© 1990-2006 J. Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture0003.ppt 1 BMS 602A/631 - Lecture 3 Light and Matter J. Paul Robinson, PhD

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© 1990-2006 J. Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture0003.ppt

BMS 602A/631 - Lecture 3

Light and Matter

J. Paul Robinson, PhD

Professor of Immunopharmacology and Bioengineering

Reading materials:

(Shapiro 3rd ed. Pp 75-93; 4th Ed. Shapiro pp 101-136)

Note: The web version of these slides were converted to web slides by Microsoft PowerPoint directly. Microsoft made such a bad job of this process that all text boxes had to be eliminated because they did not translate at all – so forgive the problems – they are mostly bad Microsoft programming - - - thanks Bill!

All materials used in this course are available for download on the web at

http://tinyurl.com/2wkpp

Slide last modified January 9, 2006

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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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Light and Matter• Energy

– joules, radiant flux (energy/unit time)– watts (1 watt=1 joule/second)

• Angles– steradians - sphere radius r - circumference is 2r2;

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 4r2. 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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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=h and E=h/l [-frequency, l-wavelength, h-Planck's constant 6.63 x 10-34 joule-seconds] • Energy - higher at short wavelengths - lower at longer wavelengths.

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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 () in hertz (Hz) or its wavelength (l) in meters from

E=h and E=hc/

= wavelengthh = Planck’s constant

(6.63 x 10-34 joule-secondsc = speed of light (3x108 m/s)3rd Ed Shapiro p 76

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Electromagnetic Spectrum

• We can see from about 400 nm to 700 nmThis is known as the visible spectrum

http://www.lbl.gov/MicroWorlds/ALSTool/EMSpec/EMSpec2.html

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The intensity of the radiation is inversely proportional to the square of the distance traveled

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Laser power

• One photon from a 488 nm argon laser has an energy of

6.63x10-34 joule-seconds x 3x108

• 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

E=h and E=hc/

= 4.08x10-19 J

3rd Ed. Shapiro p 77

4th Ed Shapiro p 109

488 x 10-3E=

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325 x 10-3

What about a UV laser?

E= 6.63x10-34 joule-seconds x 3x108

= 6.12 x 10-19 J so 1 Joule at 325 nm = 1.63x1018 photons

What about a He-Ne laser?

633 x 10-3

E= 6.63x10-34 joule-seconds x 3x108

= 3.14 x 10-19 J so 1 Joule at 633 nm = 3.18x1018 photons


4th Ed Shapiro p 109

E=h and E=hc/

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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


4th Ed. Shapiro p 104

Wavelength (period T)

Axis of

Magnetic F

ield

Axis of Propagation

Axi

s of

Ele

ctri

c F

ield

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Interference

ConstructiveInterference

DestructiveInterference

A

B

C

D

A+B

C+D

Am

plitude

0o 90o 180o 270o 360o Wavelength

Figure modified from Shapiro 3rd Ed “Practical Flow Cytometry” Wiley-Liss, p79


Here we have a phase difference of 180o (2 radians) so the waves cancel each other out

The frequency does not change, but the amplitude is doubled

Shapriro 4th Ed P105

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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





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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 the sky looks blue because the gas molecules scatter more light at shorter (blue) rather than longer wavelengths (red)light at shorter (blue) rather than longer wavelengths (red)

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Reflection and Refraction• Snell’s Law: The angle of

reflection (Ør) is equal to the angle of incidence (Øi) regardless of the surface material

• The angle of the transmitted beam (Øt) is dependent upon the composition of the material





t

i

r

Reflected Beam

Incident Beam

Transmitted(refracted)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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Refraction & Dispersion

Light is “bent” and the resultant colors separate (dispersion).Red is least refracted, violet most refracted.

dispersion

Short wavelengths are “bent” more than long wavelengths

refraction

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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

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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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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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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

– (n=the lowest refractive index between the object and first objective element) (hopefully 1)

– is 1/2 the angular aperture of the objective

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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

2 x NA

.000532 x 1.00= 0.265 m

.000531.00 = 0.53 m

• With a cone of light filling the entire aperture the theoretical resolution is…(fully open condenser)..

=

=

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A

NA=n(sin )

Light cone

(n=refractive index)

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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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Images reproduced from:

http://micro.magnet.fsu.edu/

Please go to this site and do the tutorials

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Refraction

Light is “bent” and the resultant colors separate (dispersion).Red is least refracted, violet most refracted.

dispersion

Short wavelengths are “bent” more than long wavelengths

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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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Absorption ChartColor in white light Color of light absorbed

red

blue

green

magenta

cyan

yellow

blue

blue

blue

blue

green

green

green

green

red

red

red

redblack

gray green bluepink

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Light absorption

Absorption

Control

No blue/green light red filter

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Light absorption

white light blue light red light green light

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The light spectrumWavelength = Frequency

Blue light

488 nm

short wavelength

high frequency

high energy (2 times the red)

Red light

650 nm

long wavelength

low frequency

low energy

Photon as a wave packet of energy

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Lecture Summary

• Principles of light and matter

• Basic Optics to take into consideration

• Essentials of light measurement

• Absorption and optical properties

Documents

© 1990-2006 J. Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture0003.ppt 1 BMS 602A/631 - Lecture 3 Light and Matter J. Paul Robinson, PhD