Glossary of Fluorescence Spectroscopy Terms | Edinburgh Instruments

Glossary of Fluorescence Spectroscopy Terms

Edinburgh Instruments has compiled a glossary of all the key terms related to Fluorescence Spectroscopy. Please browse the glossary bellow to find the definition for the term you are interested in.

Term Definition
Absorbance Measure of the amplitude of absorbed energy in a spectrum related to the concentration of an analyte. This term is also referred to as the negative log (base 10) of Transmittance (-log₁₀T = log₁₀[1/T]). This is also represented as the product of absorptivity (extinction coefficient), path length, and concentration, written as A=ɛbc. Absorbance = log₁₀ (1/reflectance) or absorbance = log₁₀ (1/transmittance).
Bandpass Filter A filter that is designed to provide transmission for a particular band of frequencies, but to attenuate (reduce) transmission of frequencies above (or above and below) the specified transmission band. Specifications include a half width or full width at half-maximum (FWHM) of the transmission band, the transmission peak position, and the attenuation transmission specifications with respect to frequency or wavelength.
Bandwidth This term refers to the full width at half-maximum (FWHM), the line width, or the spectral resolution, of a spectrophotometer. It also refers to the range of frequencies (wave-lengths) over which a particular instrument is designed to function within specified performance limits. Also, it refers to the limits or extreme difference between the specified frequencies of a measured spectral range (that is, high versus low frequencies over a spectral range).
Detector A device sensitive to electromagnetic radiation at the wavelength region of interest. The output of the device is usually an electrical signal proportional to the intensity of the electromagnetic energy input. The detector generally produces an analog signal proportional to the radiation striking the detector element. This analog signal is converted into a digital signal by means of an analog-to-digital (AID) converter and amplifier circuit. Usually, a simple silicon diode, a more sensitive photomultiplier tube (PMT), or various photodetector systems are used to detect the light energy.
Electromagnetic Spectrum The continuum of frequencies that contains electromagnetic radiation (EMR). Instruments measure the intensity of radiation within a defined range of the spectrum, and usually present the results of their measurements as a set of values of some function, or the measured intensity at (usually) evenly spaced intervals within the range. The energy throughout the electromagnetic spectrum ranges from gamma radiation (most energetic and highest frequency) to radio waves (least energetic and lowest frequency). The spectrum is expressed in terms of wavelength, wavenumber, frequency, or energy.
Fluorescence Photons with energies in the ultraviolet (that is, 190-360 nm) to the blue-green visible (that is, 350—500 nm) spectral regions will excite an electronic transition for atoms in molecules that fluoresce (that is, fluorophores). Fluorescence is an electronic transition from a ground state to the excited state With the emission of a photon to return to the ground state. After the molecule is excited, it relaxes (Stokes Shift) to the ground state while emitting a photon within a femtosecond (10ˉ15s) to picosecond (10ˉ1²s) timeframe. The Stokes shift indicates a lower energy of the fluorescence photons than the energy of the excitation photons. The fluorescence typically has a lifetime (or duration) of nanoseconds (10ˉ⁹s) per transition. A fluorescence spectrophotometer normally has an excitation monochromator that defines the excitation energy, and an emission monochromator that provides a full spectrum of the fluorescence emission.
Grating A reflective surface covered with evenly spaced, microscopic grooves, whose purpose is to separate individual wavelengths from broadband energy. The distance between grooves and the angle of the faces is determined by the wavelengths to be separated. The grating (except for diode arrays) is rotated at a set angle and speed, and the desired wavelength is diffracted through an exit slit onto the sample and detector (or detectors). It is used to disperse light of various wavelengths and orders from its surface. It disperses zero order as specular reflected light, and first, second, and higher orders as diffracted light. When the diffracted light interacts in a phonon effect it decreases the energy dispersed from the surface, resulting in a phenomenon termed Wood’s anomalies.
Highpass filter A dielectric composite filter that enables energy above a certain cut-on wavelength or frequency to pass at nearly 100% transmission, whereas energy at wavelengths or frequencies below the cut-on wavelength will not pass or is nearly 0% transmissive.
Holographic granting A grating made by using a holographic process where a photoresist is placed on an optical surface such as a glass blank. A series of interference fringes corresponding to the grooves of the desired grating model are recorded on this photosensitive layer by projecting a hologram of light and dark lines. Subsequent chemical treatments are used to erode either the exposed or unexposed photoresist material (there are different types of treatments such as positive and negative photoresist processes) on the surface to produce a grooved diffraction grating of specified groove shapes and lines per millimetre.
Integrating sphere A hollow sphere, coated with a diffuse reflecting material and provided with opening for incident beam, specimen, and detector. It is primarily used for measuring the most accurate diffuse reflectance or transmittance of objects. In most versions, the sample is measured outside of the sphere.
Interference filter A filter that controls the spectral composition of radiant energy passing through it by the effects of interference. Frequently, these filters are made up of thin layers of metals and dielectrics, and provide narrow bandpass and high transmittances.
Monochromator An instrument used to provide an incident light beam with a narrow wavelength range. A Monochromator becomes a spectrophotometer when it is combined with a light source, slits, grating, detectors, an amplifier, and an output energy measuring device.
Order-sorting filter A diffraction grating creates multiple orders of spectra from its surface. As more than one spectrum is created, those spectra beyond the first order (that is, second order and higher) overlap with one another. Order-sorting filters remove the wavelengths above and below the desired measurement wavelength. They are also termed high-pass and low-pass filters as the designation for which wavelength region they are transparent to. The filter is placed in front of a broadband detector, and only the wavelengths permitted through the filter are measured. The other dispersion orders are eliminated.
Slit An aperture, usually rectangular in shape with a large length-to-Width ratio, and a fixed or adjustable shape through which radiation enters or leaves a Monochromator-based instrument. The slit aperture is usually quite small relative to the light source. For monochromators, there is an entrance and exit slit for each grating; for interferometers there is a J-stop aperture, which in effect acts as a slit adjusting the effective resolution.
Slit width The size of opening of slit through which light passes. The slit width depends on the wavelength range, separation ability of the wavelength selector (for example, grating), and desired isolation of the specific wavelength (that is, line width). Slits are either fixed or automatically programmed for width variation.
Stray light Any radiation reaching the detector that is not remitted or transmitted from, or through, the sample at the selected measurement wavelength.
Wavelength Electromagnetic energy is transmitted in the form of a sinusoidal wave; the wavelength is the physical distance covered by one cycle of this wake. It is defined as the distance from one crest of an electromagnetic wave to the same position on the subsequent wave. Peak-to-peak distance is generally measured in nanometres or wavenumber for use in molecular spectroscopy.