1/30/14
The Nature of Light
Light and Energy
-  dependent on energy from the
sun, directly and indirectly
- solar energy intimately associated with existence of life
-light absorption:
dissipate as heat
emitted at longer wavelength
trigger chemical reaction
- putting together by light
-CO2+H2O = Glc + O2
- chlorophyll: reflects green,
absorbs violet-blue and redorange
1
1/30/14
Electromagnetic Radiation
Electron microscope
Atoms
Amino acids
Proteins
Light microscope
Viruses
Bacteria
Red blood cells
Human eye
Epithelial cells
Light: Particle or Wave
Wave nature of light is our main concern, however photon
(particle) energies are important when they excite visual
pigments in rods and cones.
Photons
Quanta of electromagnetic
radiation with detectable
photons
Waves
Main visualization of light
Electric and magnetic fields
that oscillate
=> when captured
2
1/30/14
Sources of Visible Light
-  Body
…. all organisms do
not visible
= 1000x weaker than
eyes
by-product of
biochemical processes
related to circadian
rhythm
- Sun
energy production in
form of thermal
radiation
(5,500 degrees C or
nearly 10,000 degrees
F)
= superheated
3
1/30/14
- Bioluminescence
yellow-green light produced by insects,
blue-green in marine organisms
luciferase, luciferin and oxygen
Uses?
-  Chemiluminescence
emission of light as a result of
chemical reaction
Liquid or gas reaction
4
1/30/14
- Visible light source
= incandescent light
Electrical current passing through
until light filaments glow
- Fluorescent light
= mercury or LED lights
EPA concerns
5
1/30/14
Use of Light in Microscopy
Detector
Filter
Mirror
Filter
Light
Source
Specimen
Incandescence
Traditional light source
Filament becomes hot
= light emission
Fluorescence
Fluorescent light +
filters + mirrors
= stained specimen
Bioluminescence
Fluorescent light +
filters + mirrors
= autofluorescence
Speed of Light
- in 45 years episodes of your favorite TV show will be
broadcasting somewhere in deep space
-186,000 miles/sec in a vacuum
-slows down when in a dense medium
=> reason why lenses work
- speed of light changes with every transfer into a different
medium = specimen, objectives, air etc.
6
1/30/14
Human Vision
Photoreceptors:
1.  Rods
- majority of photoreceptors
- more light sensitive
- contain rhodopsin
- when bleached causes
temporary blindness
2. Cones
- minority of photoreceptors
- contained close to the fovea
- bright light sensors
7
1/30/14
Types of vision
Scotopic = vision at low light levels
not mediating color vision
rods
Photopic = vision at bright light levels
color vision
cones
Image Formation in the Eye
8
1/30/14
Light Manipulation in the Microscope
Lenses
- lens: component of glass or transparent
plastic, circular in diameter, with two primary
surfaces that are ground and polished
9
1/30/14
Planoconvex
Biconvex
Planococave
Planar
In your Student
Microscopes
high degrees of corrections
spherical
chromatic = more than 4 colors
generally 11 lens elements
10
1/30/14
Mirrors
- different mirrors in microscopy, dependent on type of
microscope => generally planar in shape
Specimen
Microscope
Objective
Excitation
Emission
Dichroic
Mirror
Mirror
Regular upright microscope
Fluorescent inverted microscope
Prisms and Beamsplitters
- essential components
- glass/ transparent materials
- cut/ground to specific angles
Beamsplitters
- redirect portion of light
- remainder continues on path
DIC
Prism
- light dispersal
=> dissection into
components
- image rotation, deviation &
displacement
- lucite prism = true color
illumination
11
1/30/14
Light Filters/Lenses
- most are absorbing or reflecting unwanted
light
- dichroic filter = fluorescence
- polarizing filter (changes the orientation of
oscillation)
- heat absorbing filters limit infrared
- neutral density filters limit intensity of all
wavelength
=> synthetic gels and colored filter glass
Lasers
-conceptualized
in the late 1950 s
- Light Amplification by the
Stimulated Emission of Radiation
- optical amplification => photon perturbation of any
matter leads to creation of a new photo
- perturbing photon not disturbed in process
- in optical microscopy: high density monochromatic light
sources
- confocal microscopy
12
1/30/14
13