Exploring optically active defects in wide-bandgap materials using fluorescence microscopy

Defects in solid-state systems can be both detrimental, deteriorating the quality of materials, or desired, thanks to the novel functionality they bring. Optically active point defects, producing fluorescent light, are a great example of the latter. Naturally existing in various materials, of which the so-called wide-bandgap materials constitute a major part, they can be used as sensors, single-photon emitters or even quantum bits. As the defects preferentially absorb only specific wavelengths of light, the whole material can acquire a visible macroscopic color, becoming the more intense the more there are defects in its lattice. Due to this fact such defects are commonly referred to as "color centers".The most famous example of color centers is the nitrogen-vacancy (NV) center in diamond, consisting of a nitrogen atom that substitutes carbon next to a vacancy (a missing carbon atom) in the diamond lattice. From the 1990s NV centers have been at the forefront of the second quantum revolution, enabling countless experimental demonstrations of quantum phenomena, even at room temperature. Being currently widely used everywhere from secured telecommunication networks to living cells, diamond NV centers have sparked a persistent interest into novel fluorescent defects, both in diamond (e.g. silicon-, germanium- and tin-vacancy centers) and in other wide-bandgap materials.Very recently a novel class of material platforms hosting fluorescent defects has emerged -- namely layered van der Waals (vdW) materials, which can be thinned down to an ultimate single-atom thickness, opening the door into the realm of two dimensional (2D) materials. This area of research has virtually exploded after the discovery of graphene in 2004, followed by continuous reports of the superb mechanical, electrical and optical properties of graphene-based devices. The whole family of graphene-like vdW materials was rapidly and continuously expanding with new members (graphene oxide, fluorographene, borophene, transition-metal dichalcogenides (TMDCs), layered perovskites, etc.), each of which was enabling various functionalities. This thesis explores the properties of newly discovered color centers in a layered vdW wide-bandgap semiconductor - hexagonal boron nitride (hBN). These optically-active defects have shown themselves as exceptionally bright single-photon emitters (SPEs) and optically-addressable spin defects that hold a great promise for quantum sensing and quantum information processing. In this work I have shown how optical super-resolution techniques (specifically, the single-molecule localization microscopy, SMLM) can be used to study the properties of emitters in hBN, including their spectra and temporal dynamics. By engineering a specialized waveguide-based imaging platform I managed to overcome certain limitations of SMLM-based imaging and further showed how the very same imaging platform can be used for the nanophotonic on-chip integration of hBN via direct growth. In addition, I have explored the behaviour of hBN defects in aqueous solutions and how there they can be used as nanoscale charge sensors, tracking the diffusion of single protons. Finally, I developed a novel method for the deterministic engineering of optically-active defects in hBN via focused ion beam (FIB) irradiation. All together these findings pave the way for the use of optically-active defects in hBN for applications in nanophotonics, nanofluidics and nanoscale sensing

Spin-valley optoelectronics based on two-dimensional materials

Dmitrii Unuchek

Two-dimensional (2D) materials, in particular graphene and transition metal dichalcogenides (TMDC), have attracted great scientific interest over the last decade, revealing exceptional mechanical, electrical and optical properties. Owing to their layered nature, subnanometer-thick single-layer forms of these crystals can be chemically grown or mechanically isolated, representing an ultimately scaled-down platform for further miniaturization of electronic devices. Indeed, the large family of 2D materials contains hundreds of members, including metals, semiconductors, insulators, and others, covering the whole spectrum of potential applications. Availability of all necessary building blocks for the construction of all-2D solid-state devices makes this platform ideal for fabrication of ultrathin, transparent and flexible devices. Extensively investigated graphene has demonstrated excellent electrical properties. However, the absence of a bandgap is an obstacle for its interaction with light and therefore limits applications in optics. In this aspect, atomically thin TMDC semiconductors appear to be an alternative, more promising platform, as they possess a direct band gap in the visible range in the monolayer form. Despite being only three atoms thick, these semiconductors demonstrate exceptionally large light absorption, efficient light emission, and strong light-matter interaction, attracting justified interest from the optics community. We will focus on their potential applications for optoelectronics in Chapter 4. Even more, broken inversion symmetry and strong spin-orbit coupling in single-layer TMDC crystals reveal a new quantum number, the so-called valley index. Indeed, this unique degree of freedom of charge carriers is known for some materials since the 1970s. However, in the case of 2D semiconductors, spin-valley locking mechanism and valley contrasting optical selection rules open new ways for addressing, manipulation and sensing of this pseudospin, opening the whole new field of valleytronics. Due to the large splitting of the valence band, spin and valley degree of freedom become locked in these atomically thin materials. We employ this unique feature in Chapter 5 for indirect injection of spins into graphene by pumping valley polarized carriers in an adjacent TMDC monolayer. Being gapless, graphene does not allow direct optical injection of spins. Another striking feature of 2D materials is their unique ability to be stacked in vertical heterostructures with strong electrical coupling between layers. The weak van der Waals force, which keeps components together, provides freedom in the assembly process, so that a lattice mismatch or a twist angle becomes unimportant in the first approximation. This provides a novel platform for harvesting complementary properties of the constituent materials, allowing the engineering of new artificial meta-materials as we demonstrate in Chapter 6. Furthermore, synergistic effects in van der Waals heterostructures enable completely new properties and phenomena, which do not exist in the single materials, with twist angle being an important knob for tuning these effects. We observe and exploit such novel phenomena arising in heterostructures of 2D semiconductors in the last chapter of this thesis. On the way to the realization of practical optoelectronic and valleytronic applications, this thesis studies fundamental aspects of rich spin-valley physics of atomically thin semiconductors

EPFL2019

Single molecule detection and fluorescence correlation spectroscopy on surfaces

Kai Hassler

In this thesis a new approach for single molecule detection and analysis is explored. This approach is based on the combination of two well established methods, fluorescence correlation spectroscopy (FCS) and total internal reflection fluorescence microscopy (TIRFM). In contrast to most existing fluorescence spectroscopy techniques, the subject of primary interest in FCS is not the fluorescence intensity itself but the random intensity fluctuation around the mean value. Intensity fluctuations are induced by thermal noise in a minute observation volume, which is in classical FCS the confocal volume of a confocal microscope. E.g. FCS is commonly utilized to investigate diffusion. In this case, diffusing fluorescent molecules entering or leaving the observation volume cause intensity fluctuations, which are analyzed by calculating the temporal autocorrelation of the observed signal. The autocorrelation is a measure for the self-similarity of a signal and contains information about the average fluctuation strength and duration. The confocal observation volume, i.e. the measurement volume that is actually seen by the detector is approximately given by the product of the optical transfer function with the fluorescence exciting intensity distribution of a focused laser beam. To achieve a high signal-to-background ratio a small observation volume is absolutely essential, first of all because the background from e.g. scattered light increases with the size of the observation volume. Second, a small volume assures for a small average number of fluorophores inside the observation volume and therefore for a high fluctuation amplitude i.e. FCS signal. This thesis proposes and discusses an alternative to confocal FCS specially conceived for measurements on surface-bound molecular systems, such as biological receptors or immobilized enzymes. In contrast to confocal FCS, fluorescence is excited within an evanescent field generated by total internal reflection (TIR) of a laser beam at the interface between a microscope coverslip and the sample. This is achieved by focusing the laser beam off-axis at the back focal plane of a high NA oil-immersion objective. The collimated beam that emerges from the objective is incident at an oblique angle at the coverslip-sample interface and totally internal reflected. In contrast to confocal FCS, the generated observation volume is completely confined to the surface and background fluorescence as well as scattered light from the bulk is efficiently suppressed. Our method, called objective-type TIR-FCS in the following, features an increased collection efficiency compared to existing techniques that combine evanescent wave excitation and FCS. Existing techniques use total internal reflection on the surface of a prism to generate an evanescent field. This leads to a configuration where the choice of objectives is limited to air or water-immersion objectives. In our system we use a high NA oil-immersion objective, specially conceived for TIR applications, which collects light efficiently. The collection efficiency is further enhanced by a naturally occurring change of the emission properties of fluorophores close to interfaces between dielectric media. The presence of the interface favors emission into the optically denser medium so that about 60% of the emitted light can be collected. These factors, together with a reduced observation volume lead to a very sensitive method with a high potential for applications in single molecule detection and analysis. The performance of the proposed method was experimentally shown for measurements on molecules subject to Brownian motion and binding to modified coverslips. In particular, it was experimentally shown that objective-type TIR-FCS features high signal-to-background ratio on a single molecule level. In this thesis, concise derivations of analytical expressions for autocorrelation functions for diffusion and most important, the case of ligands reversibly binding to a single and localized binding site are presented. The derived model allows for the quantitative determination of binding rates for a single receptor. We strongly believe that the application of these results in the context of investigations of receptor-ligand binding kinetics will allow for deeper understanding of cellular signaling. Moreover, this thesis discusses the applicability of the proposed method in enzymology. Enzymes, as most proteins are subject to continuous changes of their structure or conformation. These conformational changes are correlated with the function of the enzyme. In the discussed example the enzyme catalyzes an oxidation where the product is fluorescent but the substrate is not. The function of the enzyme i.e. the recurring product formation leads to observable intensity fluctuations. Since function and conformational states are correlated, conformational fluctuations can be investigated by means of FCS as was already shown for confocal FCS. A technique closely related to FCS is fluorescence lifetime spectroscopy (where lifetime refers to the mean lifetime of the electronic excited state). Whereas in FCS the relaxation after random deviations from thermal equilibrium is investigated, relaxation of excited fluorophores towards their electronic ground-state is investigated in lifetime spectroscopy. The technique is used for e.g. discrimination between fluorophores with different lifetimes. Lifetime spectroscopy combined with imaging is used in many domains of life-science, including microarray reading and medical diagnostics. In preliminary work we developed a novel approach to perform lifetime imaging that is based on a multiplexing technique. The proposed method requires no mechanical scanning stage and only a single-point detector. Furthermore, noise is reduced under certain circumstances if the signal is low. Characteristics of this technique as well as advantages and disadvantages are shortly discussed.

EPFL2006

Toxicity study of nanostructures

Lenke Horváth

The great achievement of nanotechnology is the controlled synthesis of a large variety of nanometer-size materials like needle-formed nanotubes, nanowires and two-dimensional graphene flakes. Due to their unique physico-chemical properties, these nanostructures are considered to be of great benefit for many applications in engineering, electronics, alternative energies and nanomedicine. Since the expectations for the improvement of our everyday life through engineered nanomaterials are high, there is rapid expansion of their manufacturing, which makes it likely that intentional and unintentional human and environmental exposure will increase in the near future. Consequently, the concern grows, related to their possible health hazards, as some of them strongly resemble asbestos. Motivated by this issue, we have investigated the in vitro acute cellular toxicity associated with four model nanomaterials: carbon nanotubes, boron nitride nanotubes, titanium dioxide nanofilaments and graphene oxide. This study focused on the toxic effect these nanomaterials had on cell types found in the respiratory system, where exposure to these materials is most prominent. These are lung epithelial cells, macrophages, and fibroblasts, but also other cell types like kidney cells were tested. The cytotoxicity was assessed by using MTT, DNA and FMCA assays, which measure different endpoints such as metabolic activity, cell proliferation and viable cell number. The cell death was determined by the Annexin V assay. We employed various microscopic techniques: light microscopy to reveal the morphological alterations associated with the nanomaterial toxicity at the cellular level; scanning electron microscopy to study the cell-nanomaterial interactions on the surface of the cell membrane; transmission electron microscopy to examine the uptake and subsequent localization of the nanomaterials within the cytosol and cell organelles. In addition, the generation of intracellular reactive oxygen species induced by graphene oxide was detected by the DCF assay. Last but not least, the pro-inflammatory potential and the biochemical perturbations in cells exposed to boron nitride nanotubes were investigated by Western blot and Synchrotron Infrared Microspectroscopy (SIRMS), respectively. All these techniques point to the adverse effects of the investigated nanostructures: i) The toxic effect of carbon nanotubes and carbon nanoparticles showed a time- and dose-dependent impairment in the metabolic activity of the cells characteristic for each cell type. Moreover, distinct morphological alterations typical for cell death were particularly apparent in macrophages. ii) The toxic potential of boron nitride nanotubes exhibited more pronounced adverse effects than carbon nanotubes. This was demonstrated by cell viability assays combined with cytopathological and biochemical analyses, showing induction of serious morphological changes, particularly in macrophages and fibroblasts, and biochemical processes characteristic for cell death. A higher acute toxicity was determined for boron nitride nanotubes when compared to crocidolite asbestos and their pro-inflammatory potential was demonstrated by the secretion of mature IL-1β cytokine in macrophages. iii) Titanium dioxide nanofilaments were also shown to impair the metabolic activity of the studied cells and induce morphological changes pointing to cell insult. iv) Graphene oxide exhibited a mild cytotoxic action in comparison to carbon nanotubes on epithelial cells and macrophages. The interaction of the nanomaterial with the cell surface generated reactive oxygen species during the initial phase of the exposure and transmission electron microscopy showed that graphene oxide flakes are taken up via the endocytic pathway. In summary, our findings highlight important physico-chemical parameters, which are important in relation to the toxic effect of nanomaterials. These are: i) the chemical composition; ii) the surface modification including functionalized groups and structural defects; iii) the geometry: length, diameter and tortuosity. In addition to the identified nanomaterial characteristics, we pinpoint that the target cells ́ response depends on their type, which is likely to be linked to their physiological function.

EPFL2012