1	BMS 631 - Lecture 4
2	Review Scatter - Rayleigh Scatter - directly proportional to property of the scattering molecule called molecular polarizability (ie dipole formation), inversely proportional to the fourth power of the wavelength of the incident light (blue light has highest scatter - thus blue sky!) Scatter - Raman Scatter - (p 93 3rd ed) molecules undergo vibrational transitions at the same time as scatter occurs- if is transition to higher level is known as Stoke's Raman emission. Normally 1/1000th intensity of Rayleigh Scatter, but is significantly increased when using lasers for excitation.. Raman emission of water at 488 nm excitation is around 570-590 nm. Polarizations - E vectors - larger changes in E vectors not incident light plane; Mie scattering - increased scatter in the forward angle for larger particles (1/4 wavelength to tens of wavelength). (p89, 3rd ed) Incident light, reflected light, transmitted light, refractive index - note the angle of incidence = angle of reflection regardless of the material of surface. tt transmission angle depends upon the composition of material according to Snell's law of refraction n¹ sin T_i =n² sin T_t n₁, n₂ are the refractive indices respectively through which the incident beam passes (air = 1 essentially) Brewster's Angle, chromatic aberration, filters, interference, band pass, dichroic, absorption, laser blocker. Fluorescence lifetime, polarization, fluidity, anisotrophy, resonance energy transfer, quenching, bleaching (p82 3rd ed)
3	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 woul dhave 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
4	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
5	Properties of thin Lenses
6	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
7	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
8	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).
9	Depth of Field and Resolution Depth of field is designated as the longitudinal distance for the formation of a sharp image is obtained at a fixed point in the image plane Limits of resolution are diffraction limited - the diffraction image is a point is a bright central spot surrounded by what is called the Airy disk (alternating light and dark rings) at wavelength l, the radius of the Airy disk is 0.61 l Thus to resolve two points they need to be at least this distance apart (radius of the Airy disk) thus the resolution is defined as 0.61 l /NA
10	Object Resolution Example: 40 x 1.3 N.A. objective at 530 nm light
11	Köhler Köhler illumination creates an evenly illuminated field of view while illuminating the specimen with a very wide cone of light Two conjugate image planes are formed one contains an image of the specimen and the other the filament from the light
12	Köhler Illumination
13	Refraction
14	Refraction
15	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
16	Absorption Chart
17	Light absorption
18	Light absorption
19	The light spectrum Wavelength = Frequency
20	Technical Aspects of Flow Cytometry Illumination Sources
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22	Elite Cytometer with 4 Lasers
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24	Coulter Optical System - Elite The Elite optical system uses 5 side window PMTs and a number of filter slots into which any filter can be inserted
25	Coulter Optical System - Elite
26	Detection Systems Bio-Rad Bryte HS
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28	Bryte HS Optical System
29	Summary Slide Light propagation and image planes We use optical filters to separate the spectrum Each cytometer has a different optical train PMTs are used for signal collectio www.cyto.purdue.edu