With respect to the principal planes of a lens, the image-to-lens and
object-to-lens distances, as predicted by the lens equation in geometrical
optics. See also Lens equation.
of fluorescence microscopy in which a certain molecular species in a specimen
is labeled with a specific fluorescent antibody. Fluorescence emission from
excited antibodies is collected by the objective lens to form an image of the
specimen. Antibodies can be made fluorescent by labeling them directly with a
fluorescent dye (direct immunofluorescence) or with a second fluorescent
antibody that recognizes epitopes on the primary antibody (indirect
inert gas and metal filament that emits photons as the filament becomes excited
during passage of electric current. The spectrum of visible wavelengths emitted
by the filament shifts to increasingly shorter wavelengths as the amount of
excitation is increased. The output of incandescent lamps is very high at red
and infrared wavelengths.
optical design for microscope objective lenses in which the specimen is placed
at the focal length of the lens. Used by itself, the image rays emerge from the
lens parallel to the optic axis and the image plane is located at infinity. In
practice, a tube lens or Telan lens located in the body of the microscope acts
together with the objective to form an image in the real intermediate image
plane. This optical design relaxes constraints on the manufacture of the
objective lens itself and allows for placement of bulky accessory equipment
such as fluorescence filter cubes in the space between the objective and the
brightness or flux of light energy perceived by the eye. By universal
agreement, the term intensity, meaning the flow of energy per unit area per
unit time, is being replaced by the word irradiance, a radiometric term
indicating the average energy (photon flux) per unit area per unit time, or
watts/meter2. As a term describing the strength of light, intensity is
proportional to the square of the amplitude of an electromagnetic wave.
interacting electromagnetic waves. Two waves can interfere only if a component
of the E vector of one wave vibrates in the plane of the other wave. Resultant
waves with amplitudes greater or less than the constituent waves are said to
represent constructive and destructive interference, respectively.
results from removal of a band of visible wavelengths from a source of white
alternating layers of different dielectric materials or layers of a dielectric
material and thin metal film that transmits a specific band of wavelengths. The
spacings between the layers of one-quarter or one-half wavelength allow constructive
interference and reinforce propagation through the filter of a particular
wavelength λ. All other wavelengths give destructive interference and are
absorbed or reflected and do not propagate through the filter.
gas or plasma between two electrodes that radiates visible wavelengths when
excited by an electric current. Arc lamps used in light microscopy usually
contain mercury vapor or xenon gas.
radiometrically correct term for light intensity. Irradiance is the radiant
flux incident per surface unit area and is given as watts/meter2. Irradiance is
a measure of the concentration of power.
properties of an object or propagation medium, having identical properties in
the energy levels occupied by an excited electron in an atom or molecule as
steps on a vertical ladder. Singlet and triplet excited states are shown
separately as ladders standing next to each other.
method for illuminating specimens in the light microscope, whereby a collector
lens near the light source is used to focus an image of the light source in the
front aperture of the condenser. The microscope condenser is focused to position the conjugate image of
the light source in the back focal plane (diffraction plane) of the objective
lens. The method provides bright, even illumination across the diameter of the
the equation 1/f 1/a 1/b describing the
relationship between the object distance a and the image distance b for a lens
of focal length f.
employing light as an analytic probe and optics based on glass lenses to
produce a magnified image of an object specimen.
light in which the E vectors of the constituent waves vibrate in planes that
are mutually parallel. Linearly polarized light need not be coherent or
interference filter that transmits (passes) long wavelengths and blocks short
objective lens having a working distance many times greater than that of a
conventional objective lens of the same magnification. A long working distance
lens is sometimes easier to employ and focus, can look deeper into transparent
specimens, and allows the operator greater working space for employing
micropipettes or other equipment in the vicinity of the object. However, the NA
and resolution are less than those for conventional lenses of comparable
the flux through a unit solid angle (steradian) from a uniform point source of
1 candle intensity.
lumen per square meter.
mode of light microscope optics in which a transparent phase object is made
visible by providing unilateral oblique illumination and employing a mask in
the back aperture of the objective lens that blocks one sideband of diffracted
light and partially attenuates the 0th-order undeviated rays. In both MCM and
DIC optics, brightly illuminated and shadowed edges in the three-dimensional
relief-like image correspond to optical path gradients (phase gradients) in the
specimen. Although resolution and detection sensitivity are somewhat reduced
compared with DIC, the MCM system produces superior images at low
magnifications, allows optical sectioning, and lets you examine cells on
birefringent plastic dishes.
composed of just one wavelength, but in practice, light that is composed of a
narrow band of wavelengths. Owing to Heisenberg’s uncertainty principle, true
monochromatic light does not exist in nature. Even the monochromatic emission
from a laser or an excited atomic source has a measurable bandwidth. Therefore,
while the light produced by a narrow bandpass interference filter is called
monochromatic, this is just an approximation.
objective lens whose spherical aberration is corrected for use by immersion in
media of various refractive indices, including water, glycerin, and oil. A
focusable lens element used to minimize spherical aberration is adjusted by
rotating a focus ring on the barrel of the objective.
filter set for simultaneous viewing or photography of multiple fluorescent
signals. The transmission profile of each filter in the set contains multiple
peaks and troughs for the reflection and transmission of the appropriate
excitation and emission wavelengths as in a conventional single-fluorochrome
filter set. Because of constraints on the widths of bandwidths, the steepness
of transmission profiles, and the inability to reject certain wavelengths, the
performance is somewhat less than that of individual filter sets for specific
the removal of a certain band of visible wavelengths. Thus, white light minus
blue gives the negative color yellow, because simultaneous stimulation of red
and green cone cells results in this color perception. Similarly, the mixture
of cyan pigment (absorbs red wavelengths) and yellow pigment (absorbs blue
wavelengths) gives green, because green is the only reflected wavelength in the
beam of parallel incident rays. A simple negative lens is thinner in the middle
than at the periphery and has at least one concave surface. It does not form a
real image, and when held in front of the eye, it reduces or demagnifies.
contrast optics, the term applies to systems employing a negative phase plate
that retards the background 0th-order light by /4 relative to the diffracted
waves. Since the diffracted light from an object is retarded /4 relative to the
phase of the incident light, the total amount of phase shift between background
and diffracted waves is 0 and interference is constructive, causing objects to
appear bright against a gray background.
filter that reduces equally the amplitudes of all wavelengths across the
visible spectrum. The glass substrate contains light-absorbing colloids or is
coated on one surface with a thin metal film to reduce transmission. Neutral
density filters are labeled according to their absorbance or fractional
thin opaque disk with thousands of minute pinholes, which when rotated at high
speed provides parallel scanning of the specimen with thousands of minute
diffraction-limited spots. The return fluorescence emission is refocused at the
same pinhole in the disk, which provides the same function in rejecting
out-of-focus light as does a single pinhole in a conventional confocal
microscope. Nipkow disk confocal microscopes produce a real image that can be
inspected visually or recorded on a high-resolution CCD camera, whereas images
of single-spot scanning microscopes are reconstructed from signals from a PMT
and are displayed on a computer monitor.
describing the angular aperture of objective and condenser lenses. NA is
defined as n sin, where n is the refractive index of the medium between the
object and the lens, and , the angle of light collection, is the apparent half-angle
subtended by the front aperture of the lens as seen from a point in the
of the microscope responsible for forming the real intermediate image located
in the front apertures of the eyepieces.
measure of the time or distance (measured in wavelengths) defining the path
taken by a wave between two points. Optical path length is defined as n t, where n is the refractive index and t
indicates the thickness or geometrical distance. A complex optical path
composed of multiple domains of different refractive index and thickness is
given as n1t1 n2t2 .
. . niti.
difference in the optical path lengths of two waves that experience refractive
index domains of different value and thickness. In interference optics,
differences in optical path length determine the relative phase shift and thus
the degree of interference between 0th-order and higher-order diffracted waves
that have their origins in a point in the object.
booster lens that can be rotated into the optical path to further increase the
magnification provided by the objective by a small amount.
optics, the member of a ray pair that obeys normal laws of refraction and whose
velocity remains constant in different directions during transmission through a
birefringent medium. See also Extraordinary ray.
aperture condenser for dark-field microscopy having a reflective surface that
is a segment of a figure of revolution of a parabola. The steeply pitched
illumination cone produced by the condenser is suitable for darkfield
examination with high-power oil immersion objectives.
same distance between the specimen and the objective turret of the microscope.
With parfocal lenses, one can focus an object with one lens and then switch to
another lens without having to readjust the focus dial of the microscope.
other modes of interference microscopy, the wave (P wave) that results from
interference between diffracted and surround waves in the image plane, and
whose amplitude is different from that of the surrounding background, allowing
it to be perceived by the eye. See also Diffracted wave and Surround wave.
interference microscopy that transforms differences in optical path in an
object to differences in amplitude in the image, making transparent phase
objects appear as though they had been stained. Surround and diffracted rays
from the specimen occupy different locations in the diffraction plane at the
back aperture of the objective lens where their phases are differentially
manipulated in order to generate a contrast image. Two special pieces of
equipment are required: a condenser annulus and a modified objective lens
containing a phase plate. Because the method is dependent on diffraction and
scattering, phase contrast optics differentially enhance the visibility of
small particles, filaments, and the edges of extended objects. The technique
allows for examination of fine details in transparent specimens such as live
microscopy, the gradient of phase shifts in an image corresponding to optical
path differences in the object.
phase of light as opposed to those that absorb light (amplitude objects) as the
basis for image formation. See also Amplitude object.
microscopy, a transparent plate with a semitransparent raised or depressed
circular annulus located at the rear focal plane of a phase contrast objective.
The annulus reduces the amplitude of background (0th order) waves and advances
or retards the phase of the 0th-order component relative to diffracted waves.
Its action is responsible for the phase contrast interference image.
s) emission of photons after excitation of a material by light or other
sheet of linear polarizing film (dichroic filter or Polaroid filter) and
particularly to its use as a polarizer or analyzer in producing and analyzing
a property describing the strength of interaction of light with molecules in a
manner that depends on the orientation of atomic bonds. Light waves interact
more strongly with molecules when their E vectors are oriented parallel to the
axis defining light-deformable (polarizable) covalent bonds such as the axes of
long-chain hydrocarbon polymers like polyvinyl alcohol, cellulose, and
collagen. This geometry is supported when an incident light ray is
perpendicular to the long axis of the polymer. Interaction of light with
molecules along their polarizable axis retards wave propagation and accounts
for the direction-dependent variability in their refractive index, a property
known as birefringence.
microscopy, the appearance of a dark upright cross in the back aperture of the
objective lens under conditions of extinction with two crossed polars. Ideally,
the back aperture is uniformly dark under this condition, but the
depolarization of light by the curved lens surfaces of the condenser and
objective lenses causes brightenings in four quadrants and hence the appearance
of a cross.
light microscopy based on the unique ability of polarized light to interact
with polarizable bonds of ordered molecules in a directionsensitive manner.
Perturbations to waves of polarized light from aligned molecules in an object
result in phase retardations between sampling beams, which in turn allow
interference-dependent changes in amplitude in the image plane. Typically the
microscope contains a polarizer and analyzer, and a retardation plate or
compensator. Image formation depends
critically on the existence of ordered molecular arrangements and a property
known as double refraction or birefringence.
vectors vibrate in plane-parallel orientation at any point along the axis of
propagation. Polarized light can be linearly polarized (vibrations at all
locations are plane parallel) or elliptically or circularly polarized
(vibration axis varies depending on location along the propagation axis).
Polarized light need not be monochromatic or coherent.
random light and transmits linearly polarized light. In microscopy, polarizers
are made from sheets of oriented dichroic molecules (Polaroid filter) or from
slabs of birefringent crystalline materials.
from mixing different wavelengths of light. The equal mixture of red and green
wavelengths results in the perception of yellow, a positive color.
beam of parallel incident rays. A simple positive lens is thicker in the middle
than at the periphery, and has at least one convex surface. A positive lens
forms a real image and enlarges or magnifies when held in front of the eye.
contrast optics, the term applies to systems employing a positive phase plate
that advances the background wave by /4 relative to the diffracted wave. Since
the diffracted light from an object is retarded /4 relative to the phase of the
incident light, the total phase shift between background and diffracted waves
is /2 and interference is destructive, causing objects to appear dark against a
lens, the plane within the lens and perpendicular to the optic axis from which
the focal length is determined. Thick simple lenses have two principal planes
separated by an intervening distance. Complex compound lenses may have multiple
The criterion commonly used to define spatial resolution in a lens-based imaging
device. Two point sources of light are considered to be just barely resolved
when the diffraction spot image of one point lies in the first-order minimum of
the diffraction pattern of the second point. In microscopy, the resolution
limit d is defined, d m/1.22 λ/(NAobjective
NAcondenser), where λ is the wavelength of light and NA is the numerical
aperture of the objective lens and of the condenser.
when projected on a screen or recorded on a piece of film.
focused by the objective lens in the vicinity of the oculars of the microscope.
propagation (bending) experienced by a beam of light that passes from a medium
of one refractive index into another medium of different refractive index when
the direction of propagation is not perpendicular to the interface of the
ellipsoid. An ellipsoid is the figure of revolution of an ellipse. When
rotated about its major axis, the surface of the ellipsoid is used to describe
the surface wavefront locations of E waves propagating outward from a central
point through a birefringent material. The same kind of figure is used to
describe the orientation and magnitude of the two extreme refractive index
values that exist in birefringent uniaxial crystals and ordered biological
optics, the relative shift in phase between two waves expressed in fractions of
lens in an imaging system placed between the objective and the real
intermediate image. In video, so-called TV lenses increase the magnification of
the image projected on the camera 2- to 8-fold.
interference filter that transmits (passes) short wavelengths and blocks long
single lens element and distinct from a compound lens having multiple lens
selectively manipulates a location in an image such as an aperture in a field
plane of a microscope or a sharpening or blurring filter in image processing.
the distance between two objects (periods/ distance).
selectively manipulates a location in the diffraction plane in a microscope
(aperture plane masks in modulation contrast microscopy) or a mask applied to
Fourier transforms to manipulate low and high spatial frequency information in
component features in an image. In optical systems, resolution is directly
proportional to the wavelength and inversely proportional to the angular
aperture. The practical limits on wavelength and angular aperture determine the
limit of spatial resolution, which is approximately one-half the wavelength of
wavelengths, or bandwidth, under consideration.
typical of lenses with spherical surfaces that causes paraxial rays incident on
the center and periphery of a lens to be focused at different locations in the
image plane. The degree of aberration increases with the decreasing focal ratio
of the lens. The aberration can be corrected in simple lenses by creating
nanometers between the peak excitation and peak emission wavelengths of a
small compared to its focal length. A line through the center of the lens (a
plane representing the two coincident principal planes of the lens) provides a
reasonably accurate reference plane for refraction and object and lens distance
measurements. Lenses are assumed to be thin when demonstrating the principles
of graphical ray tracing.
lens in the body of the microscope, which in conjunction with an infinity focus
objective lens forms the real intermediate image. The Telan lens provides some
of the correction for chromatic aberration, which lessens constraints on the
manufacture of the objective lens.
crystal characterized by having a single optic axis.
perceived by the eye or imaged by a converging lens, but that cannot be focused
on screen or recorded on film as can be done for a real image. The image perceived
by the eye when looking in a microscope is a virtual image.
cycle of an electromagnetic wave. Also, the distance between two successive
points at which the phase is the same on a periodic wave. The wavelength of
light is designated λ and is given in nanometers.
microscopy, a beam splitter made of two wedgeshaped slabs of birefringent
crystal such as quartz. In differential interference contrast (DIC) microscopy,
specimens are probed by pairs of closely spaced rays of linearly polarized
light that are generated by a Wollaston prism acting as a beam splitter. An
important feature of the prism is its interference plane, which lies inside the
prism (outside the prism in the case of modified Wollaston prism designs).
front lens surface of the objective lens and the coverslip. Lenses with high
NAs typically have short working distances (60–100 micro m). Lenses with longer
working distances allow you to obtain focused views deep within a specimen.