Thorsten Staudt

Molecular orientation affects localization accuracy in superresolution far-field fluorescence microscopy.

We investigate the cooperative effect of molecular tilt and defocus on fluorophore localization by centroid calculation in far-field superresolution microscopy based on stochastic single molecule switching. If tilt angle and defocus are unknown, the localization contains systematic errors up to about ±125 nm. When imaging rotation-impaired fluorophores of unknown random orientation, the average localization accuracy in three-dimensional samples is typically limited to about ±32 nm, restricting the attainable resolution accordingly.

Quantum Dot Blueing and Blinking Enables Fluorescence Nanoscopy

We demonstrate superresolution fluorescence imaging of cells using bioconjugated CdSe/ZnS quantum dot markers. Fluorescence blueing of quantum dot cores facilitates separation of blinking markers residing closer than the diffraction barrier. The high number of successively emitted photons enables ground state depletion microscopy followed by individual marker return with a resolving power of the size of a single dot (∼12 nm). Nanoscale imaging is feasible with a simple webcam.

Direct Light‐Driven Modulation of Luminescence from Mn‐Doped ZnSe Quantum Dots

Quantum dot (QD) nanocrystals remain at the forefront of fluorescence microscopy as they have the advantages of enhanced photostability, high quantum yield, and macromolecular size. Furthermore, the ability to tune the QD fluorescence, either by changing their size or by doping, allows for multiplexed imaging. The range of applications extends well beyond the realm of microscopy: QDs may also play a major role in developing novel photonic devices including lasers, light-emitting diodes, and displays. Despite significant advancements in nanocrystal research, the inability to directly modulate the fluorescence from QDs has precluded their implementation in several areas. In particular, emerging far-field diffraction-unlimited microscopy techniques uniquely benefit from the capability to reversibly modulate/switch fluorescent ensembles from a bright “on” state to a dark “off” state. This activation must occur as a response to optical stimuli which do not contain spectral components within the excitation kernel of the fluorescent markers. With the need for optical control over QD fluorescence, indirect methods have been conceived by using hybrid QD structures that incorporate a photochromic activator/quencher. Although the concept has been clearly established, hybrid QD structures suffer from inherent drawbacks, such as inadequate photostability, limited fluorescence quenching, and sensitivity to local environment/ solvent. Herein we report on the direct light-driven modulation of QD fluorescence. The mechanism for the fluorescence modulation relies only on internal electronic transitions within Mn-doped ZnSe quantum dots (Mn-QDs). It is demonstrated that the fluorescence of the QD can be reversibly depleted with efficiencies of over 90% by using continuous-wave optical intensities of approximately 1.9 MWcm . Time-domain measurements during the modulation indicate that the number of fluorescent on–off cycles exceeds 10 before a significant reduction in the fluorescence quantum efficiency occurs. Such robust nanometric probes having remotely controllable optical transitions are useful in many areas of research, particularly in far-field nanoscopy based on reversible saturable or switchable optical fluorescence transitions (RESOLFT). Consequently, we show that implementation of Mn-QDs for imaging leads to an increase in the resolution by a factor of 4.4 over that of confocal microscopy. A schematic diagram of the electronic transitions involved in light-modulated fluorescence from Mn-QDs is shown in Figure 1a. Initially, electrons are photoexcited from the

Distinct Subsets of Syt-IV/BDNF Vesicles Are Sorted to Axons versus Dendrites and Recruited to Synapses by Activity

BDNF plays a critical role in the regulation of synaptic strength and is essential for long-term potentiation, a phenomenon that underlies learning and memory. However, whether BDNF acts in a diffuse manner or is targeted to specific neuronal subcompartments or synaptic sites to affect circuit function remains unknown. Here, using photoactivation of BDNF or syt-IV (a regulator of exocytosis present on BDNF-containing vesicles) in transfected rat hippocampal neurons, we discovered that distinct subsets of BDNF vesicles are targeted to axons versus dendrites and are not shared between these compartments. Moreover, syt-IV- and BDNF-harboring vesicles are recruited to both presynaptic and postsynaptic sites in response to increased neuronal activity. Finally, using syt-IV knockout mouse neurons, we found that syt-IV is necessary for both presynaptic and postsynaptic scaling of synaptic strength in response to changes in network activity. These findings demonstrate that BDNF-containing vesicles can be targeted to specific sites in neurons and suggest that syt-IV-regulated BDNF secretion is subject to spatial control to regulate synaptic function in a site-specific manner.

Far-field optical nanoscopy with reduced number of state transition cycles.

We report on a method to reduce the number of state transition cycles that a molecule undergoes in far-field optical nanoscopy of the RESOLFT type, i.e. concepts relying on saturable (fluorescence) state transitions induced by a spatially modulated light pattern. The method is exemplified for stimulated emission depletion (STED) microscopy which uses stimulated emission to transiently switch off the capability of fluorophores to fluoresce. By switching fluorophores off only if there is an adjacent fluorescent feature to be recorded, the method reduces the number of state transitions as well as the average time a dye is forced to reside in an off-state. Thus, the photobleaching of the sample is reduced, while resolution and recording speed are preserved. The power of the method is exemplified by imaging immunolabeled glial cells with up to 8-fold reduced photobleaching.

Fluorescence nanoscopy of single DNA molecules by using stimulated emission depletion (STED).

Lens-based (far-field) fluorescence microscopy has played a key role in the life sciences, but for most of the time the resolution has been limited to about Δr=λ/(2ΝΑ)>200 nm, with λ denoting the wavelength of light and NA the numerical aperture of the lens. However, since the 1990s microscopy concepts have emerged providing diffraction-unlimited resolution by inhibiting the fluorescence of the dye such that features closer than the diffraction limit Δr are forced to fluoresce sequentially.[1, 2] Depending on how this fluorescence inhibition is implemented, the techniques broadly fall into two groups. In the group encompassing stimulated emission depletion (STED) microscopy,[2] the coordinate where the fluorophores are allowed to fluorescence is predetermined by a pattern of light in which the intensity reaches zero at a controllable position in space; in STED microscopy this light pattern typically has a doughnut shape. The second group of techniques enables the emission of fluorophores stochastically in space, such that just a single fluorophore is able to emit within a region of diameter Δr=λ/(2ΝΑ); the random emission coordinate is found by imaging the fluorescence with a camera, and then performing a centroid calculation.[3, 4] In both groups, images below the diffraction limit are obtained by consecutively allowing a representative number of dye molecules to fluoresce.[1] While most of these techniques have been applied to biological systems including DNA, high quality nanoscopy of DNA molecules has remained elusive.[5–7] This situation is unfortunate because many of DNAs functions, such as gene expression, are known to be regulated by bending, looping, supercoiling, and other conformational changes at subdiffraction length scales.[8] Many conformational changes of DNA appear in the range of 100–1000 basepairs, approximately 35–350 nm, with the persistence length of DNA (typically around 50 nm) defining a fundamental length scale. Additionally, to study the conformational changes and variations present in DNA, the given structure has to be not only uniformly labeled but also uniformly recorded. In particular, it is essential to be able to distinguish integral single strands of DNA from a strand that has been broken up into pieces or from multiple overlaid strands.[9] These requirements for far-field optical nanoscopy of DNA strands stained with standard intercalating dyes, such as YOYO-1 (YOYO), suggest that the deterministic nature of STED nanoscopy may have an inherent advantage over the stochastic approach termed stochastic ground-state depletion followed by individual molecular return (GSDIM, later also called dSTORM).[10–12] Whereas stochastic techniques rely quadratically on the number of photons to localize an emitter with increased resolution, in STED nanoscopy a few photons from the sample are sufficient to identify a molecule. Also, for the stochastic methods, the localization accuracy decreases for slightly defocused dyes with fixed dipole moment,[13] which could be relevant for YOYO molecules, the transition dipole moment of which is linked to the helical pitch of the DNA by intercalation.[14] Moreover, the depletion of the ground state underlying GSDIM entails pumping the dye to a more reactive state,[10–12] potentially harming or breaking the DNA strand (e.g. through electron transfer).[5–7] In contrast, STED is designed to disallow excited states, thus protecting the molecule from photoreactions.[15] Last but not least, to ensure that all but one of the fluorophores are transferred to a dark state within a diffraction limited volume in GSDIM, the dye concentration has to be matched to the lifetime of the dark-state. Fulfilling this condition is challenging because the dye can assume a wide range of dark states along the DNA strand, featuring a broad spectrum of lifetimes.[5–7] Not matching them, results in discontinuously imaged DNA strands and hence in unreliable information about DNA conformation. This problem is especially true for DNA bending and looping points, where nanoscale resolution is critical. For all these reasons, we decided to explore STED nanoscopy for imaging single DNA molecules. STED nanoscopy was performed by overlaying a pulsed excitation beam with a doughnut-shaped STED beam thus prohibiting the fluorescence of all the dye molecules exposed to the excitation light, except those lying within the center of the doughnut. Scanning the interlocked beams across the sample makes the object details fluoresce sequentially. Images were taken using two different pulsed wavelengths (568 nm and 647 nm) for STED. The asymmetrical dimeric cyanine dye, YOYO, is often used for single-molecule DNA studies owing to its brightness and its fluorescence enhancement (ca. 500-fold) upon DNA binding. On the other hand, intercalating cyanines tend to promote photodamage of the DNA–dye complex, manifested by elevated bleaching and breaking (photonicking) of the DNA. Photonicking can be drastically reduced by removing oxygen in the buffer but the effect of oxygen on photobleaching remains unclear, although oxidation of DNA basepairs is believed to contribute to the observed bleaching.[16] We found that adding β-mercaptoethanol (BME) was effective in preventing both photonicking and bleaching. In the STED recordings, photostability was found to be highest for 20–50 photon counts per pixel (pixel size ca. 25 nm) at a pixel dwell time of 100 μs. Using STED at 568 nm we obtain a five- to sixfold improvement in resolution over standard confocal microscopy (Figure 1) that in turn already provides a marked improvement in contrast and resolution over epifluorescence microscopy (Figure 2 c). In Figure 1, note the excellent correspondence of the variation in intensity along the DNA strands between the STED and confocal images. To explore the range of STED wavelengths that can be applied in our system we also used STED at 647 nm where the YOYO emission is a mere 3 % of its maximum. The result is a three- to fourfold improvement in resolution over standard confocal microscopy (Figure 2), thus demonstrating the applicability of STED over a range of over 80 nm. Kinks occur along DNA and can be sequence specific or due to the binding of proteins or small molecules. Figure 2 shows how STED, but not confocal microscopy can readily be used to identify these subtle structures along the DNA. Figure 1 a) Confocal image of YOYO stained λ-DNA (basepair:dye 5:1). b) The corresponding STED image taken with λSTED=568 nm (raw data). The STED image was acquired before the confocal counterpart. Scale bars: 1 μm. c) Average of three ... Figure 2 Typical raw STED images of YOYO stained λ-DNA (basepair:dye 5:1) using a) λSTED=568 nm and b) λSTED=647 nm. Scale bars in (a) and (b): 1 μm. c) Graph showing the average of 11 line profiles of a single DNA strand, with ... To investigate the photodamage inflicted by the STED beam on the DNA–dye complex (basepair:dye 5:1), a confocal image, an STED image (λSTED=568 nm), and then a confocal image were acquired one after another. While the second confocal image displayed a (50±9) % lower fluorescence level because of bleaching, photonicking was not observed, neither in the STED nor in the second confocal recording. Another series with three consecutive confocal images revealed a reduction of the fluorescence level by (34±16) %. Thus with STED the difference to photobleaching from standard confocal imaging is not significant. For details regarding imaging using a lower dye ratio (compatible with single-molecule investigations of DNA–enzyme interactions) see Supporting Information. In conclusion, we have demonstrated STED nanoscopy for DNA imaging at a resolution of approximately 45 nm, which is comparable to the persistence length, the fundamental length scale of the polymer physics of DNA. The variation in fluorescence signal over the DNA molecule corresponds well with that obtained by confocal microscopy, demonstrating the viability of STED for imaging single DNA molecules and a future potential use for comparison of signal variations caused by sequence specific dye binding or partial melting. The demonstrated combination of resolution and uniformity of imaging along the DNA strand is critical for visualizing small conformational changes as well as for optical mapping of DNA.[9] Importantly, STED can be applied over a relatively large wavelength range (at least 80 nm), with longer wavelengths being generally less prone to inducing photodamage, while still providing a marked resolution improvement. By employing molecular transitions between the two most basic states of a fluorophore, that is, the ground and the first electronically excited state, we anticipate STED will become the preferred optical pathway to exploring DNA at the molecular level.

4Pi microscopy with negligible sidelobes

The coherent addition of the wavefronts of two opposing high- angle lenses provides an axial (z) resolution improvement by 5-7-fold in far- field fluorescence microscopy. However, all microscopy concepts based on this principle have so far required mathematical deconvolution of the acquired data. This stems from the fact that the decrease of the axial width of the effective point spread function (EPSF) is accompanied by a substantial elevation of the side maxima of the EPSF along the optical axis. Here, we realize an EPSF with negligible lobes and gain axially superresolved images just through the physical phenomena involved. The constructive interference of the added wavefronts can be controlled through the image brightness which greatly simplifies the operation of the system.

Tissue multicolor STED nanoscopy of presynaptic proteins in the Calyx of Held.

The calyx of Held, a large glutamatergic terminal in the mammalian auditory brainstem has been extensively employed to study presynaptic structure and function in the central nervous system. Nevertheless, the nanoarchitecture of presynaptic proteins and subcellular components in the calyx terminal and its relation to functional properties of synaptic transmission is only poorly understood. Here, we use stimulated emission depletion (STED) nanoscopy of calyces in thin sections of aldehyde-fixed rat brain tissue to visualize immuno-labeled synaptic proteins including VGluT1, synaptophysin, Rab3A and synapsin with a lateral resolution of approximately 40 nm. Excitation multiplexing of suitable fluorescent dyes deciphered the spatial arrangement of the presynaptic phospho-protein synapsin relative to synaptic vesicles labeled with anti-VGluT1. Both predominantly occupied the same focal volume, yet may exist in exclusive domains containing either VGluT1 or synapsin immunoreactivity. While the latter have been observed with diffraction-limited fluorescence microscopy, STED microscopy for the first time revealed VGluT1-positive domains lacking synapsins. This observation supports the hypothesis that molecularly and structurally distinct synaptic vesicle pools operate in presynaptic nerve terminals.