Mechanisms and advancement of antifading agents for fluorescence microscopy and single-molecule spectroscopy

Cordes, Thorben; Maiser, Andreas; Steinhauer, Christian; Schermelleh, Lothar; Tinnefeld, Philip

doi:10.1039/c0cp01919d

Cited by 81 publications

(80 citation statements)

References 41 publications

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“…[7a] In contrast, the effect of replacing uridine with an electron-deficient 1,2,4-triazine core ( 1 versus 2 ) on the 2P cross section appears to be small, despite the 20-fold difference in emission quantum yield. [6] Samples, 4 and 5 , contain either a 5-membered or 6-membered ring, respectively, fused to a uridine core.…”

Section: Resultsmentioning

confidence: 99%

Two‐Photon‐Induced Fluorescence of Isomorphic Nucleobase Analogs

et al. 2014

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Five isomorphic fluorescent uridine mimics have been subjected to two-photon (2P) excitation analysis to investigate their potential applicability as non-perturbing probes for the single-molecule detection of nucleic acids. We find that small structural differences can cause major changes in the two-photon excitation probability, with the 2P cross sections varying by over one order of magnitude. Two of the probes, both furan-modified uridine analogs, have the highest 2P cross sections (3.8 GM and 7.6 GM) reported for nucleobase analogs, using a conventional Ti:sapphire laser for excitation at 690 nm; they also have the lowest emission quantum yields. In contrast, the analogs with the highest reported quantum yields have the lowest 2P cross sections. The structure-photophysical property relationship presented here is a first step towards the rational design of emissive nucleobase analogs with controlled 2P characteristics. The results demonstrate the potential for major improvements through judicious structural modifications.

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Section: Resultsmentioning

confidence: 99%

Two‐Photon‐Induced Fluorescence of Isomorphic Nucleobase Analogs

et al. 2014

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“…Therefore, a large number of potentially useful reagents have been phenomenologically screened to reduce blinking and improve photostability. Among the typical reagents were thiols such as β-mercaptoethanol, β-mercaptoehylamine as well as l-glutathione, propyl galate, ascorbic acid, and diazabicyclo[2,2,2]octane (27, 51, 52). …”

Section: Photophysics Of Organic Fluorophorementioning

confidence: 99%

Photophysics of Fluorescent Probes for Single-Molecule Biophysics and Super-Resolution Imaging

Tinnefeld

2012

Annu. Rev. Phys. Chem.

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623

684

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Single-molecule fluorescence spectroscopy and super-resolution microscopy are important elements of the ongoing technical revolution to reveal biochemical and cellular processes in unprecedented clarity and precision. Demands placed on the photophysical properties of the fluorophores are stringent and drive the choice of appropriate probes. Such fluorophores are not simple light bulbs of certain color and brightness but instead have their own ‘personalities’ regarding spectroscopic parameters, redox properties, size and water solubility, photostability and several more. Here, we review the photophysics of fluorescent probes, both organic fluorophores and fluorescent proteins, used in applications such as particle tracking, single molecule FRET, stoichiometry determination, and super-resolution imaging. Of particular interest is the thiol-induced blinking of Cy5, a curse for single molecule biophysical studies which was later overcome using Trolox through reducing/oxidizing system, but a boon for super-resolution imaging due to the controllable photoswitching. Understanding photophysics is critical in design and interpreting single molecule experiments.

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“…Fluorescent dyes are susceptible to photo-bleaching and there are several commercial antifades and mounting mediums available that may prevent weakening of the fluorescent signal 37 . There are also choices between hard-set mounting media and soft-set ones.…”

Section: Discussionmentioning

confidence: 99%

<em>Porphyromonas gingivalis</em> as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Wunsch

Lewis

2015

JoVE

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Citation: Wunsch, C.M., Lewis, J.P. Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells. J. Vis. Exp. (106), e53408, doi:10.3791/53408 (2015). AbstractAnaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth. The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2',7'-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal-spatial visualization of bacteria. Methods used in this study can be applied to any cultivable anaerobe and any eukaryotic cell type. Video LinkThe video component of this article can be found at

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Mechanisms and advancement of antifading agents for fluorescence microscopy and single-molecule spectroscopy

Cited by 81 publications

References 41 publications

Two‐Photon‐Induced Fluorescence of Isomorphic Nucleobase Analogs

Two‐Photon‐Induced Fluorescence of Isomorphic Nucleobase Analogs

Photophysics of Fluorescent Probes for Single-Molecule Biophysics and Super-Resolution Imaging

<em>Porphyromonas gingivalis</em> as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

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