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      Photobleaching of organic fluorophores: quantitative characterization, mechanisms, protection.

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      Methods and applications in fluorescence
      IOP Publishing

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          Abstract

          Photochemical stability is one of the most important parameters that determine the usefulness of organic dyes in different applications. This Review addresses key factors that determine the dye photostability. It is shown that photodegradation can follow different oxygen-dependent and oxygen-independent mechanisms and may involve both 1S1-3T1 and higher-energy 1Sn-3Tn excited states. Their involvement and contribution depends on dye structure, medium conditions, irradiation power. Fluorescein, rhodamine, BODIPY and cyanine dyes, as well as conjugated polymers are discussed as selected examples illustrating photobleaching mechanisms. The strategies for modulating and improving the photostability are overviewed. They include the improvement of fluorophore design, particularly by attaching protective and anti-fading groups, creating proper medium conditions in liquid, solid and nanoscale environments. The special conditions for biological labeling, sensing and imaging are outlined.

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          Most cited references261

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          A general method to improve fluorophores for live-cell and single-molecule microscopy

          Specific labeling of biomolecules with bright fluorophores is the keystone of fluorescence microscopy. Genetically encoded self-labeling tag proteins can be coupled to synthetic dyes inside living cells, resulting in brighter reporters than fluorescent proteins. Intracellular labeling using these techniques requires cell-permeable fluorescent ligands, however, limiting utility to a small number of classic fluorophores. Here, we describe a simple structural modification that improves the brightness and photostability of dyes while preserving spectral properties and cell permeability. Inspired by molecular modeling, we replaced the N,N-dimethylamino substituents in tetramethylrhodamine with four-membered azetidine rings. This addition of two carbon atoms doubles the quantum efficiency and improves the photon yield of the dye in applications ranging from in vitro single-molecule measurements to super-resolution imaging. The novel substitution is generalizable, yielding a palette of chemical dyes with improved quantum efficiencies that spans the UV and visible range.
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            Two-photon laser scanning fluorescence microscopy.

            Molecular excitation by the simultaneous absorption of two photons provides intrinsic three-dimensional resolution in laser scanning fluorescence microscopy. The excitation of fluorophores having single-photon absorption in the ultraviolet with a stream of strongly focused subpicosecond pulses of red laser light has made possible fluorescence images of living cells and other microscopic objects. The fluorescence emission increased quadratically with the excitation intensity so that fluorescence and photo-bleaching were confined to the vicinity of the focal plane as expected for cooperative two-photon excitation. This technique also provides unprecedented capabilities for three-dimensional, spatially resolved photochemistry, particularly photolytic release of caged effector molecules.
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              Photoredox Catalysis in Organic Chemistry

              In recent years, photoredox catalysis has come to the forefront in organic chemistry as a powerful strategy for the activation of small molecules. In a general sense, these approaches rely on the ability of metal complexes and organic dyes to convert visible light into chemical energy by engaging in single-electron transfer with organic substrates, thereby generating reactive intermediates. In this Perspective, we highlight the unique ability of photoredox catalysis to expedite the development of completely new reaction mechanisms, with particular emphasis placed on multicatalytic strategies that enable the construction of challenging carbon–carbon and carbon–heteroatom bonds.
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                Author and article information

                Journal
                Methods Appl Fluoresc
                Methods and applications in fluorescence
                IOP Publishing
                2050-6120
                2050-6120
                Feb 20 2020
                : 8
                : 2
                Affiliations
                [1 ] Palladin Institute of Biochemistry, Leontovicha st. 9, Kyiv 01030, Ukraine. Yuriy Fedkovych National University, Chernivtsi, 58012, Ukraine.
                Article
                10.1088/2050-6120/ab7365
                32028269
                78ac7499-4f27-4b96-bf19-f88a76bd1074
                History

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