Fluorescent Probes and Delivery Methods for Single‐Molecule Experiments

Lymperopoulos, Konstantinos; Kiel, Alexander; Seefeld, Anne; Stöhr, Katharina; Herten, Dirk‐Peter

doi:10.1002/cphc.200900359

Cited by 14 publications

(6 citation statements)

References 102 publications

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“…An open question is how well the method can be applied to living cells. Besides the problems of selective labeling with organic dyes in living cells, [122] there remains the problem whether the conditions in a living cell are compatible with redox blinking of organic fluorophores. The cell medium itself contains many redox active substances such as sulfides and disulfides (e.g.…”

Section: Blink Microscopy For Imaging Biological Specimensmentioning

confidence: 99%

Make them Blink: Probes for Super‐Resolution Microscopy

et al. 2010

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In recent years, a number of approaches have emerged that enable far-field fluorescence imaging beyond the diffraction limit of light, namely super-resolution microscopy. These techniques are beginning to profoundly alter our abilities to look at biological structures and dynamics and are bound to spread into conventional biological laboratories. Nowadays these approaches can be divided into two categories, one based on targeted switching and readout, and the other based on stochastic switching and readout of the fluorescence information. The main prerequisite for a successful implementation of both categories is the ability to prepare the fluorescent emitters in two distinct states, a bright and a dark state. Herein, we provide an overview of recent developments in super-resolution microscopy techniques and outline the special requirements for the fluorescent probes used. In combination with the advances in understanding the photophysics and photochemistry of single fluorophores, we demonstrate how essentially any single-molecule compatible fluorophore can be used for super-resolution microscopy. We present examples for super-resolution microscopy with standard organic fluorophores, discuss factors that influence resolution and present approaches for calibration samples for super-resolution microscopes including AFM-based single-molecule assembly and DNA origami.

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Section: Blink Microscopy For Imaging Biological Specimensmentioning

confidence: 99%

Make them Blink: Probes for Super‐Resolution Microscopy

et al. 2010

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“…Microarray hybridization is thus independent from target sequences since the SCISSOHR oligonucleotide serves as an indexing probe between aqueous phase targets and solid-support capture probes. A fluorophore and a quencher moieties are present on the SCISSOHR probe and are within FRET distance in the sequence and secondary structure 27, 28 . This proximity ensures sufficient quenching in all conformations.…”

Section: Methodsmentioning

confidence: 99%

“…A uorophore and a quencher moieties are present on the SCIS-SOHR probe and are within FRET distance in the sequence and secondary structure. 27,28 This proximity ensures sufficient quenching in all conformations. Finally, a phosphate moiety is added at the 3 0 -end of T1.2 to prevent elongation.…”

Section: Function Of Scissohr Probesmentioning

confidence: 98%

Structured oligonucleotides for target indexing to allow single-vessel PCR amplification and solid support microarray hybridization

Girard

Boissinot

Peytavi

et al. 2015

Analyst

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The combination of molecular diagnostic technologies is increasingly used to overcome limitations on sensitivity, specificity or multiplexing capabilities, and provide efficient lab-on-chip devices. Two such techniques, PCR amplification and microarray hybridization are used serially to take advantage of the high sensitivity and specificity of the former combined with high multiplexing capacities of the latter. These methods are usually performed in different buffers and reaction chambers. However, these elaborate methods have a high complexity cost related to reagent requirements, liquid storage and the number of reaction chambers to integrate into automated devices. Furthermore, microarray hybridizations have a sequence dependent efficiency not always predictable. In this work, we have developed the concept of a structured oligonucleotide probe which is activated by cleavage from polymerase exonuclease activity. This technology is called SCISSOHR for Structured Cleavage Induced Single-Stranded Oligonucleotide Hybridization Reaction. The SCISSOHR probes enable indexing the target sequence to a tag sequence. The SCISSOHR technology also allows the combination of nucleic acid amplification and microarray hybridization in a single vessel in presence of the PCR buffer only. The SCISSOHR technology uses an amplification probe that is irreversibly modified in presence of the target, releasing a single-stranded DNA tag for microarray hybridization. Each tag is composed of a 3-nucleotidesequence-dependent segment and a unique “target sequence-independent” 14-nucleotide segment allowing for optimal hybridization with minimal cross-hybridization. We evaluated the performance of five (5) PCR buffers to support microarray hybridization, compared to a conventional hybridization buffer. Finally, as a proof of concept, we developed a multiplexed assay for the amplification, detection, and identification of three (3) DNA targets. This new technology will facilitate the design of lab-on-chip microfluidic devices, while also reducing consumable costs. At term, it will allow the cost-effective automation of highly multiplexed assays for detection and identification of genetic targets.

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“…To track intracellular POI, the delivery of fluorescent probes appeared to be a critical aspect. To break the limitations of lipophilic cell membrane toward large, charged molecules, about five kinds of approaches have been reported for living cell imaging (Lymperopoulos et al, 2010). Among them, electroporation enabled a high efficiency of fluorophores and proteins with notable size (up to 60 kDa) into living E. coli cells.…”

Section: Molecular Modifications and Delivery Majorizationmentioning

confidence: 99%

Recognition of Proteins by Metal Chelation-Based Fluorescent Probes in Cells

Jiang

Sun

2019

Front. Chem.

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Fluorescent probes such as thiol-reactive and Ni 2+ -nitrilotriacetate (NTA) based probes provide a powerful toolbox for real-time visualization of a protein and a proteome in living cells. Herein, we first went through basic principles and applications of thiol-reactive based probes in protein imaging and recognition. We then summarize a family of metal-NTA based fluorescence probes in the visualization of His 6 -tagged protein and identification of metalloproteins at proteome-wide scale. The pros and cons of the probes, as well as ways to optimize them, are discussed.

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Fluorescent Probes and Delivery Methods for Single‐Molecule Experiments

Cited by 14 publications

References 102 publications

Make them Blink: Probes for Super‐Resolution Microscopy

Make them Blink: Probes for Super‐Resolution Microscopy

Structured oligonucleotides for target indexing to allow single-vessel PCR amplification and solid support microarray hybridization

Recognition of Proteins by Metal Chelation-Based Fluorescent Probes in Cells

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