Properties of Light II Vision

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Properties of Light II / Vision

• September 22, 2008
• Overview of Microscopy
• Dr. Behonick

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Topics for today

• Properties of Light II
• Resolution
• Vision

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Properties of Light II
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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

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Measuring Waves
A
amount of time required to complete T
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Visible Light Spectrum
wavelengths 400 700 nm constitute visible light
for humans
shorter wavelength higher frequency higher energy
longer wavelength lower frequency lower energy
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Light interacting with matter

• when light hits matter it can be
• transmitted
• reflected
• partially absorbed
• refracted
• diffracted

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Properties of Light
absorbed
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Properties of Light
transmitted
reflected
absorbed
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Microscopy Techniques
objective
light source
transmitted bright field phase DIC
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Objects and transmitted light
light wave
amplitude object
seen as color
phase object
not seen as color
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Transmitted Light

• amplitude object interferes w/ wave of
  transmitted light such that it changes amplitude
  of wave
• object has color

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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!

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Microscopy Techniques
objective
light source
transmitted bright field
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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

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Phase Shift

• when a wave changes from its original rhythm

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in-phase waves
out-of-phase waves
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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)

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Reflection
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Reflection
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Reflection

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

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Microscopy Techniques
light source/objective
reflected dissecting microscope
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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

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Fluorescence

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

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optical filters example
not a filter
white light
white light
red filter
blue/green light absorbed RED PASSES THROUGH
white light
modifications by GG
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Optical filters- examples
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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)

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Jablonski diagram
www.molecularprobes.com/handbook/figures/0664.html
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Microscopy Techniques
light source/objective
incident/epi-illumination fluorescent
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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

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Refraction
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Refraction
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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

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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

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Diffraction of waves
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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)

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Dispersion
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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

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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)

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Putting it all together

• opaque objects absorb light
• transparent objects transmit light
• reflective objects reflect light
• scattering objects diffract light

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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

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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

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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)

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Resources

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

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Resolution
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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

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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

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Same magnification, different resolving power
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Proper illumination of a diatom
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Resolution light vs. electrons
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Vision
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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

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VitreousHumor
Retina
Lens Muscle
Sclera
Ligaments
Choroid
Iris
Fovea
Lens
Pupil
Cornea
AqueousHumor
Optic Nerve
eyelid
Blind Spot
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Refraction

• refraction bending of light when it passes from
  a medium of one density into a medium of another
  density

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Refraction

• result image is inverted on retina

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Refraction
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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

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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

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Iris

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

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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
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Photoreceptors Rods Cones
Optic Nerve
light
Signal-ProcessingNeurons
GanglionCell
Membrane discs bearingrhodopsin
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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

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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

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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

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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

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Blind Spot
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Fovea

• contains only cones
• no convergence
• best visual acuity
• sharp central vision

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Fovea
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Convergence

• 120 million rods
• 6 million cones
• only 1.2 million axons enter the optic nerve

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Convergence
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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

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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)

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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

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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.

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Vision-related Movie

• http//www.macula.org/anatomy/anatomy.html

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Other nifty stuff
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Synesthesia

• humans with unusual, linked senses
• colored words
• colored sounds
• tasty shapes

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Dr. Bs synesthetic alphabet
abcdefghijklmnopqrstuvwxyz 1234567890
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References

• Giorgi, G. Lecture 7. Merritt College Biology
  035, 12 February 2008.