Improved measurement of electric fields by nanobeam precession electron diffraction

Jean-Luc Rouvière
2020
Article

Résumé

The electric field in a silicon p-n junction has been measured using pixelated scanning transmission electron microscopy. By using a convergence angle of 3.2mrad, a spatial resolution better than 1nm can be achieved leading to a rigid shift of the transmitted beam as it passes through an electric field. By precessing the beam around the optical axis at an angle of 0.1 degrees, the effects of dynamical diffraction can be reduced. This leads to an improved measurement of the electric field from the shift of the transmitted beam. Different algorithms have been used to measure this shift, and template matching leads to a more accurate measurement of the electric field than the often-used center of mass method.

Official source

https://infoscience.epfl.ch/record/278003?ln=fr

À propos de ce résultat

Cette page est générée automatiquement et peut contenir des informations qui ne sont pas correctes, complètes, à jour ou pertinentes par rapport à votre recherche. Il en va de même pour toutes les autres pages de ce site. Veillez à vérifier les informations auprès des sources officielles de l'EPFL.

Concepts associés

Chargement

Publications associées

Chargement

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

Chargement

Electro-static or electro-dynamic fields generated by micro- or nano-electrodes are today widely used in many different fields, such as micro- and nanogripping, "lab on a chip", and "lab in a cell", for the purpose of manipulation, separation, or analysis of micron-sized particles, cells, or single molecules. Commonly numerical simulations and Electrostatic Force Microscopy (EFM) methods, based on Atomic Force Microscopy (AFM), are used to study and predict the electrostatic force fields above produced structures. However, this measurement methods have several disadvantages. These disadvantages range from simple limitations (e.g. it is not possible to measure in three dimensions or expensive new equipment is needed) to much more serious disadvantages (e.g. missing compensation for known AFM problems, which results in wrong measurements). In this thesis we propose a new EFM method based on simple static force distance curves, which allows measuring accurately, and simultaneously the topography, vertical electrostatic force field and attraction forces. The method is numerical simulated in 3D to study in detail, with simulations and measurements, electrostatic force fields above nanoelectrodes. To do this, we show for the first time the fabrication of interdigitated nanoelectrodes with pitches down to 50 nm with gas assisted focused ion beam milling. We present as well for the first time the fabrication of Pt coated AFM tips with radii between 50 and 600 nm, together with their calibration. These tips not only close the gap between conventional tips with tip radii of about 10 nm and cantilevers with attached spheres. They show as well high mechanical stability, which solves a common problem in EFM, the mechanic stability of the tip. The shape of the tip greatly influences the measurement. We demonstrate this using numerical simulations, and show that already small tip discrepancies can change the measured force up to 50 %. Moreover, we proved that changes in the relative humidity result in electrostatic force changes of up to 45 %. We discovered that the resolution of EFM measurements on nanoelectrodes can be enhanced by using a tip radius of 2 to 2.5 times the pitch of the measured interdigitated nanoelectrode. Based on this and its influence on topography measurements, which are usually made before, in between or at the end of EFM measurements, we give hints for measurements with different tip radii. Beside these important general improvements, discoveries and hints for EFM, we show as well some applications of our new EFM method and the before obtained results. We show that our new method can distinguish the force field caused by trapped charges in SiO2 from the force field caused by nanoelectrodes below it. This enables to study the influence of charge trapping in future semiconductor chips, which use charge trapping to store information. Furthermore, we used our new EFM method and some gripping experiments to study electrostatic micro-and nanogripping. This experiments lead to some propositions for improvements in electrostatic micro-and nanogripping. Such as a new gripper design with gripping object size independent centering effect, which is studied using 3D numerical simulations.

EPFL2008

Chargement

EPFL2008

Single molecule detection and fluorescence correlation spectroscopy on surfaces

Kai Hassler

EPFL2006