Staining is a technique used to enhance contrast in samples, generally at the microscopic level. Stains and dyes are frequently used in histology and in the medical fields of histopathology, hematology, and cytopathology that focus on the study and diagnoses of disease at a microscopic level. Stains may be used to define biological tissues, cell populations, or organelles within individual cells.
Flow cytometry (FC) is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles.
Fluorescein is an organic compound and dye. It is available as a dark orange/red powder slightly soluble in water and alcohol. It is widely used as a fluorescent tracer for many applications.
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.
Rhodamine is a family of related dyes, a subset of the triarylmethane dyes. They are derivatives of xanthene. Important members of the rhodamine family are Rhodamine 6G, Rhodamine 123, and Rhodamine B. They are mainly used to dye paper and inks, but they lack the lightfastness for fabric dying.
Hoechst stains are part of a family of blue fluorescent dyes used to stain DNA. These Bis-benzimides were originally developed by Hoechst AG, which numbered all their compounds so that the dye Hoechst 33342 is the 33,342nd compound made by the company. There are three related Hoechst stains: Hoechst 33258, Hoechst 33342, and Hoechst 34580. The dyes Hoechst 33258 and Hoechst 33342 are the ones most commonly used and they have similar excitation–emission spectra.
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.
Fluorescein isothiocyanate (FITC) is a derivative of fluorescein used in wide-ranging applications including flow cytometry. First described in 1942, FITC is the original fluorescein molecule functionalized with an isothiocyanate reactive group (-N=C=S), replacing a hydrogen atom on the bottom ring of the structure. It is typically available as a mixture of isomers, fluorescein 5-isothiocyanate (5-FITC) and fluorescein 6-isothiocyanate (6-FITC). FITC is reactive towards nucleophiles including amine and sulfhydryl groups on proteins. It was synthesized by Robert Seiwald and Joseph Burckhalter in 1958.
Metachromasia is a characteristical change in the color of staining carried out in biological tissues, exhibited by certain dyes when they bind to particular substances present in these tissues, called chromotropes. For example, toluidine blue becomes dark blue when bound to cartilage. Other widely used metachromatic stains are the haematological Giemsa and May-Grunwald stains that also contain thiazine dyes. The white cell nucleus stains purple, basophil granules intense magenta, whilst the cytoplasms stains blue. The absence of color change in staining is named orthochromasia.
Cyanines, also referred to as tetramethylindo(di)-carbocyanines are defined as "synthetic dyes with the general formula R2N[CH=CH]nCH=N+R2↔R2N+=CH[CH=CH]nNR2 in which the nitrogen and part of the conjugated chain usually form part of a heterocyclic system, such as imidazole, pyridine, pyrrole, quinoline and thiazole." Cyanines are used in industry biotechnology.
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.
6-Carboxyfluorescein (6-FAM) is a fluorescent dye with an absorption wavelength of 495 nm and an emission wavelength of 517 nm. A carboxyfluorescein molecule is a fluorescein molecule with a carboxyl group added. They are commonly used as a tracer agents. It is used in the sequencing of nucleic acids and in the labeling of nucleotides.
Calcein, also known as fluorexon, fluorescein complex, is a fluorescent dye with excitation and emission wavelengths of 495/515 nm, respectively, and has the appearance of orange crystals. Calcein self-quenches at concentrations above 70mM and is commonly used as an indicator of lipid vesicle leakage. It is also used traditionally as a complexometric indicator for titration of calcium ions with EDTA, and for fluorometric determination of calcium.
Carboxyfluorescein succinimidyl ester (CFSE) is a fluorescent cell staining dye. CFSE is cell permeable and covalently couples, via its succinimidyl group, to intracellular molecules, notably, to intracellular lysine residues and other amine sources. Due to this covalent coupling reaction, fluorescent CFSE can be retained within cells for extremely long periods. Also, due to this stable linkage, once incorporated within cells, the dye is not transferred to adjacent cells.
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.
Virus quantification involves counting the number of viruses in a specific volume to determine the virus concentration. It is utilized in both research and development (R&D) in commercial and academic laboratories as well as production situations where the quantity of virus at various steps is an important variable. For example, the production of viral vaccines, recombinant proteins using viral vectors and viral antigens all require virus quantification to continually adapt and monitor the process in order to optimize production yields and respond to ever changing demands and applications. Examples of specific instances where known viruses need to be quantified include clone screening, multiplicity of infection (MOI) optimization and adaptation of methods to cell culture. This page discusses various techniques currently used to quantify viruses in liquid samples. These methods are separated into two categories, traditional vs. modern methods. Traditional methods are industry-standard methods that have been used for decades but are generally slow and labor-intensive. Modern methods are relatively new commercially available products and kits that greatly reduce quantification time. This is not meant to be an exhaustive review of all potential methods, but rather a representative cross-section of traditional methods and new, commercially available methods. While other published methods may exist for virus quantification, non-commercial methods are not discussed here.
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.
Calcofluor-white or CFW is a fluorescent blue dye that is used to bind to the polysaccharide polymers of amebic cysts. It functions by being able to bind to 1-3 beta and 1-4 beta polysaccharides on chitin and cellulose that is present in cell walls on fungi, plants, and algae.
Fluorochromasia, is a cellular phenomenon characterized by immediate appearance of bright green fluorescence inside viable cells upon exposure to certain membrane-permeable fluorogenic substrates such as fluorescein diacetate, fluorescein dibutyrate and fluorescein dipropionate. The phenomenon is widely used to measure cellular viability of many different species including animals, plants, and microorganisms. Moreover, fluorochromasia has been observed within organs, embryos, and zebrafish. Fluorochromasia has many applications including histocompatibility testing, measurement of cytotoxic antibodies, in vitro chemo sensitivity testing of tumors, and fluorochrome intercellular translocation. It has been applied with plants, bacteria, mammalian oocytes, mouse embryos, and human tumor cells.
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