Fluorescence microscopeA fluorescence microscope is an optical microscope that uses fluorescence instead of, or in addition to, scattering, reflection, and attenuation or absorption, to study the properties of organic or inorganic substances. "Fluorescence microscope" refers to any microscope that uses fluorescence to generate an image, whether it is a simple set up like an epifluorescence microscope or a more complicated design such as a confocal microscope, which uses optical sectioning to get better resolution of the fluorescence image.
Green fluorescent proteinThe green fluorescent protein (GFP) is a protein that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. The label GFP traditionally refers to the protein first isolated from the jellyfish Aequorea victoria and is sometimes called avGFP. However, GFPs have been found in other organisms including corals, sea anemones, zoanithids, copepods and lancelets. The GFP from A. victoria has a major excitation peak at a wavelength of 395 nm and a minor one at 475 nm.
FluorescenceFluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, than the absorbed radiation. A perceptible example of fluorescence occurs when the absorbed radiation is in the ultraviolet region of the electromagnetic spectrum (invisible to the human eye), while the emitted light is in the visible region; this gives the fluorescent substance a distinct color that can only be seen when the substance has been exposed to UV light.
Fluorescent tagIn molecular biology and biotechnology, a fluorescent tag, also known as a fluorescent label or fluorescent probe, is a molecule that is attached chemically to aid in the detection of a biomolecule such as a protein, antibody, or amino acid. Generally, fluorescent tagging, or labeling, uses a reactive derivative of a fluorescent molecule known as a fluorophore. The fluorophore selectively binds to a specific region or functional group on the target molecule and can be attached chemically or biologically.
FluorophoreA fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with several π bonds. Fluorophores are sometimes used alone, as a tracer in fluids, as a dye for staining of certain structures, as a substrate of enzymes, or as a probe or indicator (when its fluorescence is affected by environmental aspects such as polarity or ions).
ImmunofluorescenceImmunofluorescence is a technique used for light microscopy with a fluorescence microscope and is used primarily on biological samples. This technique uses the specificity of antibodies to their antigen to target fluorescent dyes to specific biomolecule targets within a cell, and therefore allows visualization of the distribution of the target molecule through the sample. The specific region an antibody recognizes on an antigen is called an epitope.
SpectrophotometrySpectrophotometry is a branch of electromagnetic spectroscopy concerned with the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength. Spectrophotometry uses photometers, known as spectrophotometers, that can measure the intensity of a light beam at different wavelengths. Although spectrophotometry is most commonly applied to ultraviolet, visible, and infrared radiation, modern spectrophotometers can interrogate wide swaths of the electromagnetic spectrum, including x-ray, ultraviolet, visible, infrared, and/or microwave wavelengths.
Optical microscopeThe optical microscope, also referred to as a light microscope, is a type of microscope that commonly uses visible light and a system of lenses to generate magnified images of small objects. Optical microscopes are the oldest design of microscope and were possibly invented in their present compound form in the 17th century. Basic optical microscopes can be very simple, although many complex designs aim to improve resolution and sample contrast. The object is placed on a stage and may be directly viewed through one or two eyepieces on the microscope.
Fluorescent lampA fluorescent lamp, or fluorescent tube, is a low-pressure mercury-vapor gas-discharge lamp that uses fluorescence to produce visible light. An electric current in the gas excites mercury vapor, which produces short-wave ultraviolet light that then causes a phosphor coating on the inside of the lamp to glow. A fluorescent lamp converts electrical energy into useful light much more efficiently than an incandescent lamp. The typical luminous efficacy of fluorescent lighting systems is 50–100 lumens per watt, several times the efficacy of incandescent bulbs with comparable light output.
Ultraviolet–visible spectroscopyUV spectroscopy or UV–visible spectrophotometry (UV–Vis or UV/Vis) refers to absorption spectroscopy or reflectance spectroscopy in part of the ultraviolet and the full, adjacent visible regions of the electromagnetic spectrum. Being relatively inexpensive and easily implemented, this methodology is widely used in diverse applied and fundamental applications. The only requirement is that the sample absorb in the UV-Vis region, i.e. be a chromophore. Absorption spectroscopy is complementary to fluorescence spectroscopy.
Fluorescence spectroscopyFluorescence spectroscopy (also known as fluorimetry or spectrofluorometry) is a type of electromagnetic spectroscopy that analyzes fluorescence from a sample. It involves using a beam of light, usually ultraviolet light, that excites the electrons in molecules of certain compounds and causes them to emit light; typically, but not necessarily, visible light. A complementary technique is absorption spectroscopy. In the special case of single molecule fluorescence spectroscopy, intensity fluctuations from the emitted light are measured from either single fluorophores, or pairs of fluorophores.
LaserA laser is a device that emits light through a process of optical amplification based on the stimulated emission of electromagnetic radiation. The word laser is an anacronym that originated as an acronym for light amplification by stimulated emission of radiation. The first laser was built in 1960 by Theodore Maiman at Hughes Research Laboratories, based on theoretical work by Charles H. Townes and Arthur Leonard Schawlow. A laser differs from other sources of light in that it emits light that is coherent.
Total internal reflection fluorescence microscopeA total internal reflection fluorescence microscope (TIRFM) is a type of microscope with which a thin region of a specimen, usually less than 200 nanometers can be observed. TIRFM is an imaging modality which uses the excitation of fluorescent cells in a thin optical specimen section that is supported on a glass slide. The technique is based on the principle that when excitation light is totally internally reflected in a transparent solid coverglass at its interface with a liquid medium, an electromagnetic field, also known as an evanescent wave, is generated at the solid-liquid interface with the same frequency as the excitation light.
Compact fluorescent lampA compact fluorescent lamp (CFL), also called compact fluorescent light, energy-saving light, compact fluorescent tube and chloro fluorescent lamp is a fluorescent lamp designed to replace an incandescent light bulb; some types fit into light fixtures designed for incandescent bulbs. The lamps use a tube that is curved or folded to fit into the space of an incandescent bulb, and a compact electronic ballast in the base of the lamp.
Super-resolution microscopySuper-resolution microscopy is a series of techniques in optical microscopy that allow such images to have resolutions higher than those imposed by the diffraction limit, which is due to the diffraction of light. Super-resolution imaging techniques rely on the near-field (photon-tunneling microscopy as well as those that use the Pendry Superlens and near field scanning optical microscopy) or on the far-field.
Ion laserAn ion laser is a gas laser that uses an ionized gas as its lasing medium. Like other gas lasers, ion lasers feature a sealed cavity containing the laser medium and mirrors forming a Fabry–Pérot resonator. Unlike helium–neon lasers, the energy level transitions that contribute to laser action come from ions. Because of the large amount of energy required to excite the ionic transitions used in ion lasers, the required current is much greater, and as a result almost all except for the smallest ion lasers are water-cooled.
Electro-osmosisElectroosmotic flow (or electro-osmotic flow, often abbreviated EOF; synonymous with electroosmosis or electroendosmosis) is the motion of liquid induced by an applied potential across a porous material, capillary tube, membrane, microchannel, or any other fluid conduit. Because electroosmotic velocities are independent of conduit size, as long as the electrical double layer is much smaller than the characteristic length scale of the channel, electroosmotic flow will have little effect.
CuvetteIn laboratories, a cuvette (French: cuvette = "little vessel") is a small tube-like container with straight sides and a circular or square cross-section. It is sealed at one end, and made of a clear, transparent material such as plastic, glass, or fused quartz. Cuvettes are designed to hold samples for spectroscopic measurement, where a beam of light is passed through the sample within the cuvette to measure the absorbance, transmittance, fluorescence intensity, fluorescence polarization, or fluorescence lifetime of the sample.
Gaussian beamIn optics, a Gaussian beam is a beam of electromagnetic radiation with high monochromaticity whose amplitude envelope in the transverse plane is given by a Gaussian function; this also implies a Gaussian intensity (irradiance) profile. This fundamental (or TEM00) transverse Gaussian mode describes the intended output of most (but not all) lasers, as such a beam can be focused into the most concentrated spot. When such a beam is refocused by a lens, the transverse phase dependence is altered; this results in a different Gaussian beam.
Confocal microscopyConfocal microscopy, most frequently confocal laser scanning microscopy (CLSM) or laser scanning confocal microscopy (LSCM), is an optical imaging technique for increasing optical resolution and contrast of a micrograph by means of using a spatial pinhole to block out-of-focus light in image formation. Capturing multiple two-dimensional images at different depths in a sample enables the reconstruction of three-dimensional structures (a process known as optical sectioning) within an object.
