Direct Monitoring of Formation and Dissociation of Individual Metal Complexes by Single‐Molecule Fluorescence Spectroscopy

Kiel, Alexander; Kovács, János; Mokhir, Andriy; Krämer, Roland; Herten, Dirk‐Peter

doi:10.1002/anie.200604965

Cited by 63 publications

(48 citation statements)

References 17 publications

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“…Besides photobleaching, dark states represent a main concern in many biomolecular single-molecule studies. Dark states often originate from stochastic transitions to the triplet state (17), or from radical ion states formed as a result of electron transfer reactions (18)(19)(20)(21). Recently, an improved understanding of the underlying (redox) processes has led to ways to suppress photobleaching as well as triplet-and redox-blinking (22,23).…”

mentioning

confidence: 99%

Controlling the fluorescence of ordinary oxazine dyes for single-molecule switching and superresolution microscopy

Vogelsang

Cordes

Forthmann

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

253

331

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Fluorescent molecular switches have widespread potential for use as sensors, material applications in electro-optical data storages and displays, and superresolution fluorescence microscopy. We demonstrate that adjustment of fluorophore properties and environmental conditions allows the use of ordinary fluorescent dyes as efficient single-molecule switches that report sensitively on their local redox condition. Adding or removing reductant or oxidant, switches the fluorescence of oxazine dyes between stable fluorescent and nonfluorescent states. At low oxygen concentrations, the off-state that we ascribe to a radical anion is thermally stable with a lifetime in the minutes range. The molecular switches show a remarkable reliability with intriguing fatigue resistance at the single-molecule level: Depending on the switching rate, between 400 and 3,000 switching cycles are observed before irreversible photodestruction occurs. A detailed picture of the underlying photoinduced and redox reactions is elaborated. In the presence of both reductant and oxidant, continuous switching is manifested by ''blinking'' with independently controllable on-and off-state lifetimes in both deoxygenated and oxygenated environments. This ''continuous switching mode'' is advantageously used for imaging actin filament and actin filament bundles in fixed cells with subdiffraction-limited resolution.electron transfer ͉ molecular switch ͉ sensor ͉ single-molecule spectroscopy M olecular switches are single-molecule devices, and hence they are key building-blocks for future, bottom-up nanotechnological devices in computers, data storages, (bio-) sensors, and displays. Furthermore, they are exciting molecules for triggered drug-release or for superresolution imaging (1-3). Molecular switches possess at least 2 stable states and can be converted between these states by external stimuli. These external stimuli can essentially be any kind of physical or chemical trigger, such as light, electricity, or certain chemical reactions. Fluorescence is a preferred transduction mechanism because of its ease of noninvasive detection with ultimate sensitivity (i.e., single-molecule sensitivity). For future devices and for superresolution microscopy, it is also important that the molecular switches can be operated at the level of single molecules. This requirement imposes further demands with respect to reliability, reproducibility, response time, and fatigue resistance. Although the single-molecule approach provides detailed information about the properties and mechanisms, only a few examples of fluorescent switches have been demonstrated (4, 5). Among the fluorescent switches, there are systems that use 2 different wavelengths for switching (photoswitchable molecules) (6-13), those that switch the efficiency of photoinduced electron transfer (14, 15) and those that respond to pH changes (15,16).Here, we show that based on a detailed understanding of their photophysics, ordinary, fluorescent dyes can act as single-molecule switches and sensors. By adapting...

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mentioning

confidence: 99%

Controlling the fluorescence of ordinary oxazine dyes for single-molecule switching and superresolution microscopy

Vogelsang

Cordes

Forthmann

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

253

331

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“…Additionally, the 5'-end of the TMR-labelled oligonucleotide was conjugated with biotin to allow immobilization on streptavidincoated surfaces (Figure 4 a). [48] After imaging and localising individual probes by raster line scanning with the confocal microscope (Figure 4 b), individual probes were positioned one after another in the focal volume for time-resolved acquisition of their fluorescence as shown in Figure 4 c. [48,49] While the fluorescence traces in absence of Cu 2 + typically show constant fluorescence until photobleaching, the presence of Cu 2 + induces flickering of the fluorescence emission due to individual binding events on the bipyridine residue (Figure 4 c). This was the first time that single-molecule fluorescence spectroscopy was used to directly monitor individual association and dissociation events of metal-ion coordination to an organic ligand.…”

Section: Metal(ii) Coordination On Bipyridin Ligands Probed By Electrmentioning

confidence: 99%

Fluorescent Probes and Delivery Methods for Single‐Molecule Experiments

et al. 2010

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The recent explosion in papers utilising single-molecule experiments pushes the envelope further for increased spatial and temporal resolution. In order to achieve this, a combination of novel fluorescent probes and spectroscopy techniques are required. Herein, we provide an overview on our contribution to developments in the field of fluorescent probes along with a palette of alternative delivery methods for introducing the probes into living cells. We discuss probe requirements arising from the use of single-molecule spectroscopy methods and the customisation of probes that depends on the target molecule, the chemical state of the molecule as well as the distance and the type of interaction between sensor and ligand. We explain how Förster resonance energy transfer (FRET) and photon-induced electron transfer (PET) can increase the probe customisation. We also discuss additional requirements that arise when performing experiments in living cells like toxicity and cell permeability. Regarding the latter, we devote a special paragraph on the different ways to introduce the desired probe into the cell and how the different properties of each probe and cell type may require different delivery methods. We offer insights based on our experience working with a variety of single-molecule methods, fluorescent probes and delivery systems. Overall, we encompass the latest developments on probe design and delivery and illustrate that the wealth of information provided by single-molecule studies goes along with increased complexity.

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“…The rates derived from the exponential fit are 16.6 s -1 for the complex formation and 4 s -1 for the complex dissociation, which is in good accordance to the experimental data derived with the TMR sensor. 7 Dividing the examined rate by the applied concentration of copper (II) the association rate constant is derived k a = 5.5 x 10 6 M -1 s -1 . As the dissociation is a first order reaction the observed rate equals the rate constant k d = 4 s -1 .…”

Section: Single-molecule Studies With Atto620 Labeled Probementioning

confidence: 99%

“…6 Very recently, we developed a metal sensor using tetramethylrhodamine (TMR) as reporter dye that was strongly quenched by the formation of a Cu 2+ -complexes in close vicinity. 7 We determined a quenching constant of 4.2 x 10 6 M -1 using fluorescence ensemble spectroscopy. For the first time, we demonstrated that SMFS, a method which has proven its advantages to investigate processes in biological samples 8 and photophysical properties of chromophores [9][10][11] , was used to determine rate constants of complex formation and dissociation.…”

Section: Introductionmentioning

confidence: 99%

Single-molecule studies on individual metal complexes

et al. 2007

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Some fluorescent dyes, like Atto635 or Cy5, exhibit alterations in fluorescence emission in the presence of various metal ions and metal ion complexes. We used this effect to design dye-ligand conjugates that can be immobilized on glass surfaces and allow studying metal-ion binding using time-resolved single-molecule fluorescence spectroscopy (SMFS). Double-stranded DNA served as a rigid scaffold carrying 2,2'-bipyridene-4,4'-dicarboxylic acid as chelating ligand and a fluorescent dye as reporter, placed in close vicinity to the ligand. In the absence of metal ions, the probes showed high fluorescence quantum yield, whereas strong fluorescence quenching upon binding of Cu 2+ -ions was observed. Timeresolved single-molecule measurements revealed stochastic switching between a highly fluorescent ("on") and a low fluorescent ("off") state. The coordination of the metal ion to the ligand is thus indicated by intramolecular fluorescence quenching of the dye. We screened various fluorescent dyes for their sensitivity to Cu 2+ -coordination, and found that both Atto620 and MR121 are well-suited for this application. Ensemble studies of the fluorescence lifetimes of metalsensors with Atto620 showed only small dependence on the metal-ion concentration, while single-molecule studies reported strong changes in the fluorescence lifetimes which were correlated with the observed on-and off-states. Our results further indicate that the fluorescence of Atto620 is not completely quenched upon association of the metal-ion complex; either because a less fluorescent complex is formed or because of intramolecular collisional quenching due to conformational changes of the C6-linker used for covalent coupling of the fluorescent dye.

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Direct Monitoring of Formation and Dissociation of Individual Metal Complexes by Single‐Molecule Fluorescence Spectroscopy

Cited by 63 publications

References 17 publications

Controlling the fluorescence of ordinary oxazine dyes for single-molecule switching and superresolution microscopy

Controlling the fluorescence of ordinary oxazine dyes for single-molecule switching and superresolution microscopy

Fluorescent Probes and Delivery Methods for Single‐Molecule Experiments

Single-molecule studies on individual metal complexes

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