Know your tool - Microscope

Image distance and object distance. 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.

Immunofluorescence microscopy. A mode 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 immunofluorescence).

Incandescent lamp. A bulb containing an 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.

Infinity corrected optics. The latest 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 tube lens.

Intensity of light. Qualitatively, 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.

Interference. The sum of two or more 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.

Interference color. The color that results from removal of a band of visible wavelengths from a source of white light.

Interference filter. A filter made from 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.

Ion arc lamp. Lamps containing an ionized 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.

Irradiance of light. The 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.

Isotropic. In describing the optical properties of an object or propagation medium, having identical properties in different directions.

Jablonski diagram. A diagram showing 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.

Koehler illumination. The principal 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 specimen.

Lens equation. In geometrical optics, 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.

Light microscope. A microscope employing light as an analytic probe and optics based on glass lenses to produce a magnified image of an object specimen.

Linearly polarized light. A beam of 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 monochromatic.

Long-pass filter. A colored glass or interference filter that transmits (passes) long wavelengths and blocks short ones.

Long working distance lens. An 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 magnification.

Lumen. A unit of luminous flux equal to the flux through a unit solid angle (steradian) from a uniform point source of 1 candle intensity.

Lux. A unit of illumination equal to 1 lumen per square meter.

Modulation contrast microscopy (MCM). A 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.

Monochromatic. In theory, light 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.

Multi-immersion objective lens. An 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.

Multiple fluorescence filter set. A 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 fluorochromes.

Negative colors. Colors resulting from 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 pigment mixture.

Negative lens. A lens that diverges a 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.

Negative phase contrast. In phase 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.

Neutral density (ND) filter. A light-attenuating 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 transmission.

Nipkow disk. In confocal microscopy, a 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.

Numerical aperture (NA). The parameter 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 specimen plane.

Objective lens. The image-forming lens of the microscope responsible for forming the real intermediate image located in the front apertures of the eyepieces.

Optical path length. In wave optics, a 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.

Optical path length difference. The 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.

Optovar. A built-in magnification booster lens that can be rotated into the optical path to further increase the magnification provided by the objective by a small amount.

Ordinary ray or O ray. In polarization 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.

Paraboloid condenser. A high numerical 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.

Parfocal. The property of having the 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.

Particle wave. In phase contrast and 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.

Phase contrast microscopy. A form of 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 cells.

Phase gradient. In interference microscopy, the gradient of phase shifts in an image corresponding to optical path differences in the object.

Phase object. Objects that shift the phase of light as opposed to those that absorb light (amplitude objects) as the basis for image formation. See also Amplitude object.

Phase plate. In phase contrast 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.

Phosphorescence: The relatively slow (9 s) emission of photons after excitation of a material by light or other radiation source.

Polar: The common term applied to a sheet of linear polarizing film (dichroic filter or Polaroid filter) and particularly to its use as a polarizer or analyzer in producing and analyzing polarized light.

Polarizability. In polarization optics, 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.

Polarization cross. In polarization 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.

Polarization microscopy. A mode of 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.

Polarized light. Light waves whose E 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.

Polarizer. A device that receives 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.

Positive colors. Colors that result from mixing different wavelengths of light. The equal mixture of red and green wavelengths results in the perception of yellow, a positive color.

Positive lens. A lens that converges a 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.

Positive phase contrast. In phase 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 gray background.

Principal plane. For a simple thin 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 principal planes.

Rayleigh criterion for spatial resolution. 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.

Real image. An image that can be viewed when projected on a screen or recorded on a piece of film.

Real intermediate image. The real image focused by the objective lens in the vicinity of the oculars of the microscope.

Refraction. The change in direction of 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 second medium.

Refractive index ellipsoid and wavefront 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 materials.

Relative retardation. In polarization optics, the relative shift in phase between two waves expressed in fractions of a wavelength.

Relay lens. An intermediate magnifying 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.

Short-pass filter. A colored-glass or interference filter that transmits (passes) short wavelengths and blocks long ones.

Simple lens. A lens consisting of a single lens element and distinct from a compound lens having multiple lens elements.

Spatial filter. A filter that 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.

Spatial frequency. The reciprocal of the distance between two objects (periods/ distance).

Spatial frequency filter. A filter that 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 image processing.

Spatial resolution. The resolution of 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 light.

Spectral range. The range of wavelengths, or bandwidth, under consideration.

Spherical aberration. A lens aberration 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 aspherical surfaces.

Stokes shift. The distance in nanometers between the peak excitation and peak emission wavelengths of a fluorescent dye.

Thin lens. A lens whose thickness is 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.

Tube lens or Telan lens. An auxiliary 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.

Uniaxial crystal. A birefringent crystal characterized by having a single optic axis.

Virtual image. An image that can be 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.

Wavelength. The distance of one beat 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.

Wollaston prism. In interference 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).

Working distance. The space between the 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.