Olaf Schulz

Aqueous synthesis of zinc blende CdTe/CdS magic-core/thick-shell tetrahedral-shaped nanocrystals with emission tunable to near-infrared.

We demonstrate the synthesis of near-IR-emitting zinc blende CdTe/CdS tetrahedral-shaped nanocrystals with a magic-sized (approximately 0.8 nm radius) CdTe core and a thick CdS shell (up to 5 nm). These high-quality water-soluble nanocrystals were obtained by a simple but reliable aqueous method at low temperature. During the growth of the shell over the magic core, the core/shell nanocrystals change from type I to type II, as revealed by their enormous photoluminescence (PL) emission peak shift (from 480 to 820 nm) and significant increase in PL lifetime (from approximately 1 to approximately 245 ns). These thick-shell nanocrystals have a high PL quantum yield, high photostability, compact size (hydrodynamic diameter less than 11.0 nm), and reduced blinking behavior. The magic-core/thick-shell nanocrystals may represent an important step toward the synthesis and application of next-generation colloidal nanocrystals from solar cell conversion to intracellular imaging.

Resolution doubling in fluorescence microscopy with confocal spinning-disk image scanning microscopy

Significance Fluorescence microscopy is an enormously powerful tool for investigating structural organization and dynamical processes on the cellular level because of its noninvasiveness, sensitivity, and specificity. However, classical fluorescence microscopy is limited in resolution by the diffraction of light. In recent years, structural illumination microscopy has succeeded in doubling this resolution without requiring any special sample preparation or labeling dyes. However, it is technically very challenging and complex. Here, we present an alternative that achieves the same resolution enhancement by using a standard confocal spinning-disk microscope with minimal modifications. This method, in principle, allows one to double the resolution of any existing confocal microscope. We demonstrate how a conventional confocal spinning-disk (CSD) microscope can be converted into a doubly resolving image scanning microscopy (ISM) system without changing any part of its optical or mechanical elements. Making use of the intrinsic properties of a CSD microscope, we illuminate stroboscopically, generating an array of excitation foci that are moved across the sample by varying the phase between stroboscopic excitation and rotation of the spinning disk. ISM then generates an image with nearly doubled resolution. Using conventional fluorophores, we have imaged single nuclear pore complexes in the nuclear membrane and aggregates of GFP-conjugated Tau protein in three dimensions. Multicolor ISM was shown on cytoskeletal-associated structural proteins and on 3D four-color images including MitoTracker and Hoechst staining. The simple adaptation of conventional CSD equipment allows superresolution investigations of a broad variety of cell biological questions.

Tip induced fluorescence quenching for nanometer optical and topographical resolution

Progress in nanosciences and life sciences is closely related to developments of high resolution imaging techniques. We introduce a technique which produces correlated topography and fluorescence lifetime images with nanometer resolution. Spot sizes below 5 nm are achieved by quenching of the fluorescence with silicon probes of an atomic force microscope which is combined and synchronized with a confocal fluorescence lifetime microscope. Moreover, we demonstrate the ability to locate and resolve the position of two fluorescent molecules separated by 20.7 nm on a DNA origami triangle with 120 nm side length by correlating topography and fluorescence data. With this method, we anticipate applications in nano- and life sciences, such as the determination of the structure of macromolecular assemblies on surfaces, molecular interactions, as well as the structure and function of nanomaterials.

Simultaneous single molecule atomic force and fluorescence lifetime imaging

The combination of atomic force microscopy (AFM) with single-molecule-sensitive confocal fluorescence microscopy enables a fascinating investigation into the structure, dynamics and interactions of single biomolecules or their assemblies. AFM reveals the structure of macromolecular complexes with nanometer resolution, while fluorescence can facilitate the identification of their constituent parts. In addition, nanophotonic effects, such as fluorescence quenching or enhancement due to the AFM tip, can be used to increase the optical resolution beyond the diffraction limit, thus enabling the identification of different fluorescence labels within a macromolecular complex. We present a novel setup consisting of two commercial, state-of-the-art microscopes. A sample scanning atomic force microscope is mounted onto an objective scanning confocal fluorescence lifetime microscope. The ability to move the sample and objective independently allows for precise alignment of AFM probe and laser focus with an accuracy down to a few nanometers. Time correlated single photon counting (TCSPC) gives us the opportunity to measure single-molecule fluorescence lifetimes. We will be able to study molecular complexes in the vicinity of an AFM probe on a level that has yet to be achieved. With this setup we simultaneously obtained single molecule sensitivity in the AFM topography and fluorescence lifetime imaging of YOYO-1 stained lambda-DNA samples and we showed silicon tip induced single molecule quenching on organic fluorophores.

Time-resolved single molecule microscopy coupled with atomic force microscopy

Time-resolved confocal microscopy is well established to image spectral and spatial properties of samples in biology and material science. Atomic Force Microscopy (AFM) in addition enables to investigate properties which are not optically addressable or are hidden by the diffraction limited optical resolution. We present a straight forward combination of single molecule sensitive time-resolved confocal microscopy with different commercially available AFMs. Besides an extra of information about for example a cell surface, the AFM tip can also be used to manipulate the sample on a nanometer scale down to the single molecule level.

Optimizing a Time-Resolved Spectrometer for All Time Scales

Time-resolved fluorescence spectroscopy is a spectroscopists most valuable tool for the investigation of excited state dynamics in molecules, complexes, or semi-conductors. In recent years, the study of luminescence properties has gained in popularity in many scientific fields, including Chemistry, Biology, Physics, as well as in Life, Material or Environmental Sciences. The investigations to be carried out in each of these fields impose different requirements. On one side, monitoring dynamic processes in the excited state necessitates high time resolution that can be achieved by fast pulsed lasers and detectors along with appropriate time-correlated single photon counting (TCSPC) units and small monochromators. On the other hand, high spectral resolution is desirable for fluorophore characterization, requiring detectors with high quantum efficiencies, flash lamps for phosphorescence measurements and large monochromators. Up to now, spectrometers have been usually targeted towards either one of these two specifications. Spectrometers equipped with hybrid detectors, versatile TCSPC cards with optional longer time ranges, and pulsed lasers capable of working in a burst mode can offer an combined solution, covering most of the demands of either high time or spectral resolution. We will demonstrate the performance of such a spectrometer in terms of its time resolution, the ability to measure long decays and record time-gated spectra using laser drivers with burst capabilities. This type of instrument is of great value for analytical facilities in research centers, as it offers a wide range of possible spectroscopic applications in a single, easy to use instrument.

Advanced Pulse Pattern Generation and Fine Tuning for STED Microscopy

Stimulated Emission Depletion (STED) microscopy has evolved into an established imaging method offering super-resolution well beyond 50 nm. Whereas STED is now available in many laboratories, it is still in the focus of research to push the boundaries of its capabilities and applications. Time-resolved STED microscopy using time correlated single photon counting (TCSPC), is advantageous for many applications and promises further development for increased resolution and less photo-damage.Here, we show the application of established methods (e.g. gSTED) as well as emerging applications of time-resolved STED. We employ pulsed interleaved excitation (PIE), where the STED laser is pulsed at half the frequency of the excitation laser, such that STED and confocal data is taken practically at the same time. By using this approach, single molecule STED experiments can be carried out while the confocal control-experiment is performed simultaneously, allowing to account for measurement artifacts due to the high power of the STED laser. We will show examples from single molecule imaging, where blinking and bleaching are monitored using the confocal data. Furthermore, we will present STED-FCS data, where the confocal data allows insight into changes of the sample due to the STED laser. Since the control experiment for the influence of the STED laser is performed at the same time as the STED measurement, experimental parameters can be adjusted online to give highest resolution while ascertaining that the relevant information drawn from the experiment is not affected.Furthermore, we will present how electronically delaying the STED laser with respect to the excitation laser can increase resolution with no increase in photo-bleaching. By setting the arrival of the STED laser with an accuracy of about 20 ps, experimental conditions for fluorophores with different fluorescence lifetimes can be adjusted.