BMS 633-BME 695Y - Week 2
Flow Cytometry Module for Engineering Students
Properties and Applications of Light
Fluorescence, Illumination Sources, Filters
A course designed for engineering students who may not have strong biology backgrounds.
This course consists of a series of lectures and practicals that allow engineering students to
understand biotechnology and its application. At the conclusion of this 2 credit hour course
students will have an excellent understanding of the technical components and operation of
flow cytometers, understand the biological principals of operation, be familiar with the
applications of the technology and be able to intelligently discuss the technological
implications of flow cytometry with a person who is well versed in the field.
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://tinyurl.com/385ss
J. Paul Robinson
Professor of Immunopharmacology & Biomedical Engineering
last modified January 8, 2004
Purdue University
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 1
Light and Matter
•
•
Side scatter, forward angle scatter, cell volume, coulter volume:
Understand light scattering concepts; intrinsic and extrinsic parameters
•
•
Photometry:
Light - what is it - wavelengths we can see 350-700 nm, most sensitive around 550 nm.
Below 400 nm essentially measuring radiant energy. Joules (energy) radiant flux
(energy per unit time) is measured in watts (1 watt=1 joule/second).
Steradian (sphere radius r has surface area of 4 pr2; one steradian is defined as that
solid angle which intercepts an area equal to r2 on the surface.
Mole - contains Avogadro's number of molecules (6.02 x 1023) and contains a mass in
grams = molecular weight. Photons - light particles - waves - Photons are particles
which have no rest mass - pure electromagnetic energy - these are absorbed and
emitted by atoms and molecules as they gain or release energy. This process is
quantized, is a discrete process involving photons of the same energy for a given
molecule or atom. The sum total of this energy gain or loss is electromagnetic
radiation propagating at the speed of light (3 x 108 m/s). The energy (joules) of a
photon is
E=hn and E=hn/l [n-frequency, l-wavelength, h-Planck's constant 6.63 x 10-34 jouleseconds]
Energy - higher at short wavelengths - lower at longer wavelengths.
•
•
•
•
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 2
Photons and Quantum Theory
• Photons
– particles have no rest mass - composed of pure electromagnetic energy
- the absorption and emission of photons by atoms and molecules is the
only mechanism for atoms and molecules can gain or lose energy
• Quantum mechanics
– absorption and emission are quantized - i.e. discrete process of gaining
or losing energy in strict units of energy - i.e. photons of the same
energy (multiple units are referred to as electromagnetic radiation)
• Energy of a photon
– can be computed from its frequency (n)
in hertz (Hz) or its wavelength (l) in meters from
 = wavelength
h = Planck’s constant
(6.63 x 10-34 joule-seconds
c = speed of light (3x108 m/s)
E=hn and E=hc/
3rd Ref: Shapiro p 76
4th Ref: Shapiro p 103
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 3
The light spectrum
Wavelength = Frequency
Blue light
488 nm
short wavelength
high frequency
high energy (2 times
the red)
Photon as a
wave packet
of energy
Red light
650 nm
long wavelength
low frequency
low energy
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 4
Laser power
E=hn and E=hc/
• One photon from a 488 nm argon laser has an energy of
E= 6.63x10-34 joule-seconds x 3x108
488 x 10-3
= 4.08x10-19 J
• To get 1 joule out of a 488 nm laser you need 2.45 x 1018 photons
• 1 watt (W) = 1 joule/second a 10 mW laser at 488 nm is putting out
2.45x1016 photons/sec
• UV Laser at 325 nm is putting out 1.63x1018 photons/sec
• He-Ne laser at 653 nm is putting out 3.18x1018 photons/sec
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
3rd Ref: Shapiro p 77
4th Ref Shapiro p 103
Page 5
Polarization and Phase:
• Electric and magnetic fields are
vectors - i.e. they have both
magnitude and direction
• The inverse of the period
(wavelength) is the frequency in
Hz
3rd Ref: Shapiro p 78
4th Ref: Shaprio p 104
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 6
Light Scatter
• Materials scatter light at wavelengths at which they
do not absorb
• If we consider the visible spectrum to be 350-700
nm then small particles (< 1/10 ) scatter rather
than absorb light
• For larger particles (i.e. size from 1/4 to tens of
wavelengths) larger amounts of scatter occur in the
forward not the side scatter direction - this is called
Mie Scatter (after Gustav Mie) - this is how we
come up with forward scatter be related to size
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
3rd Ref: Shapiro p 79
4th Ref: Shapiro p 105
Page 7
Rayleigh Scatter
• Molecules and very small particles do not
absorb, but scatter light in the visible
region (same freq as excitation)
• Rayleigh scattering is directly proportional
to the electric dipole and inversely
proportional to the 4th power of the
wavelength of the incident light
• For small particles (molecular up to sub
micron) the Rayleigh scatter intensity at 0o
and 180o are about the same
The sky looks blue because the gas molecules scatter more
light at shorter (blue) rather than longer wavelengths (red)
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 8
Optics for forward scatter
iris
Laser
beam
Not drawn to scale
scatter
detector
blocker
Stream in air or a
round capillary
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 9
rac
More Definitions
• Absorption
– When light passes through an object the intensity is reduced depending upon the color
absorbed. Thus the selective absorption of white light produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to another
with different optical density. A ray from less to more dense medium is bent
perpendicular to the surface, with greater deviation for shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp edges - the
smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a transparent
medium - the change of refractive index with wavelength, such as the spectrum
produced by a prism or a rainbow
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 10
Reflection and Refraction
Incident Beam
i
r
• Snell’s Law: The angle of
reflection (Ør) is equal to the
Transmitted
angle of incidence (Øi)
(refracted)Beam
regardless of the surface
material
t
• The angle of the transmitted
beam (Øt) is dependent upon
the composition of the
material
Reflected Beam
n1 sin Øi = n2 sin Øt
The velocity of light in a material
of refractive index n is c/n
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
3rd Shapiro p 81
4th Shapiro p 106
Page 11
Absorption
• Basic quantum mechanics requires that molecules absorb
energy as quanta (photons) based upon a criteria specific
for each molecular structure
• Absorption of a photon raises the molecule from ground
state to an excited state
• Total energy is the sum of all components (electronic,
vibrational, rotational, translations, spin orientation
energies) (vibrational energies are quite small)
• The structure of the molecule dictates the likely-hood of
absorption of energy to raise the energy state to an excited
one
3rd Shapiro p 84
4th Shapiro p 109
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 12
Absorption Chart
(what you don’t see)
(what you see)
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
blue
blue
Page 13
Interference in Thin Films
• Small amounts of incident light are reflected at the
interface between two material of different RI
• Thickness of the material will alter the constructive or
destructive interference patterns - increasing or decreasing
certain wavelengths
• Optical filters can thus be created that “interfere” with the
normal transmission of light
3rd Shapiro p 82
4th Shapiro p 108
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 14
Interference and Diffraction:
Gratings
• Diffraction essentially describes a departure from
theoretical geometric optics
• Thus a sharp objet casts an alternating shadow of
light and dark “patterns” because of interference
• Diffraction is the component that limits resolution
3rd Shapiro p 83
4th Shapiro p 108
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 15
Lifetime
• Absorption associated with electronic transitions (electrons
changing states) occurs in about 1 femptosecond (10-15 s)
• The lifetime of a molecule depends on how the molecule
disposes of the extra energy
• Because of the uncertainty principle, the more rapidly the
energy is changing, the less precisely we can define the
energy
• So, long-lifetime-excited-states have narrower absorption
peaks, and short-lifetime-excited-states have broader
absorption peaks
3rd Shapiro p 85
4th Shapiro p 110
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 16
Extinction
• Using Beer’s law (Beer-Lambert law) for light travelling through a cuvette
thickness d cm containing n molecules/cm3
ln (Io/I) = nd
where Io and I are the light entering and leaving and  is the molecular property
called the absorption cross section
• Now we can state that
ln (Io/I) = nd where C is the concentration and a is the absorption
coefficient which reflects the capacity of the absorbing substance to absorb
light
• If there are n (molecules/cm3 ; d in cm,  must be in cm2 so if  is in
cm2/mol, C must be in mol/cm3 so C=a/103
• giving
log10 (Io/I) = d = A
where A is the absorbance or optical density
and  is the decadic molar extinction coefficient in dm3mol-1cm-1
3rd Shapiro p 86
4th Shapiro p 111
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 17
Absorbance
• O.D. units or absorbance is expressed in logarithmic terms so
they are additive.
• e.g. an object of O.D. of 1.0 absorbs 90% of the light.
Another object of O.D. 1.0 placed in the path of the 10% of
the light 10% of this light or 1% of the original light is
transmitted by the second object
• It is possible to express the absorbance of a mixture of
substances at a particular wavelength as the sum of the
absorbances of the components
• You can calculate the cross sectional area of a molecule to
determine how efficient it will absorb photons. The extinction
coefficient indicates this value
3rd Shapiro p 87
4th Shapiro p 111
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 18
Parameters
• Extinction Coefficient
– 
refers to a single wavelength (usually the absorption
maximum)
• Quantum Yield
– Qf is a measure of the integrated photon emission over
the fluorophore spectral band
– Qf = photons emitted / photons absorbed
– At sub-saturation excitation rates, fluorescence intensity is
proportional to the product of  and Qf
• Fluorescence Lifetime ()
– The time delay between the absorbance and the emission
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 19
Fluorescence
• Excitation Spectrum
– Intensity of emission as a function of exciting
wavelength
• Chromophores are components of molecules which absorb
light
• They are generally aromatic rings
• The wavelength of absorption is related to the size of the
chromophores
• Smaller chromophores, higher energy (shorter wavelength)
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 20
Fluorescence
• Stokes Shift
Fluorescnece Intensity
– is the energy difference between the lowest
energy peak of absorbance and the highest
energy of emission
Fluorescein
molecule
Stokes Shift is 25 nm
495 nm
520 nm
Wavelength
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 21
Properties of Fluorescent
Molecules









Large extinction coefficient at the region of excitation
High quantum yield
Optimal excitation wavelength
Photostability
Excited-state lifetime
Minimal perturbation by probe
Dye molecules must be close to but below saturation
levels for optimum emission
Fluorescence emission is longer than the exciting
wavelength
The energy of the light increases with reduction of
wavelength
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 22
350
300 nm
457 488 514
400 nm
500 nm
Common Laser Lines
610 632
600 nm
700 nm
PE-TR Conj.
Texas Red
PI
Ethidium
PE
FITC
cis-Parinaric acid
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 23
Excitation Saturation
• The rate of emission is dependent upon the time the molecule
remains within the excitation state (the excited state lifetime f)
• Optical saturation occurs when the rate of excitation exceeds the
reciprocal of f
• In a scanned image of 512 x 768 pixels (400,000 pixels) if scanned
in 1 second requires a dwell time per pixel of 2 x 10-6 sec.
• Molecules that remain in the excitation beam for extended periods
have higher probability of interstate crossings and thus
phosphorescence
• Usually, increasing dye concentration can be the most effective
means of increasing signal when energy is not the limiting factor
(i.e. laser based confocal systems)
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 24
Resonance Energy Transfer
• Resonance energy transfer can occur when the donor and
acceptor molecules are within 100 Å of one another
• Energy transfer is non-radiative which means the donor is
not emitting a photon which is absorbed by the acceptor
• Fluorescence RET (FRET) can be used to spectrally shift
the fluorescence emission of a molecular combination.
3rd Shapiro p 90
4th Shapiro p 115
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 25
Fluorescence
Resonance Energy Transfer
Molecule 1
Molecule 2
Fluorescence
Fluorescence
ACCEPTOR
DONOR
Absorbance
Absorbance
Wavelength
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 26
Raman Scatter
• A molecule may undergo a vibrational transition (not an
electronic shift) at exactly the same time as scattering
occurs
• This results in a photon emission of a photon differing in
energy from the energy of the incident photon by the
amount of the above energy - this is Raman scattering.
• The dominant effect in flow cytometry is the stretch of the
O-H bonds of water. At 488 nm excitation this would give
emission at 575-595 nm
3rd Shapiro p 93
4th Shapiro p 118
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 27
Quenching, Bleaching &
Saturation
• Quenching is when excited molecules relax to ground
states via nonradiative pathways avoiding fluorescence
emission (vibration, collision, intersystem crossing)
• Molecular oxygen quenches by increasing the probability
of intersystem crossing
• Polar solvents such as water generally quench fluorescence
by orienting around the exited state dipoles
3rd Shapiro p 90
4th Shapiro p 115
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 28
Illumination Sources
• Lamps
• Xenon-Mercury
• Mercury
• Lasers
•
•
•
•
•
Argon Ion (Ar)
Krypton (Kr)
Helium Neon (He-Ne)
Helium Cadmium (He-Cd)
YAG
3rd Shapiro p 98
4th Shapiro p 124
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 29
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
3rd Shapiro p 98
4th Shapiro p 126
[RFM]
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 30
Mercury Arc Lamps
Lens
Arc
Lens
© J.Paul Robinson
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
© J.Paul Robinson
Page 31
Arc Lamp Excitation Spectra
Xe Lamp
Irradiance at 0.5 m (mW m-2 nm-1)



Hg Lamp





3rd Shapiro p 99
4th Shapiro p 125
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 32
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
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 33
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 = tan -1 (n2/n1)
Ør is Brewster’s angle
3rd Shapiro p 82
4th Shapiro p 135
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 34
Brewster’s Angle
High
reflector
(back)
© J.Paul Robinson
Output
coupler
(front)
© J.Paul Robinson
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 35
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 Ref: Shapiro p 106
4th Shapiro p 136, 147
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 36
Lasers
Noncoherent light
Coherent light
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 37
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
© J.Paul Robinson
3rd Shapiro p 110
4th Shapiro 141
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 38
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
© J.Paul Robinson
3rd Ref: Shapiro p 111
4th Ref: Shapiro p 142
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 39
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)
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 40
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 Shapiro p 114
4th Shapiro p 148
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 41
Layout of Elite Cytometer
with 4 Lasers (top view)
353 nm
computer
488 nm
325 nm
UV\Beam Splitter
633 nm
He-Cd Laser 325/441
395 longPass
Argon Laser 353/488 nm
(High speed sorting)
633 Beam Splitter
He-Ne Laser 633 nm
Argon Laser 488 nm
Mirror
Optical bench
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Height Translators
Page 42
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
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 43
Image Formation
• Object plane - (originating image)
• Image plane - inverted real image
• A real image is formed whenever rays emanating
from a single point in the object plane again
converge to a single point
3rd Shapiro p 94
4th Shapiro p 119
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 44
Properties of thin Lenses
f
f
p
q
1
p
Resolution (R) = 0.61 x
(lateral)
(Rayleigh criterion)
+
1
q

NA
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
=
1
f
q
Magnification =
p
Page 45
Numerical Aperture
• Resolving power is directly related to numerical aperture.
• The higher the NA the greater the resolution
• The wider the angle the lens is capable of receiving light
at, the greater its resolving power
• Resolving power:
The ability of an objective to resolve two distinct lines
very close together
NA = n sin 
(n=the lowest refractive index between the object and first
objective element) (hopefully 1)
–  is 1/2 the angular aperture of the objective
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 46
Objectives
• 1.3 NA objective
Flow cell
Objective
Objective on the Bryte cytometer
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
© J.Paul Robinson
Page 47
Depth of Field and Resolution
• Depth of field is designated as the longitudinal distance for
the formation of a sharp image is obtained at a fixed point
in the image plane
• Limits of resolution are diffraction limited - the diffraction
image is a point is a bright central spot surrounded by what
is called the Airy disk (alternating light and dark rings)
• At wavelength , the radius of the Airy disk is 0.61  Thus
to resolve two points they need to be at least this distance
apart (radius of the Airy disk)
– Therefore: Resolution = 0.61  /NA
3rd Shapiro p 97
4th Shapiro p 124
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 48
Goals of Light Collection
•
•
•
•
•
•
Maximum signal, minimum noise
Maximum area of collection
Inexpensive system if possible
Easy alignment
Reduced heat generation
Reduced power requirement
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 49
Optical Collection systems
He-Cd Laser
Argon Lasers
He-Ne Laser
© J.Paul Robinson
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 50
Optics - Filter Properties
• When using laser light sources, filters must have very sharp cutons
and cutoffs since there will be many orders of magnitude more
scattered laser light than fluorescence
• Can specify wavelengths that filter must reject to certain tolerance
(e.g., reject 488 nm light at 10-6 level: only 0.0001% of incident
light at 488 nm gets through)
• When a filter is placed at a 45o angle to a light source, light which
would have been transmitted by that filter is still transmitted but
light that would have been blocked is reflected (at a 90o angle)
• Good optical filters
– Remove as much excitation signal as possible
– Collect as much fluorescence as possible (use concave spherical
mirrors)
• Used this way, a filter is called a dichroic filter or dichroic mirror
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 51
Optical Filters
• Interference filters:
Dichroic, Dielectric, reflective filters…….reflect the
unwanted wavelengths
• Absorptive filters:
Color glass filters…..absorb the unwanted wavelengths
Dichroic Filter/Mirror at 45 deg
Light Source
Transmitted Light
Reflected light
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
Page 52
Optical filters evaluation
• By analyzing a population of appropriately stained
particles and identifying which filters give the
maximum signal.
• Spectrofluorometers and spectrophotometers can be
used as tools for assessment of optical filters.
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
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Optical filter evaluation
reference PMT
light source
beam splitter (45o)
slit/shutter
grating
grating
Detector
PMT
Optical filter (45o)
SPECTROFLUOROMETER FOR
ASSESSMENT
OF OPTICAL FILTER REFLECTANCE
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
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Interference filters
• They are composed of transparent glass or
quartz substrate on which multiple thin
layers of dielectric material, sometimes
separated by spacer layers .
• Permit great selectivity.
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
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Dichroic Filters
•They used to direct light in different spectral region to different
detectors.
•They are interference filters , long pass or short pass.
Reflected
Light
Transmitted
Light
Filter acting as a DICHROIC
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
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Interference filters advantages
•
•
•
•
They can be used as reflectors in two and three color analysis.
They usually do not themselves produce fluorescence.
They are available in short pass versions.
They are excellent as primary barrier filters.
Interference filters: disadvantages
• Lower blocking properties
• Reduced passing properties
• Their reflecting and passing properties are not absolute, this
should be considered while dealing with multiple wavelengths
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
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Absorptive filters
• Such as colored glass filters which absorb
unwanted light.
• Consist of dye molecules uniformly suspended in
glass or plastic.
• Remove much more of the unwanted light than do
the interference filters
• Will often fluoresce (not good!)
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
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Absorbance filters: advantages
• They are inexpensive.
• They have very good blocking properties.
• They have very good transmission properties.
Absorbance filters: disadvantages
• They can only pass long wavelengths
( hence, can only block short wavelength)
• Since they are made of solution of dye and glass, they can
themselves produce fluorescence.
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
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Standard Band Pass Filters
630 nm Bandpass Filter
White Light Source
Transmitted Light
620 -640 nm Light
Standard Long and Short Pass Filters
520 nm Long Pass Filter
Light Source
Transmitted Light
>520 nm Light
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
575 nm Short Pass Filter
Light Source
Transmitted Light
<575 nm Light
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Filters transmission
• Bandpass filters: characterized by their
T max and (the Full Width at Half Maximum) FWHM
• Notch filters are band pass filters in the reverse position
• Long pass and Short pass filters: characterized by their T max
and Cuton, cutoff wavelengths.
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
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Filter transmission
Attenuation OD=-log(T)
e.g. O.D. 4.5 = 3 x 10-5 T (0.0003%T)
Cross talk level describes the minimum attenuation level of two filters placed together in series.
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
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Transmission determination
• Constructive and destructive interference
occurs between reflections from various
layers
• Transmission determined by :
– thickness of the dielectric layers
– number of these layers
– angle of incidence light on the filters
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
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Optical Design
PMT 5
PMT 4
Sample
PMT 3
Flow cell
Dichroic
Filters
Scatter
Sensor
PMT 2
PMT 1
Laser
Bandpass
Filters
This is a layout of a typical flow cytometer optical collection pathway. Dichroic mirrors
split the light path and bandpass filters specify the wavelength collected by each PMT
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
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Learning Goals
• At the conclusion of this lecture you should:
• Have an understanding of the nature of light and how it
impact flow cytometry
• Know how different light sources impact measurement.
• Understand how and what fluorescence is
• Know how an optical filter is created and used and
understand the different types of filters
• Understand how lasers are used in flow cytometry
• Have an understanding of what the optical configuration is
on common flow cytometers.
This lecture and related lectures can be found on our website
at
http://tinyurl.com/385ss
© 1988-2004 J.Paul Robinson, Purdue University BMS 633 –BME 695Y week 2.PPT
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