Constructing an Autocorrelation System to Identify Single Nitrogen Vacancy Centers in Diamond

The nitrogen-vacancy (NV) center is a defect in the structure of diamond, roughly the size of a single atom, that possesses properties that make it applicable to quantum information processing, high-resolution magnetic resonance imaging, and probing biological systems. When an NV center is excited by green light of wavelength 523 nm, it emits red light of in the range from 600-800 nm tens of nanoseconds later. A confocal microscope can be used to excite and collect emitted photons from NV centers. By detecting the intensity of the emitted red photons, we can determine the quantum state of a single NV center, which is needed to build various quantum computing, sensing and imaging devices. The autocorrelation method is used to detect antibunching in the emission of red photons and ascertain that the observed photons are being emitted from a single NV center. We have built an autocorrelation device that measures, with sub-nanosecond accuracy, the statistical distribution of the arrival times of the red photons emitted from a diamond sample at a photon counter. The device can measure fixed time values with a time resolution of 120 ps FWHM. For uncorrelated sources measured for 120 seconds at the TAC full range of 100 ns, the average counts in each histogram bin were about 0.01% of the predicted value. The autocorrelation device that we have built costs about 10 times less than commercial units used by other NV researchers

A fluorescent nanodiamond foundation for quantum sensing in cells

Free radicals play a major role in the aging process as well as a most diseases. However, we barely know anything about them. These tiny molecules have an extremely short lifespan and are difficult to measure, while their role in health related processes is considerable. Fluorescent nanodiamonds are very small diamonds which can shed a light on this research question. These diamonds emit a constant light in a controlled setting. This is possible due to a small defect in the structure of the diamond, which makes it fluorescent. When free radicals are present, the light changes, which allows measurement of the radicals. During my PhD I have laid the basis for these biological measurements. Not all cells automatically take up diamonds or the diamonds tend to aggregate in cellular medium. By changing the solutions in which we administer the diamonds, we can prevent these obstacles. I have also performed a very detailed analysis of the cellular response on diamond uptake. Conveniently, the cells hardly show any response to the diamond uptake, an important result for our future measurements. In addition, I have developed new ways of targeting the diamonds to specific places in the cells, to obtain location specific information. Finally I have determined the subcellular location of the diamonds using a new technique, based on integrated electron microscopy. During my work I have laid the foundation for promising cellular research of ageing and disease using fluorescent nanodiamonds

Novel technologies for studies of structural and functional connections

To understand the mechanisms underlying the correct functioning of an organ it is important to study its architecture and how the interactions between cells are leading to a specific function. Specifically, the connections that form in the brain are related to the pattern of activation that neurons have, and can help to understand what is the function of each region. Combining the structural knowledge with functional studies is crucial to understand how the cells communicate and propagate the depolarization. Another way to understand the mechanisms underlying tissue functionality is to try to replicate its features and inspect if the resulting behavior is similar to the original one. In this thesis I am describing different tools that we developed to inspect the cells connectivity from the architectural and functional point of view, focusing on imaging, analytic and engineering techniques. To inspect the connection within the brain, in paper I we developed a microscopy system capable of performing fast volumetric imaging of large cleared samples (called LSTM, Light Sheet Theta Microscopy). LSTM is built upon the LSM (Light Sheet Microscopy) system, but instead of illuminating the sample from the sides –which leads to a physical constrain to the samples lateral dimension or depth– the light sheet is scanned on the imaging plane from an angle smaller than 90°. Therefore this approach eliminates the constraints on the lateral size without compromising the image quality and speed. Furthermore, it overcomes the LSM limitation that leads to huge scattering on the center part of the sample. In fact, LSTM images each plane with the same intensity leading to homogeneous x-y acquisition throughout the whole dept. This system can help to create maps of long ranging connections of neurons of intact rodents organs (eg brain) and can theoretically be used to acquire un entire human brain, slab by slab, in a reasonable amount of time. In paper II we propose a tool to inspect the evolution of living cultures for an extended period of time. To do so, we developed a mini-microscope to be placed in the incubator that performs long lasting recordings and automatically detects the Regions Of Interest (ROI), calculates the intensity profiles, and compresses the data after every time-point. This system (called XDscope) is designed to limit the user interaction with the culture, minimize the light exposure and to ease the process of getting the desired information out of the experiment and store as little data as possible. Using the XDscope we performed long term monitoring of GCaMP6 expressing neurosphere (NSP) networks for over 2 weeks, showing that the cells behavior is not affected by the long acquisition. Furthermore we used the system to evaluate the uptake mechanism of p-HTMI, an LCO (Luminescent Conjugated Oligothiophene) over the NSP network, showing that the targeted cells are progenitor cells as expected, since the fluorescent cells are mainly located around the spheres. Finally we investigated further the specific target of p-HTMI within the cells performing double labeling with proteins that seemed to be in the targeted area. From the results it seems like GM130/Golga2, a protein that facilitates the transportation between ER and Golgi apparatus has a high percentage of overlap with the molecule. Finally in paper III we tried to mimic the features and the cell spatial arrangement of a leaving tissue to infer similar properties to an engineered construct. We propose an innovative strategy to integrate a patterned gold microelectrode into a flexible biomimetic hybrid actuator with double muscle-like patterned layers made using PEG (Polyethylene Glycol) and CNT-GelMA (Carbon Nano Tubes- Gelatin Methacryloyl). The CNT-GelMA patterned layer acted as a substrate for cell culture to induce maturation of cardiac muscle cells, while the PEG layer acts as the backbone of the whole membrane. The resulting muscle-like biohybrid actuator showed excellent mechanical integrity with an inserted Au microelectrode and advanced electrophysiological functions with strong muscle contractions. Therefore, we successfully fabricated a biomimetic hybrid actuator with muscle-like pattern, and controllable movement under an electrical field produced by integrated electrodes

Calibrating evanescent-wave penetration depths for biological TIRF microscopy

Roughly half of a cells proteins are located at or near the plasma membrane. In this restricted space the cell senses its environment, signals to its neighbors and ex-changes cargo through exo- and endocytotic mechanisms. Ligands bind to receptors, ions flow across channel pores, and transmitters and metabolites are transported against con-centration gradients. Receptors, ion channels, pumps and transporters are the molecular substrates of these biological processes and they constitute important targets for drug discovery. Total internal reflection fluorescence microscopy suppresses background from cell deeper layers and provides contrast for selectively imaging dynamic processes near the basal membrane of live-cells. The optical sectioning of total internal reflection fluorescence is based on the excitation confinement of the evanescent wave generated at the glass-cell interface. How deep the excitation light actually penetrates the sample is difficult to know, making the quantitative interpretation of total internal reflection fluorescence data problematic. Nevertheless, many applications like super-resolution microscopy, colocalization, fluorescence recovery after photobleaching, near-membrane fluorescence recovery after photobleaching, uncaging or photo-activation-switching, as well as single-particle tracking require the quantitative interpretation of evanescent-wave excited images. Here, we review existing techniques for characterizing evanescent fields and we provide a roadmap for comparing total internal reflection fluorescence data across images, experiments, and laboratories.Comment: 18 text pages, 7 figures and one supplemental figur

Single NV in nanodiamond for quantum sensing of protein dynamics in an ABEL trap

Enzymes are cellular protein machines using a variety of conformational changes to power fast biochemical catalysis. Our goal is to exploit the single-spin properties of the luminescent NV (nitrogen-vacancy) center in nanodiamonds to reveal the dynamics of an active enzyme complex at physiological conditions with the highest spatio-temporal resolution. Specifically attached to the membrane enzyme FoF1-ATP synthase, the NV sensor will report the adenosine triphosphate (ATP)-driven full rotation of Fo motor subunits in ten consecutive 36{\deg} steps. Conformational dynamics are monitored using either a double electron-electron resonance scheme or NV- magnetometry with optical readout or using NV- relaxometry with a superparamagnetic nanoparticle as the second marker attached to the same enzyme. First, we show how all photophysical parameters like individual size, charge, brightness, spectral range of fluorescence and fluorescence lifetime can be determined for the NV- center in a single nanodiamond held in aqueous solution by a confocal anti-Brownian electrokinetic trap (ABEL trap). Stable photon count rates of individual nanodiamonds and the absence of blinking allow for observation times of single nanodiamonds in solution exceeding hundreds of seconds. For the proposed quantum sensing of nanometer-sized distance changes within an active enzyme, we show that local magnetic field fluctuations can be detected all-optically by analyzing fluorescence lifetime changes of the NV- center in each nanodiamond in solution.Comment: 14 pages, 5 figure

Nitrogen-Vacancy Centers in Diamond for Nanoscale Magnetic Resonance Imaging Applications

The nitrogen-vacancy (NV) center is a point defect in diamond with unique properties for use in ultra-sensitive, high-resolution magnetometry. One of the most interesting and challenging applications is nanoscale magnetic resonance imaging (nano-MRI). While many review papers have covered other NV centers in diamond applications, there is no survey targeting the specific development of nano-MRI devices based on NV centers in diamond. Several different nano-MRI methods based on NV centers have been proposed with the goal of improving the spatial and temporal resolution, but without any coordinated effort. After summarizing the main NV magnetic imaging methods, this review presents a survey of the latest advances in NV center nano-MRI

Nitrogen-Vacancy Centers in Diamond for Nanoscale Magnetic Resonance Imaging Applications

The nitrogen-vacancy (NV) center is a point defect in diamond with unique properties for use in ultra-sensitive, high-resolution magnetometry. One of the most interesting and challenging applications is nanoscale magnetic resonance imaging (nano-MRI). While many review papers have covered other NV centers in diamond applications, there is no survey targeting the specific development of nano-MRI devices based on NV centers in diamond. Several different nano-MRI methods based on NV centers have been proposed with the goal of improving the spatial and temporal resolution, but without any coordinated effort. After summarizing the main NV magnetic imaging methods, this review presents a survey of the latest advances in NV center nano-MRI

Optimized, versatile diamond-based sensors : materials, fabrication and novel applications

Quantum sensing as one of the backbones of the second quantum revolution is about to enable a variety of novel applications requiring good spatial resolution and sensitivity. The atomic-sized, negatively charged nitrogen vacancy (NV) color center in single crystal diamond was found to enable magnetic field sensing at the nanoscale. Magnetic sensing using NV centers is enabled by bright photostable emission and optically addressable spin states. Due to its extraordinary coherence time, sensitivities of few nT\Hz^(1/2) can be achieved under ambient conditions. To enhance the spatial resolution of NV-based sensing, it is necessary to approach the NV center to a sample to investigate. Here, a challenging nanofabrication procedure is needed to sculpt the diamond into a photonic nanostructure usable as a scanning probe tip. In this thesis, we report on the progress towards optimizing the applicability of NV centers as quantum sensors. We investigate novel material systems promising for upscaling nanofabrication. By introducing a novel approach to enhance the adhesion of etch masks and novel plasma treatments, we optimize the reliability of the nanofabrication procedure. In addition, we study a novel near-field interaction-based sensing resource. By investigating the interaction of shallow NV centers with a monolayer of WSe2, we were able to show simultaneous near-field and magnetic field sensing using the NV center.Als eine der Säulen der zweiten Quanten-Revolution ermöglicht die Quantensensorik viele neue Anwendungen, die eine gute Ortsau ösung und Sensitivität benötigen. Das atomar kleine, negativ geladene Stickstoff-Fehlstellen (NV) Farbzentrum in einkristallinem Diamant ermöglicht das Detektieren von Magnetfeldern auf Nanomaÿstäben. Magnetfelddetektion mittels NV Zentren wird durch helle, photostabile Emission und optisch adressierbare Spin-Zustände ermöglicht. Aufgrund seiner auÿergewöhnlichen Kohärenzzeit erreicht es Sensitivitäten von einigen nT/Hz^(1/2) unter Umgebungsbedingungen. Zur Verbesserung der Ortsauflösung NV-basierter Sensorik, muss das NV-Zentrum an die zu untersuchende Probe angenähert werden. Dies erfordert einen herausfordernden Nanfabrikationsprozess, um den Diamanten in eine photonische Struktur zu formen, die als Rastersonde nutzbar ist. Diese Arbeit beschreibt Fortschritte zur Optimierung der Anwendbarkeit von NV-Zentren als Quantensensoren. Wir untersuchen neuartige Materialien, die vielversprechend für die Skalierbarkeit des Prozesses sind. Durch neue Ansätze zur Verbesserung der Adhäsion von Ätzmasken und neue Plasmabehandlungen optimieren wir die Zuverlässigkeit der Nanofabrikation. Zudem analysieren wir einen neuen, auf Nahfeldwechselwirkung beruhenden Sensorikansatz. Bei der Untersuchung der Wechselwirkung von oberflächennahen NV-Zentren mit monolagigem WSe2 konnten wir das gleichzeitige Erfassen von Nah- und magnetischen Feldern mittels NV-Zentren zeigen