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- Lamps
- Lasers
- Argon Ion (Ar)
- Krypton (Kr)
- Helium Neon (He-Ne)
- Helium Cadmium (He-Cd)
- YAG
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3
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- 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
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4
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5
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6
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- 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
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- 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
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- Coherent Enterprise laser - UV-visible
- Air cooled laser (Argon)
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- 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)
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10
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11
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12
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- 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
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- 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.
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- 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.
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- 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
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- 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)
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- 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
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- 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
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- Maximum signal, minimum noise
- Maximum area of collection
- Inexpensive system if possible
- Easy alignment
- Reduced heat generation
- Reduced power requirement
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21
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- 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
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24
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- Good optical filters
- Remove as much excitation signal as possible
- Collect as much fluorescence as possible (use concave spherical mirrors)
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25
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- Monochromators Vs Filters
- Filters are reasonably inexpensive
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- 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
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