© J. Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture0003.ppt 1 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; 4 th Ed. Shapiro pp ) 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 Slide last modified January 9, 2006

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

© J. Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture0003.ppt 3 Light and Matter Energy –joules, radiant flux (energy/unit time) –watts (1 watt=1 joule/second) Angles –steradians - sphere radius r - circumference is 2  r 2 ; 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  r 2. One steradian is defined as the solid angle which intercepts as area equal; to r 2 on the sphere surface 3 rd Ed - Shapiro p 75 4 th Ed – Shapiro p 101

© J. Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture0003.ppt 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  r 2 ; one steradian is defined as that solid angle which intercepts an area equal to r 2 on the surface. Mole - contains Avogadro's number of molecules (6.02 x ) 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 10 8 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 joule- seconds] Energy - higher at short wavelengths - lower at longer wavelengths.

© J. Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture0003.ppt 5 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/ = wavelength h = Planck’s constant (6.63 x joule-seconds c = speed of light (3x10 8 m/s) 3 rd Ed Shapiro p 76

© J. Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture0003.ppt 6 Electromagnetic Spectrum We can see from about 400 nm to 700 nm This is known as the visible spectrum

© J. Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture0003.ppt 7 The intensity of the radiation is inversely proportional to the square of the distance traveled

© J. Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture0003.ppt 8 Laser power One photon from a 488 nm argon laser has an energy of 6.63x joule-seconds x 3x10 8 To get 1 joule out of a 488 nm laser you need 2.45 x photons 1 watt (W) = 1 joule/second a 10 mW laser at 488 nm is putting out 2.45x10 16 photons/sec E=h and E=hc/ = 4.08x J 3 rd Ed. Shapiro p 77 4 th Ed Shapiro p x E=

© J. Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture0003.ppt x What about a UV laser? E= 6.63x joule-seconds x 3x10 8 = 6.12 x J so 1 Joule at 325 nm = 1.63x10 18 photons What about a He-Ne laser? 633 x E= 6.63x joule-seconds x 3x10 8 = 3.14 x J so 1 Joule at 633 nm = 3.18x10 18 photons 3 rd Ed. Shapiro p 77 4 th Ed Shapiro p 109 E=h and E=hc/

© J. Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture0003.ppt 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 3 rd Ed. Shapiro p 78 4 th Ed. Shapiro p 104 Wavelength (period T) Axis of Magnetic Field Axis of Propagation Axis of Electric Field

© J. Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture0003.ppt 11 Interference Constructive Interference Destructive Interference A B C D A+B C+D Amplitude 0o0o 90 o 180 o 270 o 360 o Wavelength Figure modified from Shapiro 3 rd Ed “Practical Flow Cytometry” Wiley-Liss, p79 4 th Ed. Shapiro p 109 Here we have a phase difference of 180 o (2  radians) so the waves cancel each other out The frequency does not change, but the amplitude is doubled Shapriro 4 th Ed P105

© J. Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture0003.ppt 12 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 0 o and 180 o 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 3 rd Ed. Shapiro p 79 4 th Ed. Shapiro p rd Ed. Shapiro p 79 4 th Ed. Shapiro p 105

© J. Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture0003.ppt 13 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 4 th 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)

© J. Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture0003.ppt 14 Reflection and Refraction Snell’s Law: The angle of reflection (Ø r ) is equal to the angle of incidence (Ø i ) regardless of the surface material The angle of the transmitted beam (Ø t ) is dependent upon the composition of the material 3 rd Ed. Shapiro p 81 4 th Ed. Shapiro p rd Ed. Shapiro p 81 4 th Ed. Shapiro p 106 tt ii rr Reflected Beam Incident Beam Transmitted (refracted)Beam n 1 sin Ø i = n 2 sin Ø t The velocity of light in a material of refractive index n is c/n

© J. Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture0003.ppt 15 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 ref rac tion

© J. Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture0003.ppt 16 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 3 rd Shapiro p 93 4 th Shapiro p rd Shapiro p 93 4 th Shapiro p 119

© J. Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture0003.ppt 17 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

© J. Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture0003.ppt 18 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

© J. Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture0003.ppt 19 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

© J. Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture0003.ppt 20 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 x 1.00 =  m = 0.53  m With a cone of light filling the entire aperture the theoretical resolution is…(fully open condenser).. = =

© J. Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture0003.ppt 21 A  NA=n(sin  ) Light cone (n=refractive index)

© J. Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture0003.ppt 22 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

© J. Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture0003.ppt 23 Images reproduced from: Please go to this site and do the tutorials

© J. Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture0003.ppt 24 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

© J. Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture0003.ppt 25 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

© J. Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture0003.ppt 26 Absorption Chart Color in white light Color of light absorbed red blue green magenta cyan yellow blue green red black gray green blue pink

© J. Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture0003.ppt 27 Light absorption Absorption Control No blue/green light red filter

© J. Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture0003.ppt 28 Light absorption white lightblue lightred lightgreen light

© J. Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture0003.ppt 29 The light spectrum Wavelength = 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

© J. Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture0003.ppt 30 Lecture Summary Principles of light and matter Basic Optics to take into consideration Essentials of light measurement Absorption and optical properties