Glossary |
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.
Angular deviation
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).
Aperture diaphragm
An adjustable diaphragm located in the illumination optics which controls
the numerical aperture of the illuminating beam and affects the brightness
of the beam.
Attenuation level
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).
Autofluorescence
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.
Average transmission
The average calculated over the useful transmission region of a filter rather
than over the entire spectrum. For a bandpass filter, this region spans the
FWHM of the transmission band.
Background
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.
Bandpass
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.
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.
Barrier filter
Blocking level
Blocking range
The range of wavelengths over which an optical filter maintains a specified
attenuation level.
Blocker
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.
Brightfield
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.
Clear aperture
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.
Critical illumination
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.
Cross-talk
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.
Darkfield
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.
Diascopic illumination
Illumination using light transmitted through the specimen, using a condenser
to focus the light.
Dichroic beamsplitter
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.
Edge filter
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.
Emission filter
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.
Emitter
Epifluorescence microscope
A fluorescence microscope that illuminates the specimen episcopically (i.e.,
with light reflected onto the specimen).
Episcopic illumination
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
Excitation filter
Also Exciter. A color filter that transmits only those wavelengths of the illumination
light that efficiently excites a specific dye. See Emission filter.
Exciter
Extinction coefficient
A measure of the absorption characteristics of a fluorochrome.
Fading
Field diaphragm
An adjustable diaphragm located in the illumination optics which controls
the area of illumination on the specimen.
Filter block
Filter cube
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.
Filter glass
Also Absorption glass. Colored glass that is manufactured for technical and
scientific applications. The most common types of filter glass used in fluores-
cence 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.
Fluorescence
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).
Fluorescent probe
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.
Fluorochrome
A fluorescent dye used either directly as a specimen stain or conjugated to an
active substance to make a fluorescent probe.
Fluorophore
Front surface
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.
FWHM
Half-cone angle
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.
Heat filter
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.
Hot mirror
Indirect fluorescence
Also Secondary fluorescence. In fluorescence microscopy, fluorescence emitted
by fluorochromes introduced into a specimen as a stain or probe. See
Primary fluorescence.
Infinity-corrected optics
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.
Interference filter
See Thin-film interference coating.
Köhler illumination
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.
Longpass (LP)
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.
Luminance
Nanometer (nm)
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.
Normal incidence
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).
Parallelism
Photobleaching
Also Fading. A photochemical reaction that causes the intensity of fluorescence
to decrease with time.
Pinholes
Small breaks in the coating of an interference filter, usually caused by the
presence of dust particles on the substrate during coating.
Polarization
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.
Polychroic
A name for a dichroic beamsplitter that has multiple reflection bands and
transmission regions.
P-plane
Primary fluorescence
In fluorescence microscopy, fluorescent emissions from the specimen itself
rather than emissions from any fluorochromes present in the specimen. See
also Autofluorescence.
Quantum efficiency
A measure of how efficiently a fluorochrome converts absorbed radiation into
emitted fluorescence.
Quenching
Any chemical process which reduces the quantum efficiency of a fluorochrome
in situ.
Radiance
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.
Registration shift
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.
Scratch/dig
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.
Secondary fluorescence
Shortpass (SP)
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.
Signal-to-noise (S/N)
A measure of the brightness of the desired fluorescence (the signal) relative to
the brightness of the background (the noise).
Slope
A measure of the sharpness of the transition from the transmitting region to
the blocking region of a color filter.
Spectrofluorimeter
An instrument used for measuring the excitation and emission spectra of a
fluorescent substance.
S-plane
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.
Stokes shift
The shift in wavelength between the peak excitation intensity and peak emission
intensity of a fluorochrome.
Substrate
The ground and polished piece of optical glass that is used as a base for the
beamsplitter coating.
Surface flatness
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).
TE-mode
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.
TM-mode
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).
Ultraviolet (UV)
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.
Wedge
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.
Wide-field
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.
