Lecture 0005

© 1990-2008 J.Paul Robinson, Purdue University Lecture0005.ppt

BMS 631 – Lecture 5

Properties and Applications of Light Sources

J. Paul Robinson, PhDProfessor of Immunopharmacology Professor of Biomedical EngineeringPurdue University

last modified February 2, 2008

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 (4th Ed)

The WEB version of these slides can be found on

http://www.cyto.purdue.edu/class


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


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

3rd Shapiro p 98

4th Shapiro p 124

3rd Shapiro p 98

4th Shapiro p 124


Optics - Light Sources Epi-ilumination 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 Shapiro p 98

4th Shapiro p 126

3rd Shapiro p 98

4th Shapiro p 126


© J.Paul Robinson© J.Paul Robinson

Mercury Arc Lamps

Arc

Lens

Lens


Arc Lamp Excitation Spectra

Irra

dia

nce

at 0

.5 m

(m

W m

-2 n

m-1)

Xe Lamp

Hg Lamp

3rd Shapiro p 99

4th Shapiro p 125

3rd Shapiro p 99

4th Shapiro p 125

Detection SystemsBio-Rad Bryte HS

Fluorescence Detectors and Optical TrainDsc00050.jpg

PMTs

Excitation dichroic filter

Fluorescence signal viewing telescope

Fluorescence emission filters

Light source

Sample Inlet

MicroscopeObjective

MicroscopeObjective

ExcitationFilterBlock

EmissionFilterBlock

Forward AngleScatter PMT

Large AngleScatter PMT

“Red”PMT

“Green” PMT

RetractableMirror

Ocular

Slit Slit

Lamp Housing

The Bryte Optical Layout

“Orange” PMT

EmissionFilterBlock

RetractableMirror

Ocular


Coulter Quanta – uses both UV lamp and laser


Lasers

Coherent light

Noncoherent light


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

3rd Shapiro p 114

4th Shapiro p 148

3rd Shapiro p 114

4th Shapiro p 148


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

3rd Ref: Shapiro p 103

4th Ref: Shapiro p 130




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)


4th Shapiro p 136, 147


4th Shapiro p 136, 147


© 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

3rd Shapiro p 110

4th Shapiro 141

3rd Shapiro p 110

4th Shapiro 141


© 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





He-Cd laser


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


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)


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. (8 h/day for 5 years)

Source: From: Howard Shapiro ([email protected])Date: Thu Feb 07 2002 - 19:38:14 EST http://www.cyto.purdue.edu/hmarchiv/current/1039.htm


Argon and Krypton Ion Lasers

• Probably most common lasers around

• Slowly being taken over by solid state lasers


Solid state Blue lasers

Coherent Laser – 1999 (20 mw version)

Diode976nmin

frequency doubler

Blue 488 out


Coherent and Spectra Physics

10, 20, 40, 50 mW

Low and high (200 mw) versionscall the SapphireTM

Lyt 200 is from Icyt Visionalry Biosceinceshttp://www.i-cyt.com/lyt200s.htm


Image from: www.kellerstudio.de/repairfaq/sam/laserssl.htm


Common Diode Lasers

• CD Player = 632 nm• DVD Player = 658 nm• Blueray DVD = 405 nm


Image from: http://an.hitchcock.org/repairfaq/sam/l54-101.gif


DPSS Lasers

• Diode-pumped solid state (DPSS) 532-nm

• DPSS 561-nm for its ability to improve PE and DsRed fluorescent protein detection sensitivity.

Reference: DPSS yellow-green 561-nm lasers for improved fluorochrome detection by flow cytometry; William Telford, Matilde Murga, Teresa Hawley, Robert Hawley, Beverly Packard, Akira Komoriya, Fred Haas, Charles Hubert, Cytometry A Volume 68A, Pages 36 - 44


How you create multi laser lines?

Laser combiners

Laser 1

Laser 2

Laser 3


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 90o

thus Ør + Øt = 90o

since sin (90-x) = cos x

Snell’s provides (sin Øi / cos Øi ) = n2/n1

Ør is Brewster’s angle

3rd Shapiro p 82

4th Shapiro p 135

3rd Shapiro p 82

4th Shapiro p 135

Ør = tan -1 (n2/n1)


Coherent’s dual beam laser – 488 and UV simultaneously


Water cooled laser power supply

Water flow is controlled by thisflow valve. It does not like high-mineral content water – and itcan be troublesome to fix

Power supplies in water cooled lasersare usually large and heavy (80-100lbs)


Fiber Lasers


Fiber Lasers• A fiber laser or fibre laser is a

laser in which the active gain medium is an optical fiber doped with rare-earth elements such as erbium, ytterbium, neodymium, dysprosium, praseodymium, and thulium. They are related to doped fiber amplifiers, which provide light amplification without lasing. Fiber nonlinearities, such as stimulated Raman scattering or four-wave mixing can also provide gain and thus serve as gain media for a fiber laser.

http://en.wikipedia.org/wiki/Image:FiberDiskLasers.jpg

3 fiber disk lasers, fiber lasers with transversal delivery of pump. the optical fiber with Yb: doped core is coiled and surrounded with the laser diodes (LDs) which deliver the pump at wavelength of order of 940nm with angle of order of 20 degree, which provides the efficient absorption after to pass few mm across the strip of collied fibers. The yellow parts are cooling systems for the LD; the white boxes are the adjusters necessary to activate each diode. The red and blue wires deliver few kW of electric power. Two black tubes provide the water cooling of the fiber coil and the laser diodes. The output is realized through two ends of the fiber; one of them is barely seen at the left side of the image. The output light at one micron wavelength is distributed among a thousand of transversal modes of the fibers and can be delivered, for example, to the cutting or welding machine without any additional coupler.


Ultra-broadband light source Spectral range: 450 - 2500 nm Spectral density up to 3 mW/nm Output power up to 6 W Maintenance Free Small footprint


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 nm633 nm

488 nm

Height TranslatorsOptical bench

computer


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 possiblestream


Laser alignment

A “translator’ can be used to move a beam in either theVertical or horizontal direction without changing the alignment

Beckman-Coulter’s Xl and MCL optical systemArgonlaser

He-Nelaser


A translator assists in spatially moving the beam in one dimension while remaining parallel


Use of Fiber Optics in Light delivery

B-D Aria optical delivery via fiber optics


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

3rd Shapiro p 93

4th Shapiro p 119

3rd Shapiro p 93

4th Shapiro p 119


Summary and Learning Objectives covered

• Each instrument has a unique light path• Some instruments use optical benches but they

typically build their own bench• Many instruments use “free space” optics and air

cooled lasers• Some are using fibers but there are problems in

delivering lower wavelengths but mostly these are overcome these days

• New lasers are small, low energy consumption but high power and very stable

Lecture 0005

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