Fluorescence 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, 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 materials cease to glow nearly immediately when the radiation source stops, unlike phosphorescent materials, which continue to emit light for some time after.
Fluorescence spectroscopy 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.
A fluorophore 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.
Nile blue is a stain used in biology and histology. It may be used with live or fixed cells, and imparts a blue colour to cell nuclei.
Nile red is a lipophilic stain. Nile red stains intracellular lipid droplets yellow. In most polar solvents, Nile red will not fluoresce; however, when in a lipid-rich environment, it can be intensely fluorescent, with varying colors from deep red to strong yellow-gold emission. The dye is highly solvatochromic and its emission and excitation wavelength both shift depending on solvent polarity and in polar media will hardly fluoresce at all.
Phycobilisomes are light harvesting antennae of photosystem II in cyanobacteria, red algae and glaucophytes. It was lost in the plastids of green algae / plants (chloroplasts).
Acridine orange is an organic compound that serves as a nucleic acid-selective fluorescent dye with cationic properties useful for cell cycle determination. Acridine orange is cell-permeable, which allows the dye to interact with DNA by intercalation, or RNA via electrostatic attractions. When bound to DNA, acridine orange is very similar spectrally to an organic compound known as fluorescein. Acridine orange and fluorescein have a maximum excitation at 502nm and 525 nm (green). When acridine orange associates with RNA, the fluorescent dye experiences a maximum excitation shift from 525 nm (green) to 460 nm (blue). The shift in maximum excitation also produces a maximum emission of 650 nm (red). Acridine orange is able to withstand low pH environments, allowing the fluorescent dye to penetrate acidic organelles such as lysosomes and phagolysosomes that are membrane-bound organelles essential for acid hydrolysis or for producing products of phagocytosis of apoptotic cells. Acridine orange is used in epifluorescence microscopy and flow cytometry. The ability to penetrate the cell membranes of acidic organelles and cationic properties of acridine orange allows the dye to differentiate between various types of cells. The shift in maximum excitation and emission wavelengths provides a foundation to predict the wavelength at which the cells will stain.
Squaraine dyes are a class of organic dyes showing intense fluorescence, typically in the red and near infrared region. They are characterized by their unique aromatic four membered ring system derived from squaric acid. Most squaraines are encumbered by nucleophilic attack of the central four membered ring, which is highly electron deficient. This encumbrance can be attenuated by the formation of a rotaxane around the dye to protect it from nucleophiles. They are currently used as sensors for ions and have recently, with the advent of protected squaraine derivatives, been exploited in biomedical imaging.
The DyLight Fluor family of fluorescent dyes are produced by Dyomics in collaboration with Thermo Fisher Scientific. DyLight dyes are typically used in biotechnology and research applications as biomolecule, cell and tissue labels for fluorescence microscopy, cell biology or molecular biology.
BODIPY is the technical common name of a chemical compound with formula C
9H
7BN
2F
2, whose molecule consists of a boron difluoride group BF
2 joined to a dipyrromethene group C
9H
7N
2; specifically, the compound 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene in the IUPAC nomenclature. The common name is an abbreviation for "boron-dipyrromethene". It is a red crystalline solid, stable at ambient temperature, soluble in methanol.
Fluorescence is used in the life sciences generally as a non-destructive way of tracking or analysing biological molecules. Some proteins or small molecules in cells are naturally fluorescent, which is called intrinsic fluorescence or autofluorescence. Alternatively, specific or general proteins, nucleic acids, lipids or small molecules can be "labelled" with an extrinsic fluorophore, a fluorescent dye which can be a small molecule, protein or quantum dot. Several techniques exist to exploit additional properties of fluorophores, such as fluorescence resonance energy transfer, where the energy is passed non-radiatively to a particular neighbouring dye, allowing proximity or protein activation to be detected; another is the change in properties, such as intensity, of certain dyes depending on their environment allowing their use in structural studies.
The FluoProbes series of fluorescent dyes were developed by Interchim to improve performances of standard fluorophores. They are designed for labeling biomolecules, cells, tissues or beads in advanced fluorescent detection techniques.
Super-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 or on the far-field. Among techniques that rely on the latter are those that improve the resolution only modestly beyond the diffraction-limit, such as confocal microscopy with closed pinhole or aided by computational methods such as deconvolution or detector-based pixel reassignment, the 4Pi microscope, and structured-illumination microscopy technologies such as SIM and SMI.
Photo-activated localization microscopy and stochastic optical reconstruction microscopy (STORM) are widefield fluorescence microscopy imaging methods that allow obtaining images with a resolution beyond the diffraction limit. The methods were proposed in 2006 in the wake of a general emergence of optical super-resolution microscopy methods, and were featured as Methods of the Year for 2008 by the Nature Methods journal. The development of PALM as a targeted biophysical imaging method was largely prompted by the discovery of new species and the engineering of mutants of fluorescent proteins displaying a controllable photochromism, such as photo-activatible GFP. However, the concomitant development of STORM, sharing the same fundamental principle, originally made use of paired cyanine dyes. One molecule of the pair, when excited near its absorption maximum, serves to reactivate the other molecule to the fluorescent state.
YOYO-1 is a green fluorescent dye used in DNA staining. It belongs to the family of monomethine cyanine dyes and is a tetracationic homodimer of Oxazole Yellow, typically available as tetraiodide salt. In aqueous buffer, free YOYO-1 dye has very low fluorescence quantum yield. However, the intensity of fluorescence increases 3200 times upon binding through bis-intercalation to double-stranded DNA.
Pacific Blue, or systematically 3-carboxy-6,8-difluoro-7-hydroxycoumarin, is a fluorophore used in cell biology. Its excitation maximum lies at 401 nm, while its emission maximum is at 452 nm. In contrast to the less acidic 7-hydroxy-3-carboxycoumarin (pKa=7.0), the high acidity of the phenol of Pacific Blue (pKa=3.7) causes its fluorescence to remain very high at neutral pH.
Fluorescence imaging is a type of non-invasive imaging technique that can help visualize biological processes taking place in a living organism. Images can be produced from a variety of methods including: microscopy, imaging probes, and spectroscopy.
3-(2-furoyl)-quinoline-2-carboxaldehyde (FQ) is a fluorogenic amine labeling dye that is not fluorescent itself, but reacts with primary amines to form fluorescent products. It was first reported in 1990. Cyanide, typically provided via KCN or NaCN salts, is a required co-substrate in the fluorogenic reaction. It has been used for the detection of amines and peptides, largely in CE-SDS, where it is recognized to reach a silver stain-like high sensitivity via laser-induced fluorescence. Once bound to protein the excitation wavelength is 480 nm (blue) and the emission wavelength is ~600 nm (orange).
3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) is a fluorogenic amine labeling dye that is not fluorescent itself, but covalently reacts with primary amines to form fluorescent products. It was first reported in 1991. Today, it is largely used in the context of quantifying peptides or proteins. Either cyanide or thiols are required as a co-substrate in the fluorogenic reaction, although thiols also react with & mask the CBQCA aldehyde thereby preventing the fluorogenic reaction against the targeted primary amines. Once bound to protein the excitation wavelength is 465 nm (blue) and the emission wavelength is ~550 nm (green).
Pyrylium-1 (Py-1) is a fluorogenic pyrylium & tetrafluoroborate salt and amine-labeling dye that is not fluorescent itself, but reacts with primary amines to form fluorescent products. It is within the "chameleon labels" class, so named due to their clear color-changing properties upon conjugation. Py-1 was first reported in 2004. It has been used for the detection of amines and peptides, largely in CE-SDS, where it is recognized to reach a high sensitivity via laser-induced fluorescence. Once bound to protein the excitation wavelength is 503 nm (green) and the emission wavelength is 603 nm (orange). Similar to FQ, these fluorescence wavelengths makes Py-1 suitable for excitation with a 488 nm argon-ion laser.
