Light and Sound are complex phenomena that are classically
explained with a simple model based on rays and
wavefronts.
The
Molecular Expressions Microscopy Primer
explores many of the aspects of visible light starting with
an introduction to electromagnetic radiation and continuing
through to human vision and the perception of color. Each
section outlined below is an independent treatise on a
limited aspect of light and color. We hope you enjoy your
visit and find the answers to your questions.
Electromagnetic Radiation - Visible light is a complex
phenomenon that is classically explained with a simple model
based on propagating rays and wavefronts, a concept first
proposed in the late 1600s by Dutch physicist Christiaan
Huygens. Electromagnetic radiation, the larger family of
wave-like phenomena to which visible light belongs (also
known as radiant energy), is the primary vehicle transporting
energy through the vast reaches of the universe. The
mechanisms by which visible light is emitted or absorbed by
substances, and how it predictably reacts under varying
conditions as it travels through space and the atmosphere,
form the basis of the existence of color in our universe.
Light:
Particle or a Wave?
- Many distinguished scientists have attempted to explain how
electromagnetic radiation can display what has now been
termed duality, or both particle-like and wave-like behavior.
At times light behaves as if composed of particles, and at
other times as a continuous wave. This complementary, or
dual, role for the properties of light can be employed to
describe all of the known characteristics that have been
observed experimentally, ranging from refraction, reflection,
interference, and diffraction, to the results with polarized
light and the photoelectric effect.
Sources of Visible Light - A wide variety of sources are
responsible for emission of electromagnetic radiation, and
are generally categorized according to the specific spectrum
of wavelengths generated by the source. Relatively long radio
waves are produced by electrical current flowing through huge
broadcast antennas, while much shorter visible light waves
are produced by the energy state fluctuations of negatively
charged electrons within atoms. The shortest form of
electromagnetic radiation, gamma waves, results from decay of
nuclear components at the center of the atom. The visible
light that humans are able to see is usually a mixture of
wavelengths whose varying composition is a function of the
light source.
Fluorescence
- The phenomenon of fluorescence was known by the middle of
the nineteenth century. British scientist Sir George G.
Stokes made the observation that the mineral fluorspar
exhibits fluorescence when illuminated with ultraviolet
light, and he coined the word "fluorescence". Stokes observed
that the fluorescing light has longer wavelengths than the
excitation light, a phenomenon that has become to be known as
the Stokes shift. Fluorescence microscopy is an excellent
method of studying material that can be made to fluoresce,
either in its natural form (termed primary or auto
fluorescence) or when treated with chemicals capable of
fluorescing (known as secondary fluorescence). The
fluorescence microscope was devised in the early part of the
twentieth century by August Köhler, Carl Reichert, and
Heinrich Lehmann, among others. However, the potential of
this instrument was not realized for several decades, and
fluorescence microscopy is now an important (and perhaps
indispensable) tool in cellular biology.
Speed
of Light
- Starting with Ole Roemer's 1676 breakthrough endeavors, the
speed of light has been measured at least 163 times by more
than 100 investigators utilizing a wide variety of different
techniques. Finally in 1983, more than 300 years after the
first serious measurement attempt, the speed of light was
defined as being 299,792.458 kilometers per second by the
Seventeenth General Congress on Weights and Measures. Thus,
the meter is defined as the distance light travels through a
vacuum during a time interval of 1/299,792,458 seconds. In
general, however, (even in many scientific calculations) the
speed of light is rounded to 300,000 kilometers (or 186,000
miles) per second.
Reflection
of Light
- Reflection of light (and other forms of electromagnetic
radiation) occurs when the waves encounter a surface or other
boundary that does not absorb the energy of the radiation and
bounces the waves away from the surface. The incoming light
wave is referred to as an incident wave and the wave that is
bounced away from the surface is called the reflected wave.
The simplest example of visible light reflection is the
glass-like surface of a smooth pool of water, where the light
is reflected in an orderly manner to produce a clear image of
the scenery surrounding the pool. Throw a rock into the pool,
and the water is perturbed to form waves, which disrupt the
image of the scene by scattering the reflected light in all
directions.
Refraction
of Light
- As light passes from one substance into another, it will
travel straight through with no change of direction when
crossing the boundary between the two substances head-on
(perpendicular, or a 90-degree angle of incidence). However,
if the light impacts the boundary at any other angle it will
be bent or refracted, with the degree of refraction
increasing as the beam is progressively inclined at a greater
angle with respect to the boundary. As an example, a beam of
light striking water vertically will not be refracted, but if
the beam enters the water at a slight angle it will be
refracted to a very small degree. If the angle of the beam is
increased even further, the light will refract with
increasing proportion to the entry angle. Early scientists
realized that the ratio between the angle at which the light
crosses the media interface and the angle produced after
refraction is a very precise characteristic of the material
producing the refraction effect.
Diffraction
of Light
- Depending on the circumstances that give rise to the
phenomenon, diffraction can be perceived in a variety of
different ways. Scientists have cleverly utilized diffraction
of neutrons and X-rays to elucidate the arrangement of atoms
in small ionic crystals, molecules, and even such large
macromolecular assemblies as proteins and nucleic acids.
Electron diffraction is often employed to examine periodic
features of viruses, membranes, and other biological
organisms, as well as synthetic and naturally occurring
materials. No lens exists that will focus neutrons and X-rays
into an image, so investigators must reconstruct images of
molecules and proteins from the diffraction patterns using
sophisticated mathematical analysis. Fortunately, magnetic
lenses can focus diffracted electrons in the electron
microscope, and glass lenses are very useful for focusing
diffracted light to form an optical image that can easily be
viewed.
Polarization
of Light
- The human eye lacks the ability to distinguish between
randomly oriented and polarized light, and plane-polarized
light can only be detected through an intensity or color
effect, for example, by reduced glare when wearing polarized
sun glasses. In effect, humans cannot differentiate between
the high contrast real images observed in a polarized light
microscope and identical images of the same specimens
captured digitally (or on film), and then projected onto a
screen with light that is not polarized. The first clues to
the existence of polarized light surfaced around 1669 when
Erasmus Bartholin discovered that crystals of the mineral
Iceland spar (more commonly referred to as calcite) produce a
double image when objects are viewed through the crystals in
transmitted light. During his experiments, Bartholin also
observed a quite unusual phenomenon. When the calcite
crystals are rotated about their axis, one of the images
moves in a circle around the other, providing strong evidence
that the crystals are somehow splitting the light into two
different beams.
Fundamentals
of Interference
- The seemingly close relationship between diffraction and
interference occurs because they are actually manifestations
of the same physical process and produce ostensibly
reciprocal effects. Most of us observe some type of optical
interference almost every day, but usually do not realize the
events in play behind the often-kaleidoscopic display of
color produced when light waves interfere with each other.
One of the best examples of interference is demonstrated by
the light reflected from a film of oil floating on water.
Another example is the thin film of a soap bubble, which
reflects a spectrum of beautiful colors when illuminated by
natural or artificial light sources.
Optical
Birefringence
- Anisotropic crystals, such as quartz, calcite, and
tourmaline, have crystallographically distinct axes and
interact with light by a mechanism that is dependent upon the
orientation of the crystalline lattice with respect to the
incident light angle. When light enters the optical axis of
anisotropic crystals, it behaves in a manner similar to the
interaction with isotropic crystals, and passes through at a
single velocity. However, when light enters a non-equivalent
axis, it is refracted into two rays each polarized with the
vibration directions oriented at right angles to one another,
and traveling at different velocities. This phenomenon is
termed double refraction or birefringence and is exhibited to
a greater or lesser degree in all anisotropic crystals.
Color
Temperature
- The concept of color temperature is of critical importance
in photography and digital imaging, regardless of whether the
image capture device is a camera, microscope, or telescope. A
lack of proper color temperature balance between the
microscope light source and the film emulsion or image sensor
is the most common reason for unexpected color shifts in
photomicrography and digital imaging. If the color
temperature of the light source is too low for the film,
photomicrographs will have an overall yellowish or reddish
cast and will appear warm. On the other hand, when the color
temperature of the light source is too high for the film,
photomicrographs will have a blue cast and will appear cool.
The degree of mismatch will determine the extent of these
color shifts, with large discrepancies leading to extremes in
color variations. Perhaps the best example is daylight film
used in a microscope equipped with a tungsten-halogen
illumination source without the benefit of color balancing
filters. In this case, the photomicrographs will have a quite
large color shift towards warmer reddish and yellowish hues.
As problematic as these color shifts may seem, they are
always easily corrected by the proper use of conversion and
light balancing filters.
Primary
Colors
- The human eye is sensitive to a narrow band of
electromagnetic radiation that lies in the wavelength range
between 400 and 700 nanometers, commonly known as the visible
light spectrum, which is the only source of color. When
combined, all of the wavelengths present in visible light,
about a third of the total spectral distribution that
successfully passes through the Earth's atmosphere, form
colorless white light that can be refracted and dispersed
into its component colors by means of a prism. The colors
red, green, and blue are classically considered the primary
colors because they are fundamental to human vision. Light is
perceived as white by humans when all three cone cell types
are simultaneously stimulated by equal amounts of red, green,
and blue light.
Light
Filters
- A majority of the common natural and artificial light
sources emit a broad range of wavelengths that cover the
entire visible light spectrum, with some extending into the
ultraviolet and infrared regions as well. For simple lighting
applications, such as interior room lights, flashlights, spot
and automobile headlights, and a host of other consumer,
business, and technical applications, the wide wavelength
spectrum is acceptable and quite useful. However, in many
cases it is desirable to narrow the wavelength range of light
for specific applications that require a selected region of
color or frequency. This task can be easily accomplished
through the use of specialized filters that transmit some
wavelengths and selectively absorb, reflect, refract, or
diffract unwanted wavelengths.
Human
Vision and Color Perception
- Human stereo color vision is a very complex process that is
not completely understood, despite hundreds of years of
intense study and modeling. Vision involves the nearly
simultaneous interaction of the two eyes and the brain
through a network of neurons, receptors, and other
specialized cells. The first steps in this sensory process
are the stimulation of light receptors in the eyes,
conversion of the light stimuli or images into signals, and
transmission of electrical signals containing the vision
information from each eye to the brain through the optic
nerves. This information is processed in several stages,
ultimately reaching the visual cortices of the cerebrum.
Light
and Energy
- Mankind has always been dependent upon energy from the
sun's light both directly - for warmth, to dry clothing, to
cook, and indirectly to provide food, water, and air. Our
awareness of the value of the sun's rays revolves around the
manner in which we benefit from the energy, but there are far
more fundamental implications from the relationship between
light and energy. Whether or not mankind devises ingenius
mechanisms to harness the sun's energy, our planet and the
changing environment contained within is naturally driven by
the energy of sunlight.
Perception
of Sound
- Sound is perceived through the sense of hearing. Humans and
many animals use their ears to hear sound, but loud sounds
and low frequency sounds can be perceived by other parts of
the body through the sense of touch. Sounds are used in
several ways, most notably for communication through speech
or, for example, music. Sound can also be used to acquire
information about properties of the surrounding environment
such as spatial characteristics and presence of other animals
or objects. For example, bats use echolocation, ships and
submarines use sonar, and humans can determine spatial
information by the way in which they perceive sounds.
The range of frequencies that humans can hear is
approximately between 20 Hz and 20,000 Hz. This range is by
definition the audible spectrum, but some people
(particularly women) can hear above 20,000 Hz. This range
varies by individual and generally shrinks with age, mostly
in the upper part of the spectrum. The ear is most sensitive
to frequencies around 3,500 Hz. Sound above 20,000 Hz is
known as ultrasound; sound below 20 Hz as infrasound.
The amplitude of a sound wave is specified in terms of its
pressure. The human ear can detect sounds with a very wide
range of amplitudes and so a logarithmic decibel amplitude
scale is used. The quietest sounds that humans can hear have
an amplitude of approximately 20 µPa (micropascals) or a
sound pressure level (SPL) of 0 dB re 20 µPa (often
incorrectly abbreviated as 0 dB SPL). Prolonged exposure to a
sound pressure level exceeding 85 dB can permanently damage
the ear, sometimes resulting in tinnitus and hearing
impairment. Sound levels in excess of 130 dB are considered
above of what the human ear can withstand and may result in
serious pain and permanent damage. At very high amplitudes,
sound waves exhibit non-linear effects including shock.
Speed of sound
The speed at which sound travels depends on the medium
through which the sound waves pass, and is often quoted as a
fundamental property of the material. In general, the speed
of sound is proportional to the square root of the ratio of
the stiffness of the medium and its density. Those physical
properties and the speed of sound change with ambient
conditions. For example, the speed of sound in air and other
gases depends on temperature. In air, the speed of sound is
approximately 345 ms-1, in water 1500 ms-1 and in a bar of
steel 5000 ms-1.
Sound pressure
Sound
pressure is the pressure deviation from the local ambient
pressure caused by a sound wave. Sound pressure can be
measured using a microphone in air and a hydrophone in water.
The SI unit for sound pressure is the pascal (symbol: Pa).
The instantaneous sound pressure is the deviation from the
local ambient pressure caused by a sound wave at a given
location and given instant in time. The effective sound
pressure is the root mean square of the instantaneous sound
pressure over a given interval of time. In a sound wave, the
complementary variable to sound pressure is the acoustic
particle velocity. For small amplitudes, sound pressure and
particle velocity are linearly related and their ratio is the
acoustic impedance. The acoustic impedance depends on both
the characteristics of the wave and the medium. The local
instantaneous sound intensity is the product of the sound
pressure and the acoustic partical velocity and is,
therefore, a vector quantity.
Introduction
to Lenses and Geometrical Optics
- The action of a simple lens, similar to many of those used
in the microscope, is governed by the principles of
refraction and reflection and can be understood with the aid
of a few simple rules about the geometry involved in tracing
light rays through the lens. The basic concepts explored in
this discussion, which are derived from the science of
Geometrical Optics, will lead to an understanding of the
magnification process, the properties of real and virtual
images, and lens aberrations or defects.
Basic
Properties of Mirrors
- Reflection of light is an inherent and important
fundamental property of mirrors, and is quantitatively gauged
by the ratio between the amount of light reflected from the
surface and that incident upon the surface, a term known as
reflectivity. Mirrors of different design and construction
vary widely in their reflectivity, from nearly 100 percent
for highly-polished mirrors coated with metals that reflect
visible and infrared wavelengths, to nearly zero for strongly
absorbing materials.
Prisms and Beamsplitters - Prisms and beamsplitters are
essential components that bend, split, reflect, and fold
light through the pathways of both simple and sophisticated
optical systems. Cut and ground to specific tolerances and
exact angles, prisms are polished blocks of glass or other
transparent materials that can be employed to deflect or
deviate a light beam, rotate or invert an image, separate
polarization states, or disperse light into its component
wavelengths. Many prism designs can perform more than one
function, which often includes changing the line of sight and
simultaneously shortening the optical path, thus reducing the
size of optical instruments.
Laser Fundamentals - Ordinary natural and artificial light is
released by energy changes on the atomic and molecular level
that occur without any outside intervention. A second type of
light exists, however, and occurs when an atom or molecule
retains its excess energy until stimulated to emit the energy
in the form of light. Lasers are designed to produce and
amplify this stimulated form of light into intense and
focused beams. The word laser was coined as an acronym for
Light Amplification by the Stimulated Emission of Radiation.
The special nature of laser light has made laser technology a
vital tool in nearly every aspect of everyday life including
communications, entertainment, manufacturing, and medicine.
FAIR USE
NOTICE: This site may contain copyrighted material, the use
of which has not been specifically authorized by the
copyright owner. This website distributes this material
without profit to those who have expressed a prior interest
in receiving the included information for research and
educational purposes. We believe this constitutes a fair use
of any such copyrighted material as provided for in 17 U.S.C
§ 107.
NOTE TO AUTHORS: If you are the author or owner of an article
or video that I have made available through THEINFOVAULT.NET
and you do not wish to have your article or video posted on
theinfovault, please contact me and I
will remove the item.