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) 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)-494 0757 Fax (765)-494 0517 Email: robinsonATflowcyt.cyto.purdue.edu WEB: http://www.cyto.purdue.edu All materials used in this course are available for download on the web at http://tinyurl.com/2wkpp Slide last modified January, 2013 1:19 AM

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

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

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

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 10-34 joule-seconds c = speed of light (3x108 m/s) 3rd Ed Shapiro p 76 1:19 AM

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 1:19 AM

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

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

What about a He-Ne laser? Laser power What about a UV laser? E=h and E=hc/ 325 x 10-3 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 3rd Ed. Shapiro p 77 4th Ed Shapiro p 109 1:19 AM

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

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

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

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

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

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

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

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

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 = = 0.265 m 2 x NA 2 x 1.00 1:19 AM

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

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 1:19 AM

Images reproduced from: http://micro.magnet.fsu.edu/ Please go to this site and do the tutorials 1:19 AM

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

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

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

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

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

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 1:19 AM

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 1:19 AM

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 1:19 AM

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

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

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

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

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

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

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

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

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

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

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

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