Beyond the diffraction limit of optical/IR interferometers I. Angular diameter and rotation parameters of Achernar from differential phases

Context. Spectrally resolved long-baseline optical/IR interferometry of rotating stars opens perspectives to investigate their fundamental parameters and the physical mechanisms that govern their interior, photosphere, and circumstellar envelope structures. Aims. Based on the signatures of stellar rotation on observed interferometric wavelength-differential phases, we aim to measure angular diameters, rotation velocities, and orientation of stellar rotation axes. Methods. We used the AMBER focal instrument at ESO-VLTI in its high-spectral resolution mode to record interferometric data on the fast rotator Achernar. Differential phases centered on the hydrogen Br gamma line (K band) were obtained during four almost consecutive nights with a continuous Earth-rotation synthesis during similar to 5h/night, corresponding to similar to 60 degrees position angle coverage per baseline. These observations were interpreted with our numerical code dedicated to long-baseline interferometry of rotating stars. Results. By fitting our model to Achernar's differential phases from AMBER, we could measure its equatorial radius R-eq = 11.6 +/- 0.3 R-circle dot, equatorial rotation velocity V-eq = 298 +/- 9 km s(-1), rotation axis inclination angle i = 101.5 +/- 5.2 degrees, and rotation axis position angle (from North to East) PA(rot) = 34.9 +/- 1.6 degrees. From these parameters and the stellar distance, the equatorial angular diameter circle divide(eq) of Achernar is found to be 2.45 +/- 0.09 mas, which is compatible with previous values derived from the commonly used visibility amplitude. In particular, circle divide(eq) and PA(rot) measured in this work with VLTI/AMBER are compatible with the values previously obtained with VLTI/VINCI. Conclusions. The present paper, based on real data, demonstrates the super-resolution potential of differential interferometry for measuring sizes, rotation velocities, and orientation of rotating stars in cases where visibility amplitudes are unavailable and/or when the star is partially or poorly resolved. In particular, we showed that differential phases allow the measurement of sizes up to similar to 4 times smaller than the diffraction-limited angular resolution of the interferometer.

Analysis of micro- and nano-structures of the corneal surface of Drosophila and its mutants by atomic force microscopy and optical diffraction.

Drosophila melanogaster is a model organism instrumental for numerous biological studies. The compound eye of this insect consists of some eight hundred individual ommatidia or facets, ca. 15 µm in cross-section. Each ommatidium contains eighteen cells including four cone cells secreting the lens material (cornea). High-resolution imaging of the cornea of different insects has demonstrated that each lens is covered by the nipple arrays--small outgrowths of ca. 200 nm in diameter. Here we for the first time utilize atomic force microscopy (AFM) to investigate nipple arrays of the Drosophila lens, achieving an unprecedented visualization of the architecture of these nanostructures. We find by Fourier analysis that the nipple arrays of Drosophila are disordered, and that the seemingly ordered appearance is a consequence of dense packing of the nipples. In contrast, Fourier analysis confirms the visibly ordered nature of the eye microstructures--the individual lenses. This is different in the frizzled mutants of Drosophila, where both Fourier analysis and optical imaging detect disorder in lens packing. AFM reveals intercalations of the lens material between individual lenses in frizzled mutants, providing explanation for this disorder. In contrast, nanostructures of the mutant lens show the same organization as in wild-type flies. Thus, frizzled mutants display abnormal organization of the corneal micro-, but not nano-structures. At the same time, nipples of the mutant flies are shorter than those of the wild-type. We also analyze corneal surface of glossy-appearing eyes overexpressing Wingless--the lipoprotein ligand of Frizzled receptors, and find the catastrophic aberration in nipple arrays, providing experimental evidence in favor of the major anti-reflective function of these insect eye nanostructures. The combination of the easily tractable genetic model organism and robust AFM analysis represents a novel methodology to analyze development and architecture of these surface formations.

Marker-free phase nanoscopy

We introduce a microscopic method that determines quantitative optical properties beyond the optical diffraction limit and allows direct imaging of unstained living biological specimens. In established holographic microscopy, complex fields are measured using interferometric detection, allowing diffraction-limited phase measurements. Here, we show that non-invasive optical nanoscopy can achieve a lateral resolution of 90 nm by using a quasi-2 pi-holographic detection scheme and complex deconvolution. We record holograms from different illumination directions on the sample plane and observe subwavelength tomographic variations of the specimen. Nanoscale apertures serve to calibrate the tomographic reconstruction and to characterize the imaging system by means of the coherent transfer function. This gives rise to realistic inverse filtering and guarantees true complex field reconstruction. The observations are shown for nanoscopic porous cell frustule (diatoms), for the direct study of bacteria (Escherichia coil), and for a time-lapse approach to explore the dynamics of living dendritic spines (neurones).

Spontane Strukturbildung in Mehrschichtsystemen unter Verwendung Molekularer Gläser

Den Schwerpunkt dieser Dissertation bildet zum einen die Entwicklung eines theoretischen Modells zur Beschreibung des Strukturbildungsprozesses in organisch/anorganischen Doppelschichtsystemen und zum anderen die Untersuchung der Übertragbarkeit dieser theoretisch gewonnenen Ergebnisse auf reale Systeme. Hierzu dienen systematische experimentelle Untersuchungen dieses Phänomens an einem Testsystem. Der Bereich der selbstorganisierenden Systeme ist von hohem wissenschaftlichen Interesse, erlaubt er doch die Realisierung von Strukturen, die nicht den Begrenzungen heutiger Techniken unterliegen, wie etwa der Beugung bei lithographischen Verfahren. Darüber hinaus liefert ein vertieftes Verständnis des Strukturbildungsprozesses auch eine Möglichkeit, im Falle entsprechender technischer Anwendungen Instabilitäten innerhalb der Schichtsysteme zu verhindern und somit einer Degradation der Bauteile entgegenzuwirken. Im theoretischen Teil der Arbeit konnte ein Modell im Rahmen der klassischen Elastizitätstheorie entwickelt werden, mit dessen Hilfe sich die Entstehung der Strukturen in Doppelschichtsystemen verstehen läßt. Der hier gefundene funktionale Zusammenhang zwischen der Periode der Strukturen und dem Verhältnis der Schichtdicken von organischer und anorganischer Schicht, wird durch die experimentellen Ergebnisse sehr gut bestätigt. Die Ergebnisse zeigen, daß es technologisch möglich ist, über die Vorgabe der Schichtdicke in einem Materialsystem die Periodizität der entstehenden Strukturen vorzugeben. Darüber hinaus liefert das vorgestellte Modell eine Stabilitätsbedingung für die Schichtsysteme, die es ermöglicht, zu jedem Zeitpunkt die dominierende Mode zu identifizieren. Ein Schwerpunkt der experimentellen Untersuchungen dieser Arbeit liegt auf der Strukturbildung innerhalb der Schichtsysteme. Das Testsystem wurde durch Aufbringen einer organischen Schicht - eines sog. Molekularen Glases - auf ein Glassubstrat realisiert, als Deckschicht diente eine Siliziumnitrid-Schicht. Es wurden Proben mit variierenden Schichtdicken kontrolliert erwärmt. Sobald die Temperatur des Schichtsystems in der Größenordnung der Glasübergangstemperatur des jeweiligen organischen Materials lag, fand spontan eine Strukturbildung auf Grund einer Spannungsrelaxation statt. Es ließen sich durch die Wahl einer entsprechenden Heizquelle unterschiedliche Strukturen realisieren. Bei Verwendung eines gepulsten Lasers, also einer kreisförmigen Wärmequelle, ordneten sich die Strukturen konzentrisch an, wohingegen sich ihre Ausrichtung bei Verwendung einer flächenhaften Heizplatte statistisch verteilte. Auffällig bei allen Strukturen war eine starke Modulation der Oberfläche. Ferner konnte in der Arbeit gezeigt werden, daß sich durch eine gezielte Veränderung der Spannungsverteilung innerhalb der Schichtsysteme die Ausrichtung der Strukturen (gezielt) manipulieren ließen. Unabhängig davon erlaubte die Variation der Schichtdicken die Realisierung von Strukturen mit einer Periodizität im Bereich von einigen µm bis hinunter zu etwa 200 nm. Die Kontrolle über die Ausrichtung und die Periodizität ist Grundvoraussetzung für eine zukünftige technologische Nutzung des Effektes zur kontrollierten Herstellung von Mikro- bzw. Nanostrukturen. Darüber hinaus konnte ein zunächst von der Strukturbildung unabhängiges Konzept eines aktiven Sensors für die optische Raster-Nahfeld-Mikroskopie vorgestellt werden, das das oben beschriebene System, bestehend aus einem fluoreszierenden Molekularen Glas und einer Siliziumnitrid-Deckschicht, verwendet. Erste theoretische und experimentelle Ergebnisse zeigen das technologische Potential dieses Sensortyps.

Atomic nanolithography patterning of submicron features: writing an organic self-assembled monolayer with cold bright Cs atom beams

Cs atom beams, transversely collimated and cooled, passing through material masks in the form of arrays of reactive-ion-etched hollow Si pyramidal tips and optical masks formed by intense standing light waves, write submicron features on self-assembled monolayers (SAMs). Features with widths as narrow as 43 ± 6 nm and spatial resolution limited only by the grain boundaries of the substrate have been realized in SAMs of alkanethiols. The material masks write two-dimensional arrays of submicron holes; the optical masks result in parallel lines spaced by half the optical wavelength. Both types of feature are written to the substrate by exposure of the masked SAM to the Cs flux and a subsequent wet chemical etch. For the arrays of pyramidal tips, acting as passive shadow masks, the resolution and size of the resultant feature depends on the distance of the mask array from the SAM, an effect caused by the residual divergence of the Cs atom beam. The standing wave optical mask acts as an array of microlenses focusing the atom flux onto the substrate. Atom 'pencils' writing on SAMs have the potential to create arbitrary submicron figures in massively parallel arrays. The smallest features and highest resolutions were realized with SAMs grown on smooth, sputtered gold substrates.

Development of 3D super-resolution tomographic STED microscopy and its application to studies on bone resorption

In this doctoral thesis, a tomographic STED microscopy technique for 3D super-resolution imaging was developed and utilized to observebone remodeling processes. To improve upon existing methods, wehave used a tomographic approach using a commercially available stimulated emission depletion (STED) microscope. A certain region of interest (ROI) was observed at two oblique angles: one at a standard inverted configuration from below (bottom view) and another from the side (side view) via a micro-mirror positioned close to the ROI. The two viewing angles were reconstructed into a final tomogram. The technique, named as tomographic STED microscopy, was able to achieve an axial resolution of approximately 70 nm on microtubule structures in a fixed biological specimen. High resolution imaging of osteoclasts (OCs) that are actively resorbing bone was achieved by creating an optically transparent coating on a microscope coverglass that imitates a fractured bone surface. 2D super-resolution STED microscopy on the bone layer showed approximately 60 nm of lateral resolution on a resorption associated organelle allowing these structures to be imaged with super-resolution microscopy for the first time. The developed tomographic STED microscopy technique was further applied to study resorption mechanisms of OCs cultured on the bone coating. The technique revealed actin cytoskeleton with specific structures, comet-tails, some of which were facing upwards and some others were facing downwards. This, in our opinion, indicated that during bone resorption, an involvement of the actin cytoskeleton in vesicular exocytosis and endocytosis is present. The application of tomographic STED microscopy in bone biology demonstrated that 3D super-resolution techniques can provide new insights into biological 3D nano-structures that are beyond the diffraction-limit when the optical constraints of super-resolution imaging are carefully taken into account.

Bioimage informatics in STED super-resolution microscopy

Optical microscopy is living its renaissance. The diffraction limit, although still physically true, plays a minor role in the achievable resolution in far-field fluorescence microscopy. Super-resolution techniques enable fluorescence microscopy at nearly molecular resolution. Modern (super-resolution) microscopy methods rely strongly on software. Software tools are needed all the way from data acquisition, data storage, image reconstruction, restoration and alignment, to quantitative image analysis and image visualization. These tools play a key role in all aspects of microscopy today – and their importance in the coming years is certainly going to increase, when microscopy little-by-little transitions from single cells into more complex and even living model systems. In this thesis, a series of bioimage informatics software tools are introduced for STED super-resolution microscopy. Tomographic reconstruction software, coupled with a novel image acquisition method STED< is shown to enable axial (3D) super-resolution imaging in a standard 2D-STED microscope. Software tools are introduced for STED super-resolution correlative imaging with transmission electron microscopes or atomic force microscopes. A novel method for automatically ranking image quality within microscope image datasets is introduced, and it is utilized to for example select the best images in a STED microscope image dataset.

Near-field mediated enhancement effects on plasmonic nanostructures

In dieser Arbeit wird eine detaillierte Untersuchung und Charakterisierung der Zwei-Photonen-induzierten Fluoreszenzverstärkung von organischen Farbstoffen auf plasmonischen Nanostrukturen vorgestellt. Diese Fluoreszenzverstärkung ist insbesondere für hochaufgelöste Fluoreszenzmikroskopie und Einzelmolekülspektroskopie von großer Bedeutung. Durch die Zwei-Photonen-Anregung resultiert eine Begrenzung des Absorptionsprozesses auf das fokale Volumen. In Kombination mit dem elektrischen Nahfeld der Nanostrukturen als Anregungsquelle entsteht eine noch stärkere Verringerung des Anregungsvolumens auf eine Größe unterhalb der Beugungsgrenze. Dies erlaubt die selektive Messung ausgewählter Farbstoffe. Durch die Herstellung der Nanopartikel mittels Kolloidlithografie wird eine definierte, reproduzierbare Geometrie erhalten. Polymermultischichten dienen als Abstandshalter, um die Farbstoffe an einer exakten Distanz zum Metall zu positionieren. Durch die kovalente Anbindung des Farbstoffs an die oberste Schicht wird eine gleichmäßige Verteilung des Farbstoffs in geringer Konzentration erhalten. rnEs wird eine Verstärkung der Fluoreszenz um den Faktor 30 für Farbstoffe auf Goldellipsen detektiert, verglichen mit Farbstoffen außerhalb des Nahfelds. Sichelförmige Nanostrukturen erzeugen eine Verstärkung von 120. Dies belegt, dass das Ausmaß der Fluoreszenzverstärkung entscheidend von der Stärke des elektrischen Nahfelds der Nanostruktur abhängt. Auch das Material der Nanostruktur ist hierbei von Bedeutung. So erzeugen Silberellipsen eine 1,5-fach höhere Fluoreszenzverstärkung als identische Goldellipsen. Distanzabhängige Fluoreszenzmessungen zeigen, dass die Zwei-Photonen-angeregte Fluoreszenzverstärkung an strukturspezifischen Abständen zum Metall maximiert wird. Elliptische Strukturen zeigen ein Maximum bei einem Abstand von 8 nm zum Metall, wohingegen bei sichelförmigen Nanostrukturen die höchste Fluoreszenzintensität bei 12 nm gemessen wird. Bei kleineren Abständen unterliegt der Farbstoff einem starken Löschprozess, sogenanntes Quenching. Dieses konkurriert mit dem Verstärkungsprozess, wodurch es zu einer geringen Nettoverstärkung kommt. Hat die untersuchte Struktur Dimensionen größer als das Auflösungsvermögen des Mikroskops, ist eine direkte Visualisierung des elektrischen Nahfelds der Nanostruktur möglich. rnrnEin weiterer Fokus dieser Arbeit lag auf der Herstellung neuartiger Nanostrukturen durch kolloidlithografische Methoden. Gestapelte Dimere sichelförmiger Nanostrukturen mit exakter vertikaler Ausrichtung und einem Separationsabstand von etwa 10 nm wurden hergestellt. Die räumliche Nähe der beiden Strukturen führt zu einem Kopplungsprozess, der neue optische Resonanzen hervorruft. Diese können als Superpositionen der Plasmonenmoden der einzelnen Sicheln beschrieben werden. Ein Hybridisierungsmodell wird angewandt, um die spektralen Unterschiede zu erklären. Computersimulationen belegen die zugrunde liegende Theorie und erweitern das Modell um experimentell nicht aufgelöste Resonanzen. rnWeiterhin wird ein neuer Herstellungsprozess für sichelförmige Nanostrukturen vorgestellt, der eine präzise Formanpassung ermöglicht. Hierdurch kann die Lage der Plasmonenresonanz exakt justiert werden. Korrelationen der geometrischen Daten mit den Resonanzwellenlängen tragen zum grundlegenden Verständnis der Plasmonenresonanzen bei. Die vorgestellten Resultate wurden mittels Computersimulationen verifiziert. Der Fabrikationsprozess erlaubt die Herstellung von Dimeren sichelförmiger Nanostrukturen in einer Ebene. Durch die räumliche Nähe überlappen die elektrischen Nahfelder, wodurch es zu kopplungs-induzierten Shifts der Plasmonenresonanzen kommt. Der Unterschied zu theoretisch berechneten ungekoppelten Nanosicheln kann auch bei den gegenüberliegenden sichelförmigen Nanostrukturen mit Hilfe des Plasmonenhybridisierungsmodells erklärt werden.

Tracking individual membrane proteins and their biochemistry: The power of direct observation

The advent of single molecule fluorescence microscopy has allowed experimental molecular biophysics and biochemistry to transcend traditional ensemble measurements, where the behavior of individual proteins could not be precisely sampled. The recent explosion in popularity of new super-resolution and super-localization techniques coupled with technical advances in optical designs and fast highly sensitive cameras with single photon sensitivity and millisecond time resolution have made it possible to track key motions, reactions, and interactions of individual proteins with high temporal resolution and spatial resolution well beyond the diffraction limit. Within the purview of membrane proteins and ligand gated ion channels (LGICs), these outstanding advances in single molecule microscopy allow for the direct observation of discrete biochemical states and their fluctuation dynamics. Such observations are fundamentally important for understanding molecular-level mechanisms governing these systems. Examples reviewed here include the effects of allostery on the stoichiometry of ligand binding in the presence of fluorescent ligands; the observation of subdomain partitioning of membrane proteins due to microenvironment effects; and the use of single particle tracking experiments to elucidate characteristics of membrane protein diffusion and the direct measurement of thermodynamic properties, which govern the free energy landscape of protein dimerization. The review of such characteristic topics represents a snapshot of efforts to push the boundaries of fluorescence microscopy of membrane proteins to the absolute limit.

Analysis of Super Imaging Properties of Spherical Geodesic Waveguide Using COMSOL Multyphisics

Negative Refractive Lens (NRL) has shown that an optical system can produce images with details below the classic Abbe diffraction limit using materials of negative dielectric and magnetic constants. Recently, two devices with positive refraction, the Maxwell Fish Eye lens (MFE) (Leonhardt et al 2000) and the Spherical Geodesic Waveguide (SGW)(Minano et all 2011) have been claimed to break the diffraction limit using positive refraction with a different meaning. In these cases, it has been considered the power transmission from a point source to a point receptor, which falls drastically when the receptor is displaced from the focus by a distance much smaller than the wavelength. Moreover, recent analysis of the SGW with defined object and image surfaces, which are both conical sections of the sphere, has shown that the system transmits images bellow diffraction limit. The key assumption is the use of a perfectly absorbing receptor called perfect drain. This receptor is capable to absorb all the radiation without reflection or scattering. Here, it is presented the COMSOL analysis of the SGW using a perfect drain that absorbs perfectly two modes. The design procedure for PD capable to absorb k modes is proposed, as well.

Perfect imaging analysis of the spherical geodesic waveguide

Negative Refractive Lens (NRL) has shown that an optical system can produce images with details below the classic Abbe diffraction limit. This optical system transmits the electromagnetic fields, emitted by an object plane, towards an image plane producing the same field distribution in both planes. In particular, a Dirac delta electric field in the object plane is focused without diffraction limit to the Dirac delta electric field in the image plane. Two devices with positive refraction, the Maxwell Fish Eye lens (MFE) and the Spherical Geodesic Waveguide (SGW) have been claimed to break the diffraction limit using positive refraction with a different meaning. In these cases, it has been considered the power transmission from a point source to a point receptor, which falls drastically when the receptor is displaced from the focus by a distance much smaller than the wavelength. Although these systems can detect displacements up to ?/3000, they cannot be compared to the NRL, since the concept of image is different. The SGW deals only with point source and drain, while in the case of the NRL, there is an object and an image surface. Here, it is presented an analysis of the SGW with defined object and image surfaces (both are conical surfaces), similarly as in the case of the NRL. The results show that a Dirac delta electric field on the object surface produces an image below the diffraction limit on the image surface.

Perfect imaging of point sources with positive refraction

The capability of a device called the Spherical Geodesic Waveguide (SGW) to produce images with details below the classic Abbe diffraction limit (super-resolution) is analyzed here. The SGW is an optical system equivalent (by means of Transformation Optics) to the Maxwell Fish Eye (MFE) refractive index distribution. Recently, it has been claimed that the necessary condition to get super-resolution in the MFE and the SGW is the use of a Perfect Point Drain (PPD). The PPD is a punctual receptor placed in the focal point that absorbs the incident wave, without reflection or scattering. A microwave circuit comprising three elements, the SGW, the source and the drain (two coaxial lines loaded with specific impedances) is designed and simulated in COMSOL. The super-resolution properties have been analyzed for different position of the source and drain and for two different load impedances: the PPD and the characteristic line impedance. The results show that in both cases super-resolution occurs only for discrete number of frequencies. Out of these frequencies, the SGW does not show SR in the analysis carried out.

Experimental evidence of super-resolution better than lambda/105 with positive refraction

Super-resolution (SR) systems surpassing the Abbe diffraction limit have been theoretically and experimentally demonstrated using a number of different approaches and technologies: using materials with a negative refractive index, utilizing optical super-oscillation, using a resonant metalens, etc. However, recently it has been proved theoretically that in the Maxwell fish-eye lens (MFE), a device made of positive refractive index materials, the same phenomenon takes place. Moreover, using a simpler device equivalent to the MFE called the spherical geodesic waveguide (SGW), an SR of up to λ/3000 was simulated in COMSOL. Until now, only one piece of experimental evidence of SR with positive refraction has been reported (up to λ/5) for an MFE prototype working at microwave frequencies. Here, experimental results are presented for an SGW prototype showing an SR of up to λ/105. The SGW prototype consists of two concentric metallic spheres with an air space in between and two coaxial ports acting as an emitter and a receiver. The prototype has been analyzed in the range 1 GHz to 1.3 GHz.

Superfocusing of high-M2 semiconductor laser beams:experimental demonstration

The focusing of multimode laser diode beams is probably the most significant problem that hinders the expansion of the high-power semiconductor lasers in many spatially-demanding applications. Generally, the 'quality' of laser beams is characterized by so-called 'beam propagation parameter' M2, which is defined as the ratio of the divergence of the laser beam to that of a diffraction-limited counterpart. Therefore, M2 determines the ratio of the beam focal-spot size to that of the 'ideal' Gaussian beam focused by the same optical system. Typically, M2 takes the value of 20-50 for high-power broad-stripe laser diodes thus making the focal-spot 1-2 orders of magnitude larger than the diffraction limit. The idea of 'superfocusing' for high-M2 beams relies on a technique developed for the generation of Bessel beams from laser diodes using a cone-shaped lens (axicon). With traditional focusing of multimode radiation, different curvatures of the wavefronts of the various constituent modes lead to a shift of their focal points along the optical axis that in turn implies larger focal-spot sizes with correspondingly increased values of M2. In contrast, the generation of a Bessel-type beam with an axicon relies on 'self-interference' of each mode thus eliminating the underlying reason for an increase in the focal-spot size. For an experimental demonstration of the proposed technique, we used a fiber-coupled laser diode with M2 below 20 and an emission wavelength in ~1μm range. Utilization of the axicons with apex angle of 140deg, made by direct laser writing on a fiber tip, enabled the demonstration of an order of magnitude decrease of the focal-spot size compared to that achievable using an 'ideal' lens of unity numerical aperture. © 2014 SPIE.