Transcript PPT - Purdue University Cytometry Laboratories

BMS 631 - LECTURE 3 Light and Matter
J. Paul Robinson
SVM Professor of Cytomics
Professor of Biomedical Engineering
Purdue University
Reading materials:
(Shapiro 3rd ed. Pp 75-93; 4th Ed. Shapiro pp 101-136)
Lab: Bindley 122
Purdue University
Office Phone: (765)-494 0757
Fax (765)-494 0517
Email: robinsonATflowcyt.cyto.purdue.edu
WEB: http://www.cyto.purdue.edu
Notice: The materials in this presentation are
copyrighted materials. If you want to use any of these
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All materials used in this course are available for download on the web
at
http://tinyurl.com/2wkpp
Slide last modified January, 2013
10:42 PM
© 1990-2012 J. Paul Robinson, Purdue University BMS 631
Slide 1
Learning Objectives
• Understand the basic properties of light
• Understand basic principles of light propagation
• Understand the constraints that are placed in
measurement systems
• Understand how image formation, numerical aperture
and absorption impact instrument design
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© 1990-2012 J. Paul Robinson, Purdue University BMS 631
Slide 2
Light and Matter
• Energy
– joules, radiant flux (energy/unit time)
– watts (1 watt=1 joule/second)
• Angles
– steradians - sphere radius r - circumference is 2r2;
the angle that intercepts an arc r along the
circumference is defined as 1 radian. (57.3
degrees) a sphere of radius r has a surface area of
4r2. One steradian is defined as the solid angle
which intercepts as area equal to r2 on the sphere
surface
3rd Ed - Shapiro p 75
4th Ed – Shapiro p 101
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© 1990-2012 J. Paul Robinson, Purdue University BMS 631
Slide 3
Terms
•
•
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 400-750 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 r2; 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.
•
•
•
•
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© 1990-2012 J. Paul Robinson, Purdue University BMS 631
Slide 4
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
E=hn and E=hc/
3rd Ed Shapiro p 76
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 = wavelength
h = Planck’s constant
(6.63 x 10-34 joule-seconds
c = speed of light (3x108 m/s)
© 1990-2012 J. Paul Robinson, Purdue University BMS 631
Slide 5
Electromagnetic Spectrum
We can see from about
370 nm to about 700 nm
This is known as the
visible spectrum
Images from: http://www.lbl.gov/MicroWorlds/ALSTool/EMSpec/EMSpec2.html
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© 1990-2012 J. Paul Robinson, Purdue University BMS 631
Slide 6
Inverse Square Law
The intensity of the radiation is inversely proportional to the square of the distance traveled
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© 1990-2012 J. Paul Robinson, Purdue University BMS 631
Slide 7
Laser power
E=hn and E=hc/
• One photon from a 488 nm argon laser has an energy of
6.63x10-34 joule-seconds x 3x108
E=
= 4.08x10-19 J
488 x 10-3
• 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
3rd Ed. Shapiro p 77
4th Ed Shapiro p 109
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© 1990-2012 J. Paul Robinson, Purdue University BMS 631
Slide 8
Laser power
What about a UV laser?
E=
E=hn and E=hc/
6.63x10-34 joule-seconds x 3x108
325 x 10-3
= 6.12 x 10-19 J so 1 Joule at 325 nm = 1.63x1018 photons
What about a He-Ne laser?
E=
6.63x10-34 joule-seconds x 3x108
633 x 10-3
= 3.14 x 10-19 J so 1 Joule at 633 nm = 3.18x1018 photons
3rd Ed. Shapiro p 77
4th Ed Shapiro p 109
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© 1990-2012 J. Paul Robinson, Purdue University BMS 631
Slide 9
Polarization and Phase: Interference
• 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 Ed. Shapiro p 78
4th Ed. Shapiro p 104
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© 1990-2012 J. Paul Robinson, Purdue University BMS 631
Slide 10
Interference
0o 90o 180o 270o 360o
Wavelength
Amplitude
A+B
A
The frequency does
not change, but the
amplitude is doubled
Constructive
Interference
B
C+D
C
Here we have a phase difference of
180o (2 radians) so the waves
cancel each other out
Destructive
Interference
D
Figure modified from Shapiro 3rd Ed
“Practical Flow Cytometry” Wiley-Liss, p79
4th Ed. Shapiro p 109
Shapiro 4th Ed P105
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© 1990-2012 J. Paul Robinson, Purdue University BMS 631
Slide 11
Reflection and Refraction
Reflected Beam
r
i
• Snell’s Law: The angle of
reflection (Ør) is equal to
Transmitted
the 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
Incident Beam
n1 sin Øi = n2 sin Øt
The velocity of light in a material
of refractive index n is c/n
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© 1990-2012 J. Paul Robinson, Purdue University BMS 631
3rd Ed. Shapiro p 81
4th Ed. Shapiro p 106
Slide 12
Refraction & Dispersion
rac
Short wavelengths are “bent”
more than long wavelengths
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
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© 1990-2012 J. Paul Robinson, Purdue University BMS 631
Slide 13
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 they come from a point source)
• Vergence is measured in diopters
3rd Shapiro p 93
4th Shapiro p 119
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© 1990-2012 J. Paul Robinson, Purdue University BMS 631
Slide 14
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
Shapiro p 94
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© 1990-2012 J. Paul Robinson, Purdue University BMS 631
Slide 15
Numerical Aperture
• The wider the angle the lens is capable of
receiving light at, the greater its resolving
power
• The higher the NA, the shorter the working
distance
Shapiro p 96
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© 1990-2012 J. Paul Robinson, Purdue University BMS 631
Slide 16
Numerical Aperture
• Resolving power is directly related to numerical
aperture.
• The higher the NA the greater the resolution
• Resolving power:
The ability of an objective to resolve two distinct lines very
close together
NA = n sin m
– (n=the lowest refractive index between the object and first
objective element) (hopefully 1)
– m is 1/2 the angular aperture of the objective
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© 1990-2012 J. Paul Robinson, Purdue University BMS 631
Slide 17
Numerical Aperture
• For a narrow light beam (i.e. closed illumination aperture
diaphragm) the finest resolution is (at the brightest point of the
visible spectrum i.e. 530 nm)…(closed condenser).

=
NA
.00053
1.00 = 0.53 mm
• With a cone of light filling the entire aperture the theoretical
resolution is…(fully open condenser)..

2 x NA
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=
.00053
2 x 1.00 = 0.265 mm
© 1990-2012 J. Paul Robinson, Purdue University BMS 631
Slide 18
A
m
Light cone
NA=n(sin m)
(n=refractive index)
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© 1990-2012 J. Paul Robinson, Purdue University BMS 631
Slide 19
Numerical Aperture
•
•
The wider the angle the lens is capable of receiving light at, the greater its
resolving power
The higher the NA, the shorter the working distance
Images reproduced from:
http://micro.magnet.fsu.edu/
Please go to this site and do the tutorials
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© 1990-2012 J. Paul Robinson, Purdue University BMS 631
Slide 20
Images reproduced from:
http://micro.magnet.fsu.edu/
Please go to this site and do the tutorials
10:42 PM
© 1990-2012 J. Paul Robinson, Purdue University BMS 631
Slide 21
Some 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
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© 1990-2012 J. Paul Robinson, Purdue University BMS 631
Slide 22
Absorption Chart
Color in white light
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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
© 1990-2012 J. Paul Robinson, Purdue University BMS 631
blue
blue
Slide 23
Light absorption
Control
Absorption
No blue/green light
red filter
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© 1990-2012 J. Paul Robinson, Purdue University BMS 631
Slide 24
Light absorption
white light
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blue light
red light
© 1990-2012 J. Paul Robinson, Purdue University BMS 631
green light
Slide 25
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
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© 1990-2012 J. Paul Robinson, Purdue University BMS 631
Slide 26
Light Scatter
• Materials scatter light at wavelengths at which they do not
absorb
• If we consider the visible spectrum to be 400-750 nm then
small particles (< 1/10 ) scatter rather than absorb light
• For small particles (molecular up to sub micron) the Rayleigh
scatter intensity at 0o and 180o are about the same
• 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
3rd Ed. Shapiro p 79
4th Ed. Shapiro p 105
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© 1990-2012 J. Paul Robinson, Purdue University BMS 631
Slide 27
Mie scattering
 Interaction between light and particles of size larger than the
wavelength
 Complete mathematical-physical theory of the scattering of
electromagnetic radiation by spherical particles
 Embraces all possible ratios of diameter to wavelength.
 Assumes a homogeneous, isotropic and optically linear material
irradiated by an infinitely extending plane wave
http://en.wikipedia.org/wiki/Mie_scattering
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© 1990-2012 J. Paul Robinson, Purdue University BMS 631
Slide 28
Rayleigh scattering
 Particles much smaller than the wavelength of the light
 short wavelengths are scattered more than long wavelengths

It occurs when light travels in transparent solids and liquids, but is
most prominently seen in gases.
 Dependent upon the size of the particles and the wavelength of the
light.
http://en.wikipedia.org/wiki/Rayleigh_scattering
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© 1990-2012 J. Paul Robinson, Purdue University BMS 631
Slide 29
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
the sky looks blue because the gas molecules scatter more
light at shorter (blue) rather than longer wavelengths (red)
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© 1990-2012 J. Paul Robinson, Purdue University BMS 631
Slide 30
Some Key issues with regard to light
• When you use a very bright light source to excite
molecules in a cell, you also produce a large amount of
reflected light
• This light is what we measure when we measure forward
and side scatter
• Typically it can be 105 or 106 times the fluorescence
signal produced
• So what does this mean?
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© 1990-2012 J. Paul Robinson, Purdue University BMS 631
Slide 31
Introduction to light scatter in flow

Light scattering is a complex phenomenon.

If the refractive index is not different from the surrounding medium (which may
be water) there will be a negligible difference between RI of cell and medium

Light scattering can be measured based on

light scattered by a particle suspension

light scattered by an individual particle

The scattered fields; scattering angle and/or wavelength are important

Particle parameters can be:

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Size, morphology, viability, structure, effective refractive index, etc.
© 1990-2012 J. Paul Robinson, Purdue University BMS 631
Slide 32
Light scattering
Scattering
(Mie, Rayleigh)
Particles
Absorption
Incident Beams
Emission
(Fluorescence)
The interaction of light with a particle in terms of scattering,
absorption, and emission.
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© 1990-2012 J. Paul Robinson, Purdue University BMS 631
Slide 33
Light scattering in flow cytometry
 Qualitative analyses of individual cells as well as particle suspension
 The angular dependence of light-scattering intensity
 Size and shape dependent: Forward scatter
 dependence of scattered intensity on particle RI and geometrical
cross-section
 Structure dependent: Side scatter
 anisotropic cell structures and multiple scattering processes
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© 1990-2012 J. Paul Robinson, Purdue University BMS 631
Slide 34
Forward & Side Scatter
 Forward Scatter (FSC): Diffracted light
FSC is related to cell surface area (at narrow angles)
FSC is detected along axis of incident light in the forward direction
BUT – the accuracy for cell sizing is not good – it is very dependent upon
the exact angle of scatter which can vary even between similar
instruments
 Side Scatter (SSC): Reflected and Refracted light
SSC is related to cell granularity and complexity
SSC is detected (mostly) at 90° to the laser beam
 Factors that affect light scatter
Shape, surface, size, granularity, internal complexity, refractive index.
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© 1990-2012 J. Paul Robinson, Purdue University BMS 631
Slide 35
Scanning flow cytometer
Scanning Flow Cytometer permits measurement of the angular
dependency of the scattered light from individual moving particles
Laser (La1, La2), beam splitter
(BS), lense (L1-L3),
hydrofocusing head (HFH), pinhole(P-H), photomultiplier tube
(PMT), objectives (O), optical
scanning cuvette (OSQ),
mirrors (M), and mask (M)
From Part. Part. Syst. Charact. 17 (2000) 225~229
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© 1990-2012 J. Paul Robinson, Purdue University BMS 631
Slide 36
Why look at FSC vs SSC
Since FSC ~ size and SSC ~ internal structure, a correlated
measurement between them provides a basis for a simple
differentiation between the major populations
Granulocytes
SSC
Lymphocytes
Monocytes
RBCs, Debris,
Dead Cells
FSC
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© 1990-2012 J. Paul Robinson, Purdue University BMS 631
Slide 37
Applications - Polymer particles
 Particle size and refractive index distribution
The polystyrene particles certified with the scanning flow cytometer
Review of Scientific Instruments, 71 (2000) 243~255
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© 1990-2012 J. Paul Robinson, Purdue University BMS 631
Slide 38
White blood cells
1000
800
600
Scatter
Forward
Side Scatter Projection
Forward Scatter Projection
Neutrophils
Forward Scatter
Projection
200
400
Monocytes
0
Lymphocytes
0
200
400
600
800
1000
90 Degree Scatter
Light Scatter Gating
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© 1990-2012 J. Paul Robinson, Purdue University BMS 631
Slide 39
Bacterial cells
Determination of the biomasses of small bacteria at low
concentrations in a mixture of species
Applied and Environmental Microbiology, 64 (1998) 3900–3909
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© 1990-2012 J. Paul Robinson, Purdue University BMS 631
Slide 40
Lecture Summary
•
•
•
•
•
•
•
Principles of light and matter
Basic optics to take into consideration
Essentials of light measurement
Absorption and optical properties
Angular dependency of light scattering intensity
Forward scatter: Diffracted light
Characterization of spherical and non-spherical
particle
• Side Scatter: Reflected and refracted light
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© 1990-2012 J. Paul Robinson, Purdue University BMS 631
Slide 41