Graphene setting the stage: tracking DNA hybridization with nanoscale resolution

In this study we use nanophotonic effects of graphene to study DNA hybridization: the z−4 nanoscale distance-dependence of the fluorescence lifetime for fluorophores located in the vicinity of graphene is for the first time used to track a DNA hybridization reaction with nanoscale resolution in real time. First, a nanostaircase with ≈2 nm steps from 0 to a total height of 48 nm is used as a nanoruler to confirm the distance dependence law. We find that the axial sensitivity is suited to determine the nanoscale surface roughness of these samples. The proof-of-concept DNA experiments in aqueous medium involve the hybridization of fluorescently labelled DNA beacons attached to CVD grown graphene with complementary (target) DNA added in solution. We track the conformational changes of the beacons statistically by determining the fluorescence lifetimes of the labelling dye and converting them into nanoscale distances from the graphene. In this way, we are able to monitor the vertical displacement of the label during DNA-beacon unfolding with an axial resolution reaching down to 1 nm. The measured distance increase during the DNA hybridization reaction of about 10 nm matches the length of the target DNA strand. Furthermore, the width of the fluorescence lifetime distributions could be used to estimate the molecular tilt angle of the hybridized ds-DNA configuration. The achieved nanoscale sensitivity opens innovation opportunities in material engineering, genetics, biochemistry and medicine.INL received support for this project from the CCDR-N via the project 'Nanotechnology based functional solutions' (Grant No. NORTE-01-0145-FEDER-000019) and from the Portuguese Foundation for Science and Technology (FCT) via the project 'ON4SupremeSens' PTDC/NAN-OPT/29417/2017. Edite Figueiras received a Marie Curie fellowship via the EU-EC COFUND program 'NanoTRAINforGrowth' (Grant No. 600375). U Minho research was partially supported by the FCT in the framework of the Strategic Funding UID/FIS/04650/2013

Computational cytometer based on magnetically modulated coherent imaging and deep learning.

Detecting rare cells within blood has numerous applications in disease diagnostics. Existing rare cell detection techniques are typically hindered by their high cost and low throughput. Here, we present a computational cytometer based on magnetically modulated lensless speckle imaging, which introduces oscillatory motion to the magnetic-bead-conjugated rare cells of interest through a periodic magnetic force and uses lensless time-resolved holographic speckle imaging to rapidly detect the target cells in three dimensions (3D). In addition to using cell-specific antibodies to magnetically label target cells, detection specificity is further enhanced through a deep-learning-based classifier that is based on a densely connected pseudo-3D convolutional neural network (P3D CNN), which automatically detects rare cells of interest based on their spatio-temporal features under a controlled magnetic force. To demonstrate the performance of this technique, we built a high-throughput, compact and cost-effective prototype for detecting MCF7 cancer cells spiked in whole blood samples. Through serial dilution experiments, we quantified the limit of detection (LoD) as 10 cells per millilitre of whole blood, which could be further improved through multiplexing parallel imaging channels within the same instrument. This compact, cost-effective and high-throughput computational cytometer can potentially be used for rare cell detection and quantification in bodily fluids for a variety of biomedical applications

Plasmonic waveguides self-assembled on DNA origami templates: from synthesis to near-field characterizations

Manipulating light by controlling surface plasmons on metals is being discussed as a means for bridging the size gap between micrometer-sized photonic circuits and nanometer-sized integrated electronics. Plasmonic waveguides based on metal nanoparticles are of particular interest for circumventing the diffraction limit, thereby enabling high-speed communication over short-range distances in miniaturized micro-components. However, scalable, inexpensive fine-tuning of particle assemblies remains a challenge and near-field probing is required to reveal plasmonic interactions. In this thesis, self-assembled waveguides should be produced on DNA scaffolds. DNA origami is an extremely versatile and robust self-assembly method which allows scalable production of nanostructures with a fine control of assemblies at the nanoscale. To form the plasmonic waveguides, six-helix bundle DNA origami nanotubes are used as templates for attachment of highly monodisperse and monocrystalline gold nanoparticles with an inter-particle distance of 1-2 nm. In the first part of this thesis, the effects of parameters which are involved in assembly reactions are systematically investigated. The assembly yield and binding occupancy of the gold nanoparticles are determined by an automated, high-throughput image analysis of electron micrographs of the formed complexes. As a result, unprecedented binding site occupancy and assembly yield are achieved with the optimized synthesis protocol. In addition, waveguides with different sizes of gold nanoparticles and different inter-particle distances, quantum dots attachments to the waveguides and multimerization of the waveguides are successfully realized. In the second part of this thesis, direct observation of energy transport through a self-assembled waveguide towards a fluorescent nanodiamond is demonstrated. High-resolution, near-field mapping of the waveguides are studied by electron energy loss spectroscopy and cathodoluminescence imaging spectroscopy. The experimental and simulation results reveal that energy propagation through the waveguides is enabled by coupled surface plasmon modes. These surface plasmon modes are probed at high spatial and spectral resolutions. The scalable self-assembly approach presented here will enable the construction of complex, sub diffraction plasmonic devices for applications in high-speed optical data transmission, quantum information technology, and sensing.Die Manipulation des Lichts durch die Kontrolle von Oberflächenplasmonen auf metallischen Oberflächen und Nanopartikeln gilt als vielversprechende Methode zur Überbrückung der Größen-Lücke zwischen Mikrometer-großen photonischen und nanometer-großen elektronischen Schaltkreisen. Plasmonische Wellenleiter basierend auf metallischen Nanopartikeln sind vom besonderen Interesse, da sie die Umgehung des Beugungslimits und somit eine Hochgeschwindigkeitskommunikation über kurze Distanzen in immer kleiner werdenden Schaltkreisen ermöglichen könnten. Allerdings ist die skalierbare und kostengünstige Anordnung von Partikeln eine große Herausforderung und es werden Nahfelduntersuchungen benötigt um plasmonische Interaktionen detektieren zu können. Das Ziel dieser Arbeit ist die Selbstassemblierung von multi-partikel Wellenleitern auf DNA Gerüsten. Die Verwendung von DNA-Origami bietet eine äußerst vielseitige Plattform zur skalierbaren Herstellung von Nanostrukturen mittels Selbstassemblierung und ermöglicht eine präzise Kontrolle der Anordnungen im Nanobereich. Für den Aufbau der plasmonischen Wellenleiter werden DNA-Origami Nanoröhren, bestehend aus sechs Helices als Templat für die Anbindung von monodispersen und monokristallinen Goldnanopartikeln mit einem interpartikulären Abstand von 1-2 nm verwendet. Im ersten Abschnitt dieser Arbeit werden die beeinflussenden Faktoren dieser Assemblierungsreaktion systematisch untersucht. Die Ausbeute der assemblierten Strukturen und die Besetzung der Bindungsstellen werden durch eine automatisierte und effiziente Bildanalyse von Elektronenmikroskopieaufnahmen ausgewertet. Durch die Entwicklung eines optimierten Syntheseprotokolls werden bisher unerreichte Assemblierungsausbeuten ermöglicht. Zusätzlich erfolgen die experimentelle Realisierung von Strukturen mit verschieden großen Goldnanopartikeln und unterschiedlichen interpartikulären Abständen, sowie die Anbindung von Quantenpunkten an die Wellenleiter und eine Verknüpfung der assemblierten Strukturen. Der zweite Abschnitt dieser Dissertation befasst sich mit der Untersuchung des Energietransports in selbstassemblierten Wellenleitern über einen fluoreszierenden Nanodiamanten. Dazu erfolgen hochaufgelöste Nahfeldmessungen der Wellenleiter mittels Elektronenenergieverlustspektroskopie und Kathodolumineszenz-mikroskopie. Die experimentellen Ergebnisse und zusätzlich durchgeführte Simulationen bestätigen eine durch gekoppelte Oberflächenplasmonenmoden induzierte Weitergabe der Energie innerhalb des Wellenleiters. Diese Oberflächenplasmonenmoden werden bei hoher räumlicher und spektraler Auflösung untersucht. Das hier umgesetzte Konzept der Selbstassemblierung wird den Aufbau komplexer plasmonischer Geräte für Anwendungen im Bereich der optischen Hochgeschwindigkeitsdatenübertragung, der Quanteninformations-technolgie und der Sensorik ermöglichen

Advances in High-Throughput Analysis: Automated Radiochemical Separations and Nanopillar based Separations and Field Enhanced Spectroscopy

Often the need to analyze a large number of samples coincide with critical time consternates. At such times, the implementation of high-throughput technologies is paramount. In this work we explore some viable pathways for high-throughput analysis and develop advancements in novel forms of detection of materials that are vital in the environmental, biological as well as national security arenas. Through the use of new protocols with high sensitivity and specificity as well as simplified chemical processing and sample preparation we aim to allow for improved throughput, fieldable detection, and rapid data acquisition of extensive sample sets. The methods developed in this work focus on unique platforms of the collection and analysis and combine them with automation and portability. Foremost, analytes of interest must be selectively isolated and concentrated by chemical and/or mechanical processes. Secondly, spectroscopic and physical properties are exploited and enhanced by employing viable detection platforms. Finally, automation and field portability are implemented through a combination of optimized robotics, minimized chemical preparation and/or unique lab on a chip type platforms. Presented are two sub areas of research. One focuses on the automation of a time consuming solid phase extraction process that is coupled to inductively coupled plasma mass spectrometry increasing sample throughput by orders of magnitude. The second focused on the fabrication and use of silicon nanopillars as a platform for separations and enhanced optical analysis. Each section of work focuses on the development of a practical, accessible, and deployable methods of analysis

Beating the reaction limits of biosensor sensitivity with dynamic tracking of single binding events

The clinical need for ultrasensitive molecular analysis has motivated the development of several endpoint-assay technologies capable of single-molecule readout. These endpoint assays are now primarily limited by the affinity and specificity of the molecular-recognition agents for the analyte of interest. In contrast, a kinetic assay with single-molecule readout could distinguish between low-abundance, high-affinity (specific analyte) and high-abundance, low-affinity (nonspecific background) binding by measuring the duration of individual binding events at equilibrium. Here, we describe such a kinetic assay, in which individual binding events are detected and monitored during sample incubation. This method uses plasmonic gold nanorods and interferometric reflectance imaging to detect thousands of individual binding events across a multiplex solid-phase sensor with a large area approaching that of leading bead-based endpoint-assay technologies. A dynamic tracking procedure is used to measure the duration of each event. From this, the total rates of binding and debinding as well as the distribution of binding-event durations are determined. We observe a limit of detection of 19 fM for a proof-of-concept synthetic DNA analyte in a 12-plex assay format.First author draf

Unraveling nanoscale alterations in liver cell fenestrations - Morphological studies via optical super-resolution microscopy approaches

The endothelium makes up the innermost cell layer of blood vessels. It consists of a thin layer of simple squamous cells, forming an interface between circulating blood and the surrounding tissue. Endothelial cells of different vascular beds are specialized according to tissue-specific functions. For this project emphasis was placed upon high-resolution methods enabling the study of liver sinusoidal endothelial cells (LSECs) below the diffraction limit of visible light (~200 nm). LSECs have unusual morphology with as much of 20% of their surface covered with cellular fenestrations - holes through the cells of 50-300 nm diameter. These allow bi-directional flow of plasma from the sinusoids to the surrounding hepatocytes, while retaining blood cells in the sinusoidal lumen. Little is known about the function of fenestrations, their regulation, and their role in the transfer of metabolites, viruses, lipoproteins and pharmaceuticals to other cells of the liver. There are two major challenges with the study of LSEC fenestrations; i) the majority have diameters smaller than the diffraction limit of visible light and; ii) they disappear rapidly in cultured LSEC, and there are no cell line alternatives that express fenestrations. To address the first challenge, the project used classical super resolution imaging technologies such as scanning electron microscopy, and two novel super-resolution optical microscopy modalities: dSTORM (direct stochastic optical reconstruction microscopy) and SIM (structured illumination microscopy) to study the in vitro effects of xanthines, sildenafil and oxidized LDL on LSEC fenestrations. One of the xanthines, theobromine, and sildenafil increased both the frequency and diameter of fenestrations in cultured LSEC. While oxidized LDL caused major disruptions in LSEC fenestration morphology. Finally, to address the second challenge, namely the rapid loss of fenestrations in LSEC, a cryopreservation method for freshly isolated LSEC was developed such that they can be used at researchers’ convenience, rather than directly after isolation from liv

A novel combined Structured Illumination and Single Molecule Localization Microscope and its application to Retinal Structures

Fluorescence microscopy methods have become an major imaging tool in biomedical lifescience. However, each method only addresses specific questions due to intrinsic limitations. A new, fully automated and user-friendly 'Combo'-microscope setup has been developed, which combines the two advanced high-resolution methods Structured Illumination and Localization Microscopy into one imaging system. As both methods complement one another, the 'Combo'-microscope will greatly extend the range of application in biomedical research. Moreover, artifacts, introduced in the course of the imaging and/or reconstruction processes, can be revealed and potential mis-interpretation of super-resolution data is limited. This work was motivated by the age-related macular degeneration (AMD), a disease, which is the leading cause of blindness in the Western world. Autofluorescent particles within age-related deposits (drusen) beneath the retinal pigment epithelium are studied on advanced resolution level. Furthermore, the newly built microscope is used for a dual-mode dual-color three-dimensional visualization of the axon initial segment, a crucial region for signal transduction in vision, of retinal ganglion cells. Finally, an in vitro study comparing the pharmaceuticals currently used for AMD-treatment is outlined

Molecular coordination of Staphylococcus aureus cell division

The bacterial cell wall is essential for viability, but despite its ability to withstand internal turgor must remain dynamic to permit growth and division. Peptidoglycan is the major cell wall structural polymer, whose synthesis requires multiple interacting components. The human pathogenStaphylococcus aureusis a prolate spheroid that divides in three orthogonal planes. Here, we have integrated cellular morphology during division with molecular level resolution imaging of peptidoglycan synthesis and the components responsible. Synthesis occurs across the developing septal surface in a diffuse pattern, a necessity of the observed septal geometry, that is matched by variegated division component distribution. Synthesis continues after septal annulus completion, where the core division component FtsZ remains. The novel molecular level information requires re-evaluation of the growth and division processes leading to a new conceptual model, whereby the cell cycle is expedited by a set of functionally connected but not regularly distributed components

Tracking neuronal motility in live murine retinal explants

The developing retina undergoes dynamic organizational changes involving significant intra-retinal motility of the encompassing cells. Here, we present a protocol for tracking retinal cell motility in live explanted mouse retinae. Although originally applied to rod and cone photoreceptors, this strategy is applicable to any fluorescently labeled cell in mouse retinae and other similar experimental retinal models. Careful tissue handling is critical for the successful acquisition of high-quality live imaging data. Further instructions for semi-automated in silico data handling are provided. For complete details on the use and execution of this protocol, please refer to Aghaizu et al. (2021)