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2
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- 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 Ti
=n2 sin Tt
- 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)
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3
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- 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
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4
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- 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
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5
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6
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- 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
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7
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- 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
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8
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- 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).
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9
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- 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
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10
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- Example:
- 40 x 1.3 N.A. objective at 530 nm light
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11
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- 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
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12
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13
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14
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15
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- 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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18
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19
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20
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21
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22
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23
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24
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- The Elite optical system uses 5 side window PMTs and a number of filter
slots into which any filter can be inserted
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25
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26
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27
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28
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29
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- 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
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