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Building a Fluorescent Hotspot When two gold nanoparticles come close together, their overlapping plasmonic fields can create a region that acts as a nanoantenna that can enhance the fluorescent emission of a molecule. Acuna et al. (p. 506 ) used a surface-anchored DNA origami structure to assemble one or two gold nanoparticles next to a dye trapped within the structure. A > 100-fold enhancement in fluorescent emission was observed when the dye molecules were located in a 23-nm gap between two 100-nm gold nanoparticles.

Recently, a new approach for super-resolution microscopy has emerged which is based on the successive localization of single molecules. The majority of molecules are prepared to reside in a nonfluorescent dark state, leaving only a few single molecules fluorescing. The single molecules can subsequently be localized on the camera image. Successive localization of all molecules allows reconstruction of a super-resolved image of the labeled structure. A variety of ways for limiting the number of locatable molecules have been developed recently which expand this current field of imaging. Here we describe a super-resolution microscopy method that employs the use of reversible, generic dark states, for example radical ion states. This method requires only a single laser source and can be carried out with many fluorescent dyes, in some cases, even in living cells. We provide a step-by-step procedure for this method, which we have called Blink Microscopy.

Recently, a new approach for super-resolution microscopy has emerged which is based on the successive localization of single molecules. The majority of molecules are prepared to reside in a nonfluorescent dark state, leaving only a few single molecules fluorescing. The single molecules can subsequently be localized on the camera image. Successive localization of all molecules allows reconstruction of a super-resolved image of the labeled structure. A variety of ways for limiting the number of locatable molecules have been developed recently which expand this current field of imaging. Here we describe a super-resolution microscopy method that employs the use of reversible, generic dark states, for example radical ion states. This method requires only a single laser source and can be carried out with many fluorescent dyes, in some cases, even in living cells. We provide a step-by-step procedure for this method, which we have called Blink Microscopy.

Response to NMETH-{"type":"entrez-nucleotide","attrs":{"text":"C14399","term_id":"1569106","term_text":"C14399"}}C14399: A battery of new mechanistic investigations will be required to provide a full explanation of the observation that individual, small-molecule compounds, referred to here as 'protective agents', have the capacity to promote dramatic enhancements to overall fluorophore performance1. As Tinnefeld and Cordes suggest, cycles of reduction (red) and oxidation (ox) - and/or the reverse -may confer a 'self-healing' property to the fluorogenic center. This could indeed be achieved by a single neighboring molecule, like Trolox, facilitating the fluorophore's rapid return to the ground state from relatively long-lived, non-fluorescent, radical intermediates through 'ping-pong' red-ox chemistry. However, this model requires that the rates of red-ox cycling be properly matched for benefits to be achieved. Further insights into the prevalence and lifetimes of charged intermediates for each molecule will need to be quantified to validate this model. Alternative mechanisms for photostabilization, which may play a significant or even dominant role, will also need to be carefully considered and quantified. Previous investigations, largely motivated by the once vigorous dye-laser industry, suggest that meaningful contributions to fluorophore performance may arise from changes in the rates of internal conversion and/or the introduction of mechanisms for triplet-triplet energy transfer and exciplex-type relaxation pathways2. Triplet-triplet energy transfer, a mechanism that would also fall into the 'self-healing' category, could provide substantial benefits to fluorophore performance by reducing the lifetime of non-fluorescent triplet states from which photobleaching and/or radical states can occur. Given our estimates of the triplet energy and red-ox potential of cyclooctatetraene (unpublished data), this mechanism may potentially be the dominant pathway of cyanine fluorophore photostabilization. Triplet-triplet energy transfer can be differentiated from red-ox-type chemistries by quantifying changes in triplet state lifetimes and charged, intermediate species. Other pathways may also exist that are distinct from the 'self-healing' variety. For example, a 'self-protecting' mechanism that reduces the probability of transitions into "damaged" states in the first place would also give rise to enhancements in fluorophore performance. Single-molecule imaging efforts can play an important role in the characterization of fluorophore performance by giving direct access to the frequency and duration of fluorescent and non-fluorescent states as well as a fluorophore’s total photon output prior to blinking or photobleaching. However, a quantitative understanding of the distinct mechanisms for enhancing fluorophore performance can only be delineated with confidence when married with bulk electrochemical and spectroscopic investigations. These combined approaches, while requiring large quantities of material, could provide grounded insights into the impact and relative weighting of 'self-healing' and 'self-protecting' mechanisms for distinct fluorophore types. Progress on this front will enable both further improvements in the performances of known protective agents and the search for new compounds with similar or improved properties. Developments of this kind may ultimately facilitate the design and synthesis of new classes of next-generation fluorophores spanning the visible spectrum that exhibit enhancements in performance even greater than those observed for the cyanine class (see Correspondence by Altman et al) and tailored properties for distinct experimental demands.

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 a certain color and brightness but instead have their own “personalities” regarding spectroscopic parameters, redox properties, size, water solubility, photostability, and several other factors. 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 that was later overcome using Trolox through a reducing/oxidizing system but a boon for super-resolution imaging owing to the controllable photoswitching. Understanding photophysics is critical in the design and interpretation of single-molecule experiments.

Modern fluorescence microscopy applications go along with increasing demands for the employed fluorescent dyes. In this work, we compared antifading formulae utilizing a recently developed reducing and oxidizing system (ROXS) with commercial antifading agents. To systematically test fluorophore performance in fluorescence imaging of biological samples, we carried out photobleaching experiments using fixed cells labeled with various commonly used organic dyes, such as Alexa 488, Alexa 594, Alexa 647, Cy3B, ATTO 550, and ATTO 647N. Quantitative evaluation of (i) photostability, (ii) brightness, and (iii) storage stability of fluorophores in samples mounted in different antifades (AFs) reveal optimal combinations of dyes and AFs. Based on these results we provide guidance on which AF should preferably be used with a specific dye. Finally, we studied the antifading mechanisms of the commercial AFs using single-molecule spectroscopy and reveal that these empirically selected AFs exhibit similar properties to ROXS AFs.

Since the development of the first techniques [1, 2] that bypass the diffraction limit postulated by Ernst Abbe in 1873, so called superresolution microscopies have spread rapidly diverging into different variants that are capable of resolving details smaller than 200 nm. This article focuses on those based on the subsequent localization of single molecules. Therein, most molecules are prepared in a non-fluorescent dark state, leaving only a few single molecules fluorescing, which can be localized by fitting a two dimensional Gaussian function to their respective point spread functions (PSF).

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.

In recent years, fluorescencemicroscopy techniques have been invented that are no longer fundamentally limited by diffraction despite using visible light focused by conventional optical elements. Contrary to earlier attempts to improve the spatial resolution, such as near-field optics and aperture filters, all far-field fluorescence ‘‘nanoscopy’’ methods known to date rely on a judicious exploitation of selected fluorophore properties. In particular, all are based on utilizing a molecular mechanism that renders the fluorophores incapable of responding with fluorescence emission to excitation light. This fluorescence inhibition mechanism is implemented in the image formation in such away that fluorophores that are closer than the diffraction limit emit sequentially in time and hence can be discerned. In stimulated emission depletion (STED) microscopy, fluorescence is inhibited by subjecting the dye molecules to an additional beam of light, thus inducing stimulated emission from the fluorescent state S1 to the ground state S0. The

Background: The various tasks of visual systems, including course control, collision avoidance and the detection of small objects, require at the neuronal level the dendritic integration and subsequent processing of many spatially distributed visual motion inputs. While much is known about the pooled output in these systems, as in the medial superior temporal cortex of monkeys or in the lobula plate of the insect visual system, the motion tuning of the elements that provide the input has yet received little attention. In order to visualize the motion tuning of these inputs we examined the dendritic activation patterns of neurons that are selective for the characteristic patterns of wide-field motion, the lobula-plate tangential cells (LPTCs) of the blowfly. These neurons are known to sample direction-selective motion information from large parts of the visual field and combine these signals into axonal and dendro-dendritic outputs. Results: Fluorescence imaging of intracellular calcium concentration allowed us to take a direct look at the local dendritic activity and the resulting local preferred directions in LPTC dendrites during activation by wide-field motion in different directions. These 'calcium response fields' resembled a retinotopic dendritic map of local preferred directions in the receptive field, the layout of which is a distinguishing feature of different LPTCs. Conclusions: Our study reveals how neurons acquire selectivity for distinct visual motion patterns by dendritic integration of the local inputs with different preferred directions. With their spatial layout of directional responses, the dendrites of the LPTCs we investigated thus served as matched filters for wide-field motion patterns. Background Fast flying insects such as flies need to integrate motion signals from their compound eyes for various tasks which include course control [1], collision avoidance and the detection of small objects. These tasks require that signals from individual ommatidia be combined into local motion signals, a process which is thought to take place in the medulla [2]. Local motion signals are then pooled and integrated on the dendrite of cells, which receive input from thousands of ommatidia. In the blowfly, this integration of retinotopic information occurs in the lobula plate, with a class of about 60 individually identifiable neurons showing direction selective responses to visual stimuli, the lobula-plate tangential cells (LPTCs). Among the best characterized of these cells are the neurons of the so called 'horizontal system' (HS), named after their strong response to progressive (front-to-back) motion, and those of the 'vertical system' (VS), which respond

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.

We present single-molecule fluorescence data of fluorescent proteins GFP, YFP, DsRed, and mCherry, a new derivative of DsRed. Ensemble and single-molecule fluorescence experiments proved mCherry as an ideally suited fluorophore for single-molecule applications, demonstrated by high photostability and rare fluorescence-intensity fluctuations. Although mCherry exhibits the lowest fluorescence quantum yield among the fluorescent proteins investigated, its superior photophysical characteristics suggest mCherry as an ideal alternative in single-molecule fluorescence experiments. Due to its spectral characteristics and short fluorescence lifetime of 1.46 ns, mCherry complements other existing fluorescent proteins and is recommended for tracking and localization of target molecules with high accuracy, fluorescence resonance energy transfer (FRET), fluorescence lifetime imaging microscopy (FLIM), or multicolor applications. [GRAPHICS] mCherry exhibits a relatively high photostability and brightness under single-molecule fluorescence imaging conditions. (C) 2008 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

The role and interplay of triplet states and radical ion states in single-molecule fluorescence spectroscopy has recently been elaborated providing us with new insights into the photophysics and photobleaching pathways of fluorescent dyes. Adjustment of fluorophore redox properties in combination with specific redox properties of the environment, i.e. addition of reducing and oxidizing agents, allows control of the emission properties: it has become possible to suppress blinking and to also induce blinking in single-molecule fluorescence transient by selectively opening and closing specific excited state pathways. Induced blinking is, for example, of interest for super-resolution fluorescence microscopy based on the subsequent localization of single fluorophores. For oxazines this control even allowed the separation of the influence of reducing and oxidizing agents, enabling switching the fluorescence of single fluorophores. Here, we study the factors that contribute to the kinetics of the photophysical pathways more closely with a focus on the photophysics of the oxazine ATTO655 labeled to DNA. Our data show that the oxazine ATTO655 interacts with DNA, shielding it efficiently from reagents in solution. Besides redox reactions, the pH also influences the blinking kinetics and especially the off-times. Moreover, we present the extension of ATTO655 as a single-molecule redox sensor to a ratiometric fluorescence-resonance-energy-transfer based sensor. Therefore, we designed FRET probes that showed the highest possible contrast of FRET changes and demonstrate reversible FRET-switching of Cy3B-ATTO655 DNA constructs.

In this paper we show that the emission of ordinary organic dyes can be controlled in order to increase photostability or to induce long off-states for superresolution microscopy. We therefore extend a recently introduced concept that utilizes triplet-state quenching via redox-reactions and recovery of the electronic ground-state by complementary redox-reactions: it is shown that different reagents in an oxidizing and reducing system (ROXS) can positively influence the fluorescence properties of organic dyes. In more detail, the effects of Trolox, a ferrocene-based compound, an oxidized quinone derivative of Trolox and nitrobenzoic acid are investigated and compared to the prototypical compounds ascorbic acid and N,N methylviologen. While the redox potential is the most important parameter for the realization of the ROXS concept it is demonstrated that also kinetic aspects have to be taken into account to explain the properties of the specific redox agents. Photostabilization and the induction of off-states are of paramount importance for fluorescence microscopy in general and especially for superresolution microscopy based on “blinking” molecules.

We used CdSe/ZnS quantum dots–ssDNA–fluorescent dye conjugates as bioprobes to detect micrococcal nuclease with high specificity and sensitivity, and further utilized the bioprobe to monitor the micrococcal nuclease activity in the culture medium of Staphylococcus aureus by fluorescence microscopy.

The wide-spread use of fluorescent dyes in molecular diagnostics and fluorescence microscopy together with new developments such as single-molecule fluorescence spectroscopy provide researchers from various disciplines with an ever expanding toolbox. Single-molecule fluorescence spectroscopy relies to a large extent on extraordinary bright and photostable organic fluorescent dyes such as rhodamine- or cyanine- derivatives. While in the last decade single-molecule equipment and methodology have significantly advanced and in some cases reached theoretical limits (e.g. detectors approaching unity quantum yields), instable emission (

In recent years Fluorescence Resonance Energy Transfer (FRET) has been widely used to determine distances, observe distance dynamics, and monitor molecular binding at the single-molecule level. A basic constraint of single-molecule FRET studies is the limited distance resolution owing to low photon statistics. We demonstrate that by confining molecules in nanopipettes (50-100 nm diameter) smFRET can be measured with improved photon statistics reducing the width of FRET proximity ratio distributions (PRD). This increase in distance resolution makes it possible to reveal subpopulations and dynamics in biomolecular complexes. Our data indicate that the width of PRD is not only determined by photon statistics (shot noise) and distance distributions between the chromophores but that photoinduced dark states of the acceptor also contribute to the PRD width. Furthermore, acceptor dark states such as triplet states influence the accuracy of determined mean FRET values. In this context, we present a strategy for the correction of the shift of the mean PR that is related to triplet induced blinking of the acceptor using reference FCS measurements.

Most biological processes are governed by assemblies of several dynamically interacting molecules. We have developed confocal multicolor single-molecule spectroscopy with optimized detection sensitivity on three spectrally distinct channels for the study of biomolecular interactions and FRET between more than two molecules. Using programmable acousto-optical devices as beamsplitter and excitation filter, we overcome some of the limitations of conventional multichroic beamsplitters and implement rapid alternation between three laser lines. This enables to visualize the synthesis of DNA three-way junctions on a single-molecule basis and to resolve seven stoichiometric subpopulations as well as to quantify FRET in the presence of competing energy transfer pathways. By comparing energy transfer of the different subpopulations, we can disentangle the reasons that lead to the occurrence of three-way junctions lacking one chromophore. A merit of the method is the ability to study correlated molecular movements by monitoring several distances within a biomolecular complex simultaneously.

Fluorescence based enzyme analysis is commonly done by FRET-probes, natural enzyme substrates flanked by two corresponding fluorophores, showing spectral changes upon distance variations between the fluorophores. However, the use of double labeled substrates displays several limitations such as reduction of sensitivity and high background signal accompanied by high costs for synthesis. Therefore, the development of new probes avoiding these factors is of general interest in enzyme research. A promising approach represents smart probes, i.e. singly labeled quenched enzyme substrates that increase fluorescence intensity upon enzymatic cleavage. Smart probes use the fact that certain rhodamine and oxazine dyes are selectively quenched upon contact formation with guanine or tryptophan residues via photoinduced electron transfer (PET). The rapid response time of the probes enables real-time monitoring of enzyme activity in ensemble as well as in single molecule measurements, which is an important prerequisite for the improved understanding of enzyme mechanisms. We present the design of smart probes for the detection of the two hydrolases, DNasel and Carboxypeptidase A (CPA) with respect to stability and substrate specificity in ensemble measurements. Furthermore, we investigate the influence of the attached fluorophore on hydrolysis efficiency in case of CPA and demonstrate first applications of smart probes in single enzyme experiments.

We have developed confocal multicolor single-molecule spectroscopy with optimized detection sensitivity on three spectrally distinct channels for the study of biomolecular interactions and FRET between more than two molecules. Using programmable acousto-optical devices as beamsplitter and excitation filter, we overcome some of the limitations of conventional multichroic beamsplitters and implement rapid alternation between three laser lines. This enables to visualize the synthesis of DNA three-way junctions on a single-molecule basis and to resolve seven stoichiometric subpopulations as well as to quantify FRET in the presence of competing energy transfer pathways. Furthermore, the ability to study correlated molecular movements by monitoring several distances within a biomolecular complex simultaneously is demonstrated.

Most fluorescence microscopy experiments today require a multicolor-capable setup, e.g. to study the interaction between different proteins. Multicolor capabilities are also well desirable for superresolution images. However, especially for STED (Stimulated Emission Depletion) microscopy, which requires two laser lines for a single color, multicolor imaging is technically challenging. Here we present a straightforward, easy-to-implement method to extend a single-color fluorescence (STED) microscope to a multichannel microscope without the need of modifying the optical setup. Therefore, we use a labeling technique based on complementary DNA sequences: a single-stranded short DNA sequence is attached to each structure to be imaged, different colors for labeling different features are represented by different sequences. Within the imaging process, the corresponding complementary sequence labeled with an organic fluorophore is added and transiently binds to the corresponding structure. After imaging, the labeled sequence is washed away and replaced by a second fluorescently labeled DNA strand complementary to the sequence bound to another feature. This way, multiplexing is achieved using only one arbitrary fluorophore, therefore aberrations are avoided.

Molecular photonic wires are one-dimensional representatives of a family of nanoscale molecular devices that transport excited-state energy over considerable distances in analogy to optical waveguides in the far-field. In particular, the design and synthesis of such complex supramolecular devices is challenging concerning the desired homogeneity of energy transport. On the other hand, novel optical techniques are available that permit direct investigation of heterogeneity by studying one device at a time. In this article, we describe our efforts to synthesize and study DNA-based molecular photonic wires that carry several chromophores arranged in an energetic downhill cascade and exploit fluorescence resonance energy transfer to convey excited-state energy. The focus of this work is to understand and control the heterogeneity of such complex systems, applying single-molecule fluorescence spectroscopy (SMFS) to dissect the different sources of heterogeneity, i.e., chemical heterogeneity and inhomogeneous broadening induced by the nanoenvironment. We demonstrate that the homogeneity of excited-state energy transport in DNA-based photonic wires is dramatically improved by immobilizing photonic wires in aqueous solution without perturbation by the surface. In addition, our study shows that the in situ construction of wire molecules, i.e., the stepwise hybridization of differently labeled oligonucleotides on glass cover slides, further decreases the observed heterogeneity in overall energy-transfer efficiency. The developed strategy enables efficient energy transfer between up to five chromophores in the majority of molecules investigated along a distance of approximately 14 nm. Finally, we used multiparameter SMFS to analyze the energy flow in photonic wires in more detail and to assign residual heterogeneity under optimized conditions in solution to different leakages and competing energy-transfer processes.

Spectrally and time-resolved single-molecule fluorescence spectroscopy was used to investigate fluctuations of the photophysical characteristics of different types of semiconductor nanocrystals (NCs) at room temperature. Correlation of photoluminescence (PL) emission maxima, decay time, and intensity of individual NCs with millisecond time resolution reveals new sources of intensity fluctuations and photophysical properties. In particular, we demonstrate that independent of quenched states spectral diffusion is associated with changes of the radiative rate constant k(r) by means of the quantum-confined Stark effect. Correlation of the different photophysical parameters revealed an intrinsic nonradiative rate and enabled the disentangling of intrinsic and extrinsic nonradiative rate constants. Moreover, it allowed us to assess the PL quantum yield of single NCs. Finally, the presented technique was successfully applied to demonstrate that the addition of antiblinking reagents such as mercaptoethylamine accelerates the observed fluctuations between different photophysical states.

High spatial resolution and low risks of photodamage make two-photon laser-scanning microscopy (TPLSM) the method of choice for biological imaging. However, the study of functional dynamics such as neuronal calcium regulation often also requires a high temporal resolution. Hitherto, acquisition speed is usually increased by line scanning, which restricts spatial resolution to structures along a single axis. To overcome this gap between high spatial and high temporal resolution we performed TPLSM with a beam multiplexer to generate multiple laser foci inside the sample. By detecting the fluorescence emitted from these laser foci with an electron-multiplying camera, it was possible to perform multiple simultaneous linescans. In addition to multiline scanning, the array of up to 64 laser beams could also be used in x-y scan mode to collect entire images at high frame rates. To evaluate the applicability of multiline TPLSM to functional in vivo imaging, calcium signals were monitored in visual motion-sensitive neurons in the brain of flies. The capacity of our method to simultaneously acquire signals at different cellular locations is exemplified by measurements at branched neurites and 'spine'-like structures. Calcium dynamics depended on branch size, but 'spines' did not systematically differ from their 'parent neurites'. The spatial resolution of our setup was critically evaluated by comparing it to confocal microscopy and the negative effect of scattering of emission light during image detection was assessed directly by running the setup in both imaging and point-scanning mode.

In the last decade emerging single-molecule fluorescence-spectroscopy tools have been developed and adapted to analyze individual molecules under various conditions. Single-molecule-sensitive optical techniques are now well established and help to increase our understanding of complex problems in different disciplines ranging from materials science to cell biology. Previous dreams, such as the monitoring of the motility and structural changes of single motor proteins in living cells or the detection of single-copy genes and the determination of their distance from polymerase molecules in transcription factories in the nucleus of a living cell, no longer constitute unsolvable problems. In this Review we demonstrate that single-molecule fluorescence spectroscopy has become an independent discipline capable of solving problems in molecular biology. We outline the challenges and future prospects for optical single-molecule techniques which can be used in combination with smart labeling strategies to yield quantitative three-dimensional information about the dynamic organization of living cells.

Light-harvesting complexes, one of nature's supreme examples of nanoscale engineering, have inspired researchers to construct molecular optical devices, such as photonic wires, which are optimised for efficient transfer of excited-state energy over large distances. The control parameters for the design and the advantages of single-molecule fluorescence spectroscopy for the study of such complex systems are discussed with respect to energy-transfer mechanisms, chromophore selection and arrangement as well as static and dynamic heterogeneity.

To obtain detailed information about the three-dimensional (3D) organization of small biomolecular assemblies with a size of less than 100 nanometers, advanced techniques are required that enable the determination of absolute 3D positions and distances between individual fluorophores well below the resolution limit of conventional light microscopy. We show how spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) can provide significant contributions and allow us to determine distances between conventional individual fluorophores (Bodipy 630/650 and Cy5.5) that are less than 20 nm apart. We take advantage of fluorescent dyes (here Cy5.5 and Bodipy 630/650) that can be efficiently excited by a single pulsed diode laser emitting at 635 nm but differ in their fluorescence lifetime and emission maxima. The potential of the method for ultrahigh colocalization studies is demonstrated by measuring the end-to-end distance between single fluorophores separated by double-stranded DNA of various lengths. Combining SFLIM with polarization-modulated excitation allows us to obtain, simultaneously, information about the relative orientation of fluorophores. Furthermore, we show that the environment-dependent photophysics of conventional fluorophores, that is, photostability, blinking pattern, and the tendency to enter irreversible nonfluorescent states, sets certain limitations to their in vitro and in vivo applications.

We demonstrate how photoinduced electron transfer (PET) reactions can be successfully applied to monitor conformational dynamics in individual biopolymers. Single-pair fluorescence resonance energy transfer (FRET) experiments are ideally suited to study conformational dynamics occurring on the nanometer scale, e.g. during protein folding or unfolding. In contrast, conformational dynamics with functional significance, for example occurring in enzymes at work, often appear on much smaller spatial scales of up to several Angstroms. Our results demonstrate that selective PET-reactions between fluorophores and amino acids or DNA nucleotides represent a versatile tool to measure small-scale conformational dynamics in biopolymers on a wide range of time scales, extending from nanoseconds to seconds, at the single-molecule level under equilibrium conditions. That is, the monitoring of conformational dynamics of biopolymers with temporal resolutions comparable to those within reach using new techniques of molecular dynamic simulations. We present data about structural changes of single biomolecules like DNA hairpins and peptides by using quenching electron transfer reactions between guanosine or tryptophan residues in close proximity to fluorescent dyes. Furthermore, we demonstrate that the strong distance dependence of charge separation reactions on the sub-nanometer scale can be used to develop conformationally flexible PET-biosensors. These sensors enable the detection of specific target molecules in the sub-picomolar range and allow one to follow their molecular binding dynamics with temporal resolution.

We report a new approach to an unidirectional photonic wire based on fluorescent dyes as chromophores and DNA as a rigid scaffold. The physical functioning of the wire is realized by dipole-dipole intreraction, i.e. resonant energy transfer, between chromophores. The use of four dyes (Alexa 430, TAMRA, Cy3.5, and Cy5) with different excited state energies creates an energy cascade constituting the driving force of the energy current and providing the unidirectionality of the device. The unique molecular properties of DNA, its scaffold-like structure, combined with straightforward synthesis methods allowed the engineering of a 30 base pair double-stranded DNA with inter-dye distances of 10 base pairs (3.4 nm), respectively, a range where electronic interactions between the chromophores can be neglected but dipole-dipole induced fluorescence resonance energy transfer (FRET) is expected to be still highly efficient. Steady-state and time-resolved ensemble spectroscopic measurements show an overall energy transfer efficiency of approximately 0.60. That is, the unidirectional transport of photonic energy over a distance of approximately 10 nm and a spectral separation of approximately 250 nm. Furthermore, pulsed diode laser excitation at 440 nm in combination with spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) was applied to characterize the effectiveness of individual photonic wires dispersed on glass coverslips.

We present a new technique for high-resolution colocalization of fluorescent dyes. The technique is based on polarization modulated excitation and spectrally-resolved fluorescence lifetime imaging microscopy (SFLIM) as well as on coincidence analysis of the detected photon counts following pulsed laser excitation. The method takes advantage of single fluorescent dyes that can be efficiently excited by a single pulsed diode laser emitting at 635 nm but differ in their emission maxima, and in their fluorescence lifetime. A combined analysis of the fractional intensities and fluorescence lifetimes recorded on two spectrally-separated detectors enables the classification of the portion of each dye per pixel in a point-spread-function (PSF) image with high accuracy, even though only a limited number (generally a few thousand) photons are detected per single dye. From these portions two separate PSF images are calculated and fitted to two-dimensional (2D) Gaussian functions to localize their centers with a precision of a few nanometers. To reveal the number of absorbing and emitting molecules polarization modulated excitation and coincidence analysis of the detected photon counts is used. We demonstrate that by the use of appropriately selected dyes, the presented technique permits (1) the counting of the number of molecules present in the observation volume, and (2) the determination of the distance between two single molecules down to approximately 30 nm with a precision of approximately 10 nm without any chromatic aberrations. The developed techniques are promising for applications in molecular biology, e.g. to determine the number of polymerase molecules active within a transcription factory and/or to measure their distances to nanscent transcripts.

The photophysics of a dendrimer containing four chromophores are investigated at the single-molecule level. First, the multichromophoric character of single dendrimers' absorption is probed by modulating the linear polarization of the excitation beam. Subsequently, using circular polarization, the same dendrimers are excited, and their fluorescence transients are recorded. Using pulsed excitation in combination with the classical Hanbury-Brown and Twiss coincidence setup the presented data demonstrate that efficient singlet−singlet annihilation ensures that always only one photon is emitted even when several excitations are generated in an individual multichromophoric molecule.

This article discusses advanced markers and labels for life science and biomedical applications.

An innovative marriage of techniques, combining the principles of common protein pull-down assays with single-molecule fluorescence microscopy, opens up new ways of visualizing cellular protein complexes. See Article p.484 Analysis of protein interactions is crucial for understanding cellular function and regulation. Here, Taekjip Ha and colleagues develop a novel method for elucidating the identity and stoichiometry of protein complexes from cells and tissues at single-molecule resolution. The method, called single molecule pull-down or SiMPull, can discriminate between multiple association states of a protein, and simultaneously allows determination of complex stoichiometry through photobleaching step analysis. The potential of the assay is demonstrated in a variety of contexts, including endogenous proteins from tissue extracts, organelles and membrane proteins.

Recent advances in photobleaching and blinking prevention have aided the advancement of single-molecule and super-resolution fluorescence microscopy. However, a common mechanism of the action of antifading agents such as Trolox is still missing. In this communication we present evidence that Trolox acts in accordance with a mechanism that involves triplet quenching through electron transfer and subsequent recovery of the resulting radical ion by the complementary redox reaction. The required oxidant for this unifying mechanism based on a reducing and oxidizing system (ROXS) is formed via (photo-) reaction with molecular oxygen. We present evidence that this oxidized form is a quinone derivative of Trolox with strong oxidizing properties. These findings shed light on many contradicting results regarding the action of antifading agents and might lead to a common mechanistic understanding of photobleaching and its prevention. Finally, a recipe on the proper use of Trolox as an antifading agent is provided.

New concepts for superresolution fluorescence microscopy by subsequent localization of single molecules using photoswitchable or photoactivatable fluorophores are rapidly emerging and provide new ways to resolve structures beyond the diffraction limit. Here, we demonstrate that superresolution imaging can be carried out with practically every single-molecule compatible, synthetic fluorophore by controlling their emission properties. We prepare dark states by removing oxygen that extends the triplet state lifetime to several milliseconds. We further increase the duration of the off-states using electron transfer reactions to create radical ion states of severalfold longer lifetimes. Imaging single molecules, actin filaments, and microtubules in fixed cells as well as simulations demonstrate that the thus created dark states are sufficiently long for resolution of approximately 50 nm.

Background In current protein research, a limitation still is the production of active recombinant proteins or native protein associations to assess their function. Especially the localization and analysis of protein-complexes or the identification of modifications and small molecule interaction partners by co-purification experiments requires a controllable expression of affinity- and/or fluorescence tagged variants of a protein of interest in its native cellular background. Advantages of periplasmic and/or homologous expressions can frequently not be realized due to a lack of suitable tools. Instead, experiments are often limited to the heterologous production in one of the few well established expression strains. Results Here, we introduce a series of new RK2 based broad host range expression plasmids for inducible production of affinity- and fluorescence tagged proteins in the cytoplasm and periplasm of a wide range of Gram negative hosts which are designed to match the recently suggested modular Standard European Vector Architecture and database. The vectors are equipped with a yellow fluorescent protein variant which is engineered to fold and brightly fluoresce in the bacterial periplasm following Sec-mediated export, as shown from fractionation and imaging studies. Expression of Strep-tag®II and Twin-Strep-tag® fusion proteins in Pseudomonas putida KT2440 is demonstrated for various ORFs. Conclusion The broad host range constructs we have produced enable good and controlled expression of affinity tagged protein variants for single-step purification and qualify for complex co-purification experiments. Periplasmic export variants enable production of affinity tagged proteins and generation of fusion proteins with a novel engineered Aequorea-based yellow fluorescent reporter protein variant with activity in the periplasm of the tested Gram-negative model bacteria Pseudomonas putida KT2440 and Escherichia coli K12 for production, localization or co-localization studies. In addition, the new tools facilitate metabolic engineering and yield assessment for cytoplasmic or periplasmic protein production in a number of different expression hosts when yields in one initially selected are insufficient.

Simultaneous single–molecule force spectroscopy and microfluorescence binding measurements are often hampered by background fluorescence from the bulk. Zero–Mode Waveguides (ZMW) restrict the excited volume but require a special design, which allows the tip of the force probing cantilever to protrude into the nanoaperture. Here, we describe the design and fabrication of such ZMW and report the first experiments where binding of fluorescent adenosine triphosphate to the force–activated enzyme titin kinase was measured while the enzyme was subjected to mechanical forces.

The bacterial periplasm is of special interest whenever cell factories are designed and engineered. Recombinantely produced proteins are targeted to the periplasmic space of Gram negative bacteria to take advantage of the authentic N-termini, disulfide bridge formation and easy accessibility for purification with less contaminating cellular proteins. The oxidizing environment of the periplasm promotes disulfide bridge formation – a prerequisite for proper folding of many proteins into their active conformation. In contrast, the most popular reporter protein in all of cell biology, Green Fluorescent Protein (GFP), remains inactive if translocated to the periplasmic space prior to folding. Here, the self-catalyzed chromophore maturation is blocked by formation of covalent oligomers via interchain disulfide bonds in the oxidizing environment. However, different protein engineering approaches addressing folding and stability of GFP resulted in improved proteins with enhanced folding properties. Recent studies describe GFP variants that are not only active if translocated in their folded form via the twin-arginine translocation (Tat) pathway, but actively fold in the periplasm following general secretory pathway (Sec) and signal recognition particle (SRP) mediated secretion. This mini-review highlights the progress that enables new insights into bacterial export and periplasmic protein organization, as well as new biotechnological applications combining the advantages of the periplasmic production and the Aequorea-based fluorescent reporter proteins.

Single-molecule experiments on immobilized molecules allow unique insights into the dynamics of molecular machines and enzymes as well as their interactions. The immobilization, however, can invoke perturbation to the activity of biomolecules causing incongruities between single molecule and ensemble measurements. Here we introduce the recently developed DNA origami as a platform to transfer ensemble assays to the immobilized single molecule level without changing the nano-environment of the biomolecules. The idea is a stepwise transfer of common functional assays first to the surface of a DNA origami, which can be checked at the ensemble level, and then to the microscope glass slide for single-molecule inquiry using the DNA origami as a transfer platform. We studied the structural flexibility of a DNA Holliday junction and the TATA-binding protein (TBP)-induced bending of DNA both on freely diffusing molecules and attached to the origami structure by fluorescence resonance energy transfer. This resulted in highly congruent data sets demonstrating that the DNA origami does not influence the functionality of the biomolecule. Single-molecule data collected from surface-immobilized biomolecule-loaded DNA origami are in very good agreement with data from solution measurements supporting the fact that the DNA origami can be used as biocompatible surface in many fluorescence-based measurements.

Recent developments in DNA nanotechnology facilitate a new degree of control to place arbitrary objects. Therefore, DNA is folded into arbitrary 2- and 3-dimensional structures on a nanometer to micrometer scale. In this so-called origami technique, introduced by Paul Rothemund in 2006, one ∼7.3 kbases long single-stranded DNA is hybridized with ∼200 short synthetic DNA “staple” strands to build a desired structure by self-assembly. Objects of interest, e.g. single fluorophores, are attached to individual incorporated DNA strands at specific positions within the structure. Several applications of this approach are shown using single-molecule fluorescence techniques.Revisiting the distance dependence of fluorescence resonance energy transfer (FRET), we used the DNA origami technique to build a spectroscopic ruler. In contrast to double stranded DNA, a commonly used spacer molecule, this technique offers distinct advantages. We designed a rigid DNA origami block, which has a higher persistence length and additionally allows placing the dye molecules all oriented in the same direction on the top surface, limiting static effects of the linker lengths. In contrast to dsDNA, for the origami block the Forster Radius R0 could directly be obtained from the distance dependence of energy transfer based on single-molecule FRET measurements.Guided by the idea to build complex spectroscopic networks by self-assembly, we used rectangular DNA origami as a molecular breadboard to precisely position individual fluorophores. In this artificial system the path of energy transfer can be manipulated on the nanoscale. Fluorophores were incorporated such that the light from the “blue” input dye could either be guided to the “red” or “IR” output dye, by a “green” dye that was placed at two alternative positions. We used a single-molecule four-color FRET approach with alternating laser excitation for analysis of the different energy transfer paths.