1. 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 )
Notice: The materials in this presentation are copyrighted materials. If you want to use any of these slides, you may do so if you credit each slide with the author’s name. It is illegal to place these notes on CourseHero or any other site.
Lab: Bindley 122
Purdue University
Office Phone: (765)
Fax (765)
robinsonATflowcyt.cyto.purdue.edu
WEB:
All materials used in this course are available for download on the web at
Slide last modified January, 2013
1:19 AM

2. 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
1:19 AM

3. Light and Matter Energy Angles 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
1:19 AM

4. 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 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 joule-seconds]
Energy - higher at short wavelengths - lower at longer wavelengths.
1:19 AM

5. Photons and Quantum Theory
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/
 = wavelength
h = Planck’s constant
(6.63 x joule-seconds
c = speed of light (3x108 m/s)
3rd Ed Shapiro p 76
1:19 AM

6. Electromagnetic Spectrum
We can see from about 370 nm to about 700 nm
This is known as the visible spectrum
Images from:
1:19 AM

7. Inverse Square Law
The intensity of the radiation is inversely proportional to the square of the distance traveled
1:19 AM

8. Laser power E= One photon from a 488 nm argon laser has an energy of
E=h and E=hc/
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=
= 4.08x10-19 J
488 x 10-3
3rd Ed. Shapiro p 77
4th Ed Shapiro p 109
1:19 AM

9. What about a He-Ne laser?
Laser power
What about a UV laser?
E=h and E=hc/
325 x 10-3
E= x10-34 joule-seconds x 3x108
= 6.12 x J so 1 Joule at 325 nm = 1.63x1018 photons
What about a He-Ne laser?
633 x 10-3
E= x10-34 joule-seconds x 3x108
= 3.14 x J so 1 Joule at 633 nm = 3.18x1018 photons
3rd Ed. Shapiro p 77
4th Ed Shapiro p 109
1:19 AM

10. 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
Axis of Electric Field
Wavelength (period T)
Magnetic Field
Axis of
Axis of Propagation
3rd Ed. Shapiro p 78
4th Ed. Shapiro p 104
1:19 AM

11. Interference A+B A B C+D C D Wavelength Amplitude Constructive
The frequency does not change, but the amplitude is doubled A
Amplitude B
Constructive
Interference
Here we have a phase difference of 180o (2 radians) so the waves cancel each other out
C+D C D
Destructive
Interference
Figure modified from Shapiro 3rd Ed “Practical Flow Cytometry” Wiley-Liss, p79
4th Ed. Shapiro p 109
Shapiro 4th Ed P105
1:19 AM

12. 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
Transmitted
(refracted)Beam
Reflected Beam r t i
Incident Beam
n1 sin Øi = n2 sin Øt
The velocity of light in a material of refractive index n is c/n
3rd Ed. Shapiro p 81
4th Ed. Shapiro p 106
1:19 AM

13. Refraction & Dispersion
Short wavelengths are “bent” more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
1:19 AM

14. 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
Light Propagation and Vergence
This slide changes the focus of the lecture from illumination sources back to more properties of light in regards to what can happen to light after it leaves its illumination source.
3rd Shapiro p 93
4th Shapiro p 119
1:19 AM

15. 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
1:19 AM

16. The higher the NA, the shorter the working distance
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
1:19 AM

17. 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
1:19 AM

18. Numerical Aperture   .00053 = = 0.53 m 1.00 NA .00053 = = 0.265 m
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). 
.00053
1.00
= 0.53 m = NA
With a cone of light filling the entire aperture the theoretical resolution is…(fully open condenser).. 
.00053 =
= m
2 x NA
2 x 1.00
1:19 AM

19. A m
Light cone
NA=n(sin m)
(n=refractive index)
1:19 AM

20. 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:
Please go to this site and do the tutorials
1:19 AM

21. Images reproduced from:
Please go to this site and do the tutorials
1:19 AM

22. Some Definitions Absorption Refraction Diffraction Dispersion
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
1:19 AM

23. Absorption Chart Color in white light Color of light absorbed blue red
green
blue
red
green
green
red
blue
blue
yellow
magenta
green
cyan
red
black
red
green
blue
gray
pink
green
blue
1:19 AM

24. Light absorption Control Absorption No blue/green light red filter
1:19 AM

25. Light absorption
white light
blue light
red light
green light
1:19 AM

26. Wavelength = Frequency
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
1:19 AM

27. Light Scatter
Materials scatter light at wavelengths at which they do not absorb
If we consider the visible spectrum to be 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
1:19 AM

28. 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
1:19 AM

29. 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.
1:19 AM

30. 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)
1:19 AM

31. 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?
1:19 AM

32. 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:
Size, morphology, viability, structure, effective refractive index, etc.
1:19 AM

33. Light scattering Scattering Absorption Incident Beams Emission
(Mie, Rayleigh)
Absorption
Emission
(Fluorescence)
Particles
Incident Beams
The interaction of light with a particle in terms of scattering, absorption, and emission.
1:19 AM 4

34. 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
1:19 AM

35. 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.
1:19 AM

36. 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), pin-hole(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
1:19 AM

37. 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
FSC
SSC
Granulocytes
Lymphocytes
Monocytes
RBCs, Debris,
Dead Cells
1:19 AM

38. The polystyrene particles certified with the scanning flow cytometer
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
1:19 AM

39. White blood cells Light Scatter Gating Side Scatter Projection
200
400
600
800
1000
Side Scatter Projection
Forward Scatter
Projection
90 Degree Scatter
Neutrophils
Lymphocytes
Monocytes
Light Scatter Gating
1:19 AM

40. 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
1:19 AM

41. 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
1:19 AM