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Properties of Light II Vision

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Title: Properties of Light II Vision


1
Properties of Light II / Vision
  • September 22, 2008
  • Overview of Microscopy
  • Dr. Behonick

2
Topics for today
  • Properties of Light II
  • Resolution
  • Vision

3
Properties of Light II
4
Light
  • functions like
  • stream of particles
  • wave
  • how fast it travels depends on what its
    traveling through
  • in vacuum 186,000 miles/sec
  • in anything else, its slowed-down

5
Measuring Waves
A
amount of time required to complete T
6
Visible Light Spectrum
wavelengths 400 700 nm constitute visible light
for humans
shorter wavelength higher frequency higher energy
longer wavelength lower frequency lower energy
7
Light interacting with matter
  • when light hits matter it can be
  • transmitted
  • reflected
  • partially absorbed
  • refracted
  • diffracted

8
Properties of Light
absorbed
9
Properties of Light
transmitted
reflected
absorbed
10
Microscopy Techniques
objective
light source
transmitted bright field phase DIC
11
Objects and transmitted light
light wave
amplitude object
seen as color
phase object
not seen as color
12
Transmitted Light
  • amplitude object interferes w/ wave of
    transmitted light such that it changes amplitude
    of wave
  • object has color

13
Absorption
  • When light hits matter it can be
  • absorbed - reduction of amplitude of one or more
    wavelengths
  • you see whatever is left as transmitted (or
    sometimes reflected) light
  • note if fully absorbed then the light is not
    transmitted!

14
Microscopy Techniques
objective
light source
transmitted bright field
15
Transmitted Light
  • amplitude object interferes w/ wave of
    transmitted light such that it changes amplitude
    of wave
  • object has color
  • phase object interferes w/ wave of transmitted
    light such that it changes phase of wave
  • causes phase shift
  • object is colorless

16
Phase Shift
  • when a wave changes from its original rhythm

17
in-phase waves
out-of-phase waves
18
Reflection
  • When light hits matter it can be
  • reflected - light bounces off the specimen
  • specular reflection - predictable reflection off
    a smooth surface which produces high fidelity
    image (e.g. mirror, polished metal, still water)
  • diffuse reflection - hard to predict reflection
    off a rough surface, creates haze and glare (e.g.
    wavy water, crumpled aluminum foil, cells)

19
Reflection
20
Reflection
21
Reflection
  • Interactive for reflection
  • http//micro.magnet.fsu.edu/primer/java/reflectio
    n/reflectionangles/index.html

22
Microscopy Techniques
light source/objective
reflected dissecting microscope
23
Absorption Emission
  • fluorescence microscopy
  • fluorophore molecule that upon absorbing energy
    can reach excited state, then emit energy
  • microscope light source irradiates sample w/
    specific wavelength of light
  • light absorbed by fluorophores in sample
  • causes electrons in fluorophores to become
    excited move up a level temporarily
  • when these temporarily excited electrons fall
    back to their original state, they emit their own
    light energy, which is the fluorescence you see
    under the microscope

24
Fluorescence
  • fluorescence process of excitation, loss of
    energy emission of light from a fluorophore

25
optical filters example
not a filter
white light
white light
red filter
blue/green light absorbed RED PASSES THROUGH
white light
modifications by GG
26
Optical filters- examples
27
Absorbed light chemistry
  • Light that was absorbed can be
  • very rapidly re-emitted as light of longer
    wavelength (fluorescence)
  • rapidly re-emitted as light of longer wavelength
    (phosphorescence)
  • slowly re-radiated as infrared waves (heat)
  • transformed into chemical energy (e.g. breaking
    chemical bonds)

28
Jablonski diagram
www.molecularprobes.com/handbook/figures/0664.html
29
Microscopy Techniques
light source/objective
incident/epi-illumination fluorescent
30
Refraction
  • When light hits matter it can be
  • refracted - bends as it passes from one material
    to another
  • refractive index (n) of material is important in
    microscopy

31
Refraction
32
Refraction
33
Refraction Resources
  • Refraction
  • http//micro.magnet.fsu.edu/primer/java/refractio
    n/criticalangle/index.html
  • Refraction w/ different wavelengths
  • http//micro.magnet.fsu.edu/primer/java/scienceop
    ticsu/refraction/refractionangles/index.html

34
Diffraction
  • When light hits matter it can be
  • diffracted - bends as it passes an edge
    (including that of a small aperture)
  • Abbes theory of microscopy shows how diffraction
    is critical for image formation

35
Diffraction of waves
36
Dispersion
  • When light hits matter it can be
  • dispersed - refraction diffraction are
    wavelength dependant, so white light gets
    separated into its constituent colors when
    refracted/diffracted
  • leads to blue/red shift ( corrections for it
    in confocal optics)

37
Dispersion
38
Scatter
  • When light hits matter it can be
  • scattered - a combination of various effects (or
    mostly diffraction or reflection in many
    directions
  • ex Why is the sky blue? Blue light is
    scattered in all directions by the molecules of
    the air, so no matter in what direction we look,
    we see blue sky)
  • scatter in a microscope light wandering off
    from desired path

39
Transmitted Light
  • transmitted light passing thru object can be
  • refracted
  • diffracted by edges of opaque portions by
    structures nearly as small as wavelengths of
    light
  • this diffraction allows us to use microscopes to
    see small structures (its not just about
    magnification)

40
Putting it all together
  • opaque objects absorb light
  • transparent objects transmit light
  • reflective objects reflect light
  • scattering objects diffract light

41
Putting it all together
  • objects usually have a combo of qualities - we
    refer to them by the predominant or most relevant
    quality
  • amplitude objects (a microscopy term) are
    somewhat opaque
  • phase objects (a microscopy term) are fairly
    transparent

42
Light and matter putting it together
  • light hitting a cell will be somewhat reflected,
    transmitted, refracted, absorbed, dispersed
    diffracted
  • leads to phase shifts changes in polarity which
    are used by microscope to form image
  • we cant see phase differences or polarity
    differences with our own eyes, but microscope can
    use them to generate contrast in image
  • basis of DIC phase microscopy
  • light is also absorbed can cause fluorescence

43
Light interacting with matter
  • When light hits matter it can
  • be absorbed and cause fluorescence
  • be diffracted/refracted and change phase
  • become polarized (or change polarization)

44
Resources
  • Refraction/reflection/
  • diffraction animation
  • http//www.lon-capa.org/mmp/kap13/cd372.htm

45
Resolution
46
Resolving Power
  • resolution ability to distinguish 2 nearby
    objects as separate objects
  • resolution limit distance between 2 objects
    below which they will appear as 1 object
  • resolving power ability of microscope to
    distinguish small separate objects as such

47
Resolving Power
  • depends on
  • wavelength of probe - light or electrons?
  • N.A. of lenses in system
  • quality of detector (CCD camera, PMT, etc.)
  • contrast in image
  • refractive index of materials

48
Same magnification, different resolving power
49
Proper illumination of a diatom
50
Resolution light vs. electrons
51
Vision
52
Eye
  • sclera - white, connective tissue
  • cornea - transparent, good at self-repair
  • pupil - central opening
  • iris - colored muscle
  • aqueous vitreous humor
  • retina - home of rods cones

53
VitreousHumor
Retina
Lens Muscle
Sclera
Ligaments
Choroid
Iris
Fovea
Lens
Pupil
Cornea
AqueousHumor
Optic Nerve
eyelid
Blind Spot
54
Refraction
  • refraction bending of light when it passes from
    a medium of one density into a medium of another
    density

55
Refraction
  • result image is inverted on retina

56
Refraction
57
Visual Acuity
  • visual acuity sharpness of vision
  • depends upon resolving power
  • resolving power ability of visual system to see
    2 closely spaced dots as separate
  • Myopia (nearsightedness)
  • image brought to focus in front of retina
  • Hyperopia (farsightedness)
  • image brought to focus behind the retina
  • Astigmatism
  • asymmetry of the cornea and/or lens, images of
    lines of circle appear blurred

58
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59
Accommodation
  • ability of eyes to keep image focused on the
    retina as distance between the eyes object
    varies
  • looking at something nearby, then looking at
    something far away - both are in focus
  • due to ciliary muscle flexibility of lens
  • Presbyopia less accommodation, usually with age

60
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61
Iris
  • bright light ? parasympathetic response
  • circularly arranged smooth muscles contract ?
    smaller pupil
  • dim light ? sympathetic response
  • radially arranged smooth muscles contract ?
    larger pupil

62
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63
Retina
  • thin layer of neural cells on back of eyeball
  • human retina is inverted
  • light passes through neurons or wires before
    hitting the photoreceptors

back of eye
front of eye
light
light
64
Photoreceptors Rods Cones
Optic Nerve
light
Signal-ProcessingNeurons
GanglionCell
Membrane discs bearingrhodopsin
65
Retina
  • photoreceptors rods cones
  • rods
  • best for night vision, low light
  • absorb blue-green light (not red)
  • cones
  • function best in bright light
  • provide color vision greater visual acuity
    (detail)
  • humans (trichromats) - blue, red, green cones
  • color of cone determined by what pigment it
    contains ? what color light it responds to

66
Retina
  • photoreceptors rods and cones
  • rods
  • light leads to bleaching reaction
  • ? rhodopsin dissociates into retinal and
    opsin
  • ? dark current stops
  • dark current Na channels open all the time and
    inhibitory NT are being released until free
    opsins are present (due to light!
  • ? inhibition of bipolar neurons stops and AP
    fire
  • Na channels close, inhibitory NT isnt released
  • bipolar cell releases excitatory NT, stimulates
    ganglion cell to fire an AP down the optic nerve!
  • cones similar
  • also other neurons present

67
Cones
  • each type of cone contains retinal plus a special
    pigment/opsin
  • gene for green and red opsins (allow perception
    of green/red) are on X chromosome
  • ? men are more likely to be colorblind

68
Blind Spot
  • optic disc ganglion nerve fibers gather as optic
    disc, exit as optic nerve
  • results in a blind spot - patch of retina that
    lacks photoreceptors (3 mm2)
  • blood vessels also enter and exit at the blind
    spot

69
Blind Spot
70
Fovea
  • contains only cones
  • no convergence
  • best visual acuity
  • sharp central vision

71
Fovea
72
Convergence
  • 120 million rods
  • 6 million cones
  • only 1.2 million axons enter the optic nerve

73
Convergence
74
Vision
  • saccadic eye movements small, quick movements
    to move focus
  • eyes appear still
  • keeps words focused at fovea as you read
  • prevents too much bleaching

75
Neural Pathways from Retina
  • RIGHT half of visual field projects to LEFT half
    of retina of both eyes
  • information ultimately sent to visual cortex
    (occipital lobe, cerebrum/telecephalon, forebrain)

76
Vision
  • binocular vision seeing with both eyes at once
  • advantages
  • you always have a spare
  • wider field of view
  • enhanced ability to see faint objects
  • depth perception
  • critical period ages 6 months - 2 years
  • activity-dependant wiring
  • use it or lose it

77
Vision
  • brain breaks down the info from your eyes, then
    reconstructs it into one image
  • cortex has neurons specific to..
  • rapid left to right movements
  • slow left to right movements
  • faces
  • edges
  • etc.

78
Vision-related Movie
  • http//www.macula.org/anatomy/anatomy.html

79
Other nifty stuff
80
Synesthesia
  • humans with unusual, linked senses
  • colored words
  • colored sounds
  • tasty shapes

81
Dr. Bs synesthetic alphabet
abcdefghijklmnopqrstuvwxyz 1234567890
82
References
  • Giorgi, G. Lecture 7. Merritt College Biology
    035, 12 February 2008.
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