hansen hall, b050 purdue university office: 494 0757 fax 494 0517
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Page 1© 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT
BMS 631 - LECTURE 5Flow Cytometry: Theory
J.Paul RobinsonProfessor of Immunopharmacology & Biomedical
EngineeringPurdue University
Hansen Hall, B050Purdue UniversityOffice: 494 0757Fax 494 0517email\; robinson@flowcyt.cyto.purdue.edu
WEB http://www.cyto.purdue.edu
Light Sources & Optical systems
Shapiro 97-115
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
3rd Ed. Shapiro p 98
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] 3rd Ed. Shapiro p 98
Page 4© 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT
Mercury Arc Lamps
Arc
Lens
Lens
Page 5© 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT
Arc Lamp Excitation SpectraIr
radi
ance
at 0
.5 m
(mW
m-2
nm
-1)
Xe Lamp
Hg Lamp
3rd Ed. Shapiro p 99
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
• Optical elements provide separationseparation of channels and wavelength selectionwavelength selection
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
3rd Ed. Shapiro p 103
Page 8© 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT
Lasers• Coherent Enterprise laser - UV-visible• Air cooled laser (Argon)
Page 9© 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT
Laser Power & NoiseLight 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
Page 10© 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT
Lasers
Images only available for in-house useNot for publication purposes
Page 11© 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT
Argon & Krypton Lasers
3rd. Ed. Shapiro p 108
Kr-Ar laser (488, 568, 647 nm lines) (Front)
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
3rd. Ed.Shapiro p 110
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.
3rd. Ed. Shapiro p 110
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 don’t matter here – these lasers do have power fluctuation problems eventually.
3rd. Ed. Shapiro p 111
He-Cd laser
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
3rd Ed. Shapiro p113
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)
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
3rd. Ed. Shapiro p 114
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
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
Page 20© 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT
Optical Collection systems
He-Cd Laser Argon Laser He-Ne Laser2nd Argon Laser
Optical layout of an Elite sorter at Purdue University
Page 21© 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT
Objectives• 1.3 NA objective
Objective
Harald Steen’s Bryte Cytometer
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
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!!!
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)
Page 25© 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT
Spectral Selection(Next lecture)
• Monochromators Vs Filters• Filters are reasonably inexpensive
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
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