Our growing list of fluorescence-related concepts and explanations.
Acousto-optical tunable filter (AOTF)
An active crystal device that works by setting up radio-frequency acoustical vibrations in the crystal and creating, in effect, a bulk transmission diffraction grating. By varying the frequency, one can rapidly tune the filter to diffract out a desired wavelength of light and transmit this wavelength out of the device.
Angle of incidence (AOI)
The angle between the optical axis of the light incident on the surface of a filter and the axis normal to this surface.
A shift in the direction of light beam from the true optical axis of the system, measured in units of angle such as arc-minutes (1/60 of a degree) or arc-seconds
(1/60 of an arc-minute).
An adjustable diaphragm located in the illumination optics which controls the numerical aperture of the illuminating beam and affects the brightness of the beam.
Also Blocking level. A measure of the out-of-band attenuation of an optical filter over an extended range of the spectrum. The attenuation level is often defined in units of optical density (see optical density).
In fluorescence microscopy, any fluorescence from substances other than fluorochromes, including primary fluorescence from the specimen, fluorescence from immersion oils and other media, and fluorescence from glass optical components within the microscope.
The average calculated over the useful transmission region of a filter ratherthan over the entire spectrum. For a bandpass filter, this region spans the FWHM of the transmission band.
Any detectable light that is not a desired primary or indirect fluorescent emission. Sources of background include cross-talk between excitation and emission filters, light leaking through pinholes in filters, and electronic noise in cameras, as well as autofluorescence.
An optical filter that has a well-defined short wavelength cut-on and long wavelength cut-off. Bandpass filters are denoted by their center wavelength and bandwidth.
Also FWHM (Full width at half of maximum transmission). For optical bandpass filters, typically the separation between the cut-on and cut-off wavelengths at 50% of peak transmission. Sometimes a bandwidth at, for example, 10% of peak transmission is specified.
The range of wavelengths over which an optical filter maintains a specified attenuation level.
A thin-film interference coating that is incorporated into a bandpass interference filter to extend the blocking range of the primary coating in the filter. A blocker is usually a very wide-band bandpass filter having high transmission in the band of the primary filter.
A kind of diascopic illumination in which the field of view is illuminated directly. Also, the type of condenser used for this kind of illumination.
Center wavelength (CWL)
For optical bandpass filters, the arithmetic mean of the cut-on and cut-off wavelengths at 50% of peak transmission.
The surface area of an optical filter which is free of any defects or obstructions. On interference filters the clear aperture is often delimited by an annulus of metal or opaque material.
A type of illumination optics in which the image of the light source is focused onto the specimen plane, in contrast to Köhler illumination optics. See also Köhler illumination.
The minimum attenuation level (over a specified wavelength range) of two filters placed together in series. The transmission spectrum of the beamsplitter is sometimes included in this evaluation.
A kind of diascopic illumination in which the specimen is illuminated obliquely, i.e., at angles that will not enter the objective directly. Also, the type of condenser used for this kind of illumination.
Illumination using light transmitted through the specimen, using a condenser to focus the light.
Also Dichroic mirror, Dichromatic beamsplitter. A special mirror housed in the filter cube that selectively reflects the excitation light filtered by the exciter and transmits the emitted fluorescence. Dichroic beamsplitters can also be found in any other part of a microscope system where light needs to be split or combined by wavelength.
Another term for either a shortpass or longpass optical filter. The term usually denotes a filter with a very sharp cut-on or cut-off.
Also Barrier filter, Emitter. A color filter that attenuates all of the light transmitted by the excitation filter and very efficiently transmits any fluorescence emitted by the specimen.
A fluorescence microscope that illuminates the specimen episcopically (i.e., with light reflected onto the specimen).
Also Incident-light illumination. Illumination with light reflected onto the specimen rather than transmitted through the specimen. The illuminating light is reflected through the objective by means of a beamsplitter that is either partially reflective or a dichroic.
Excitation and emission
Also Exciter. A color filter that transmits only those wavelengths of the illumination light that efficiently excites a specific dye. See Emission filter.
A measure of the absorption characteristics of a fluorochrome.
An adjustable diaphragm located in the illumination optics which controls the area of illumination on the specimen.
A removable cube-shaped unit that holds a matched set of fluorescence filters. This set usually includes an excitation filter and emission filter but always includes a dichroic beamsplitter.
Also Absorption glass. Colored glass that is manufactured for technical and scientific applications. The most common types of filter glass used in fluorescence microscopy are UV-transmitting “black glass” filters; IR-absorbing heat filters; and yellow, orange, and red sharp-cut longpass filters. Filter glass selectively attenuates light by absorption, so the spectral performance is dependent on the thickness of the glass.
A molecular phenomenon in which a substance absorbs light, then radiates part of this absorbed energy as light of another color, one of lower energy and thus longer wavelength. This process is known as excitation and emission. Fluorescence is distinguishable from other types of luminescence in that the process of excitation and emission occurs nearly instantaneously (i.e., on the order of nanoseconds).
Also Fluorophore. A fluorochrome that has been conjugated to an active substance, such as a protein, antibody, or nucleic acid, in order to selectively stain a targeted substance within the specimen.
A fluorescent dye used either directly as a specimen stain or conjugated to an active substance to make a fluorescent probe.
The side of a beamsplitter that is designed to face the incident light. In a filter cube this is the side that faces both the light source and the specimen. Beamsplitters generally perform better when oriented correctly.
The angle between the most oblique ray of a convergent or divergent light beam and the optical axis of the beam. See also Numerical aperture.
An optical filter that attenuates infrared radiation but transmits the visible. Attenuation is achieved by means of absorption (using filter glass), reflection (using a thin-film interference coating, often called a hot mirror), or a combination
of the two.
Also Secondary fluorescence. In fluorescence microscopy, fluorescence emitted by fluorochromes introduced into a specimen as a stain or probe. See Primary fluorescence.
An optical configuration employed by some microscopes in which the objective forms an image at infinity, and a secondary tube lens forms an image at the intermediate image plane. (This intermediate image is focused in turn by the ocular.) This configuration allows for a flexible distance between the objective and ocular because the distance between the objective and the tube lens can be varied without affecting the image-forming characteristics of the microscope. Note that objectives designed for infinity-corrected optics are not interchangeable with objectives designed for standard tube-length optics.
See Thin-film interference coating.
The type of illumination optics, usually used in wide-field epifluorescence microscopes, in which the image of the light source is completely out of focus at the specimen plane.
An optical filter that attenuates shorter wavelengths and transmits longer wavelengths over the active range of the spectrum (which depends on the specific application). LP filters are denoted by the cut-on wavelength at 50% of peak transmission.
A unit of length commonly used for measuring the wavelength of light: 1 nm = 10 angstroms (A) = 10-9 meters; and 1000 nm = 1 micron (μ) = 10-6 meters.
Near infrared (NIR)
The region of the electromagnetic spectrum ranging in wavelength from approximately 750 to 2500 nanometers.
Neutral-density (ND) filter
An optical filter that attenuates light by an amount independent of the wavelength within the useful range of the filter. Metal-coated filters typically have a wider neutral range than glass filters and are more heat-tolerant.
An angle of incidence of zero degrees.
Numerical aperture (NA)
In the microscope, a measure of the effective cone-angle of light focused onto the specimen (NA of the condenser) or light captured by the objective (NA of the objective). A high value of NA improves both the brightness and the resolution of the image. (NA = N sin(θ), where N is the refractive index of the medium surrounding the specimen and θ is the half-cone angle of the light.)
Optical density (OD)
A logarithmic unit of transmission: OD = -log (T), where T is the transmission (0 ≤ T ≥ 1).
Also Fading. A photochemical reaction that causes the intensity of fluorescence to decrease with time.
Small breaks in the coating of an interference filter, usually caused by the presence of dust particles on the substrate during coating.
Restriction of the orientation and phase of the electromagnetic field vibrations caused by propagating light. These vibrations are transverse to the direction of propagation of the light, and can be oriented at some angle around the axis of propagation. When the orientation and the phase of the vibrations change rapidly and randomly in time, the light is said to be unpolarized. When the vibrations are restricted to one particular orientation angle over an extended length of time, the light is said to be plane-polarized. Light can be partially as well as totally plane-polarized. When the relative phase of the vibrations varies with angle in a regular periodic fashion over an extended length of time, the light is said to be elliptically polarized. Circular polarization is a special case in which the amplitude of the vibrations are equal for all angles. When light is elliptically polarized, the orientation of the vibrations rotates around the propagation axis at the frequency of the light. When light strikes a specular surface at non-normal incidence, the component of the electric field vibrations parallel to the plane of incidence of the surface (P-plane) behaves differently than the component perpendicular to the plane of incidence (S-plane). This causes a polarizing effect that is aligned orthogonally to the orientation of the surface.
A name for a dichroic beamsplitter that has multiple reflection bands and transmission regions.
In fluorescence microscopy, fluorescent emissions from the specimen itself rather than emissions from any fluorochromes present in the specimen. See also Autofluorescence.
A measure of how efficiently a fluorochrome converts absorbed radiation into emitted fluorescence.
Any chemical process which reduces the quantum efficiency of a fluorochrome in situ.
A measure of the radiometric brightness of a light source. Technically, the radiance is the radiant flux emitted per unit solid angle per unit area of the light source. A common unit is watts per steradian per square centimeter. Luminance is a measure of the brightness of a light source as perceived by the human eye (i.e., a photometric measure), commonly measured in candelas per square centimeter.
A shift in the apparent position of the specimen that occurs when an optical element is inserted or removed, adjusted, or switched with another element.
A set of specifications for defining the maximum allowable size and number of scratches and digs on an optical surface. The scratch/dig values (e.g., 80/50) specify the scratch width in microns and the dig diameter in tens of microns, respectively. Although extensive evaluation procedures exist if rigorous standards must be maintained (military specification MIL-F-48616, for example), a qualitative visual assessment of the scratches and digs usually suffices.
An optical filter that attenuates longer wavelengths and transmits shorter wavelengths over the active range of the spectrum (which depends on the specific application). SP filters are denoted by the cut-off wavelength at 50% of peak transmission.
A measure of the brightness of the desired fluorescence (the signal) relative to the brightness of the background (the noise).
A measure of the sharpness of the transition from the transmitting region to the blocking region of a color filter.
An instrument used for measuring the excitation and emission spectra of a fluorescent substance.
Standard tube-length optics
An optical configuration employed by most microscopes in which the objective forms an image at the intermediate image plane. (This intermediate image is focused in turn by the ocular.) The distance between the nosepiece that holds the objective and the barrel that holds the ocular is fixed at a standard length of 160 mm so that objectives can be interchangeable between microscopes. See also Infinity-corrected.
The shift in wavelength between the peak excitation intensity and peak emission intensity of a fluorochrome.
The ground and polished piece of optical glass that is used as a base for the beamsplitter coating.
A measure of the deviation of the surface of an optical element from a perfect plane, measured in fractions or multiples of a wavelength of visible light (usually 550 or 630 nm).
Another term for S-plane polarization. (Short for “transverse-electric” mode.) See Polarization.
Thin-film interference coating
A type of optical coating composed of a stack of microscopically thin layers of material. Although each material is intrinsically colorless, the reflections created at each interface between the layers combine through wave interference to selectively reflect some wavelengths of light and transmit others. Thin-film interference coatings are the main component of interference filters, which consist of one or more coatings separated by glass substrates and frequently one or more layers of filter glass.
Another term for P-plane polarization. (Short for “transverse-magnetic” mode.) See Polarization.
Transmitted wavefront distortion (TWD)
A measure of the distortion a plane wave of light undergoes when transmitted through an optical element, measured in fractions or multiples of a wavelength of visible light (usually 550 or 630 nm).
The region of the electromagnetic spectrum ranging in wavelength from approximately 100 to 400 nanometers. Three distinct bands are: 1) near-UV, from 320 to 400 nm; 2) mid-UV, from 190 to 320 nm; and 3) vacuum-UV (VUV), below 190 nm. The terms UV-A and UV-B denote bands with distinct physiological effects: 320 to 380 nm and 280 to 320 nm, respectively.
Also Parallelism. A measure of the deviation of the outer surfaces of an optical element from perfect parallelism, usually measured in arc-minutes or arcseconds of angle.
An epifluorescence microscope in which the full field of view is illuminated, in contrast to a confocal epifluorescence microscope. The term brightfield is also used, but this might be confused with traditional diascopic brightfield illumination.