BMS 631 - LECTURE 5
Flow Cytometry: Theory
J.Paul Robinson
Professor of Immunopharmacology & Biomedical Engineering
Purdue University
Light Sources & Optical
systems
Hansen Hall, B050
Purdue University
Office: 494 0757
Fax 494 0517
email\; robinson@flowcyt.cyto.purdue.edu
Shapiro 97-115
WEB http://www.cyto.purdue.edu
© 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT
Page 1
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
© 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT
Page 2
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
© 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT
Page 3
Mercury Arc Lamps
Lens
Arc
Lens
© 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT
Page 4
Arc Lamp Excitation Spectra
Xe Lamp
Irradiance at 0.5 m (mW m-2 nm-1)



Hg Lamp





3rd Ed. Shapiro p 99
© 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT
Page 5
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
© 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT
Page 6
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
© 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT
Page 7
Lasers
• Coherent Enterprise laser - UV-visible
• Air cooled laser (Argon)
© 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT
Page 8
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)
3rd Ed. Shapiro p 106
© 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT
Page 9
Lasers
Images only available for in-house use
Not for publication purposes
© 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT
Page 10
Argon & Krypton Lasers
Kr-Ar laser (488, 568, 647 nm lines) (Front)
3rd. Ed. Shapiro p 108
© 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT
Page 11
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
© 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT
Page 12
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
© 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT
Page 13
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.
He-Cd laser
3rd. Ed. Shapiro p 111
© 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT
Page 14
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
© 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT
Page 15
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)
© 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT
Page 16
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
© 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT
Page 17
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
© 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT
Page 18
Goals of Light Collection
•
•
•
•
•
•
Maximum signal, minimum noise
Maximum area of collection
Inexpensive system if possible
Easy alignment
Reduced heat generation
Reduced power requirement
© 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT
Page 19
Optical Collection systems
He-Cd Laser
2nd Argon Laser
He-Ne Laser
Argon Laser
Optical layout of an Elite sorter at Purdue University
© 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT
Page 20
Objectives
• 1.3 NA objective
Objective
Harald Steen’s Bryte Cytometer
© 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT
Page 21
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
© 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT
Page 22
Optical translators
No cytometer should be without one!!!
The laser beam remains
parallel, but horizontally
translated. This reduces the
difficulty in aligning the laser.
© 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT
Page 23
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)
© 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT
Page 24
Spectral Selection
(Next lecture)
• Monochromators Vs Filters
• Filters are reasonably inexpensive
© 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT
Page 25
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
© 1990-2002J.Paul Robinson, Purdue University BMS 631 – LECTURE005.PPT
Page 26