argon Ion (Ar) Krypton (Kr) Helium Neon (He-Ne) Helium Cadmium (He-Cd) YAG
3	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
4	Mercury Arc Lamps
5	Arc Lamp Excitation Spectra
6	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
7	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(lF/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
8	Lasers Coherent Enterprise laser - UV-visible Air cooled laser (Argon)
9	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)
10	Lasers
11	Argon & Krypton Lasers
12	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
13	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.
14	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.
15	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
16	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	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
18	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	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	Optical Collection systems
21	Objectives 1.3 NA objective
22	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	Optical translators
24	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	Spectral Selection (Next lecture) Monochromators Vs Filters Filters are reasonably inexpensive
26	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