Manufacturing of orifices in glass like materials, e.g. Nanocapillaries, and objects obtained according to this process

The ability to reshape nanopores and observe their shrinkage under an electron microscope is a powerful and novel technique14,17. It increases the sensitivity of the resistive pulse sensing and enables to detect very short and small molecules12,31. However, this has not yet been shown for glass having a tubular shape, for instance nanocapillaries. In contrast to their solid-state nanopore counterparts25, nanocapillaries are cheap, easily fabricated and in the production do not necessitate clean room facilities. Nanocapillaries made out of glass-like materials such as quartz or borosilicate glass can be shrunken under a scanning electron microscope beam. Since the shrinking is caused by the thermal heating of the electrons, increasing the beam current increases the shrink rate. Higher acceleration voltage on the contrary increases the electron penetration depth and reduces the electron density causing slower shrink rates. This allows to fine control the shrink rate and to stop the shrinking process at any desired diameter. A shrunken nanocapillary may detect DNA translocation with six times higher signal amplitudes than an unmodified nanocapillary. The invention opens a new path to detect small and short molecules such as proteins or RNA with nanocapillaries and also increase the sensitivity of other techniques such as SNOM or SCIM, which also rely on conical glass capillaries.

Controllable Shrinking and Shaping of Glass Nanocapillaries under Electron Irradiation

Aleksandra Radenovic, Lorenz Jan Steinbock

The ability to reshape nanopores and observe their shrinkage under an electron microscope is a powerful and novel technique. It increases the sensitivity of the resistive pulse sensing and enables to detect very short and small molecules. However, this has not yet been shown for glass nanocapillaries. In contrast to their solid-state nanopore counterparts, nanocapillaries are cheap, easily fabricated and in the production do not necessitate clean room facilities. We show for the first time that quartz nanocapillaries can be shrunken under a scanning electron microscope beam. Since the shrinking is caused by the thermal heating of the electrons, increasing the beam current increases the shrink rate. Higher acceleration voltage on the contrary increases the electron penetration depth and reduces the electron density causing slower shrinkage. This allows us to fine control the shrink rate and to stop the shrinking process at any desired diameter. We show that a shrunken nanocapillary detects DNA translocation with six times higher signal amplitudes than an unmodified nanocapillary. This will open a new path to detect small and short molecules such as proteins or RNA with nanocapillaries.

American Chemical Society2013

Effects of base-pair sequence, nicks and gaps on DNA minicircle shapes

Arnaud Amzallag

DNA is a long polymer with the form of a double helix of about two nanometers diameter (2.10-9 meters). It is composed of nucleotides whose sequence carries the information of heredity. The four possible nucleotides have slightly different geometries, and their sequence along the DNA molecule are thought to influence the shape and the stiffness of the double helix on a scale of few tens to a few hundred base pairs, and thereby its biological activity. DNA minicircles (or miniplasmids) are closed loops with lengths of the order of a few tens or hundreds of base pairs in which the double helix bends around to close on its own tail with some number of twists. Its minimal energy shape and its energy of formation depend on the sequence of base pairs, and can be efficiently computed by polymer and rod models, if shape and stiffness parameters of DNA are provided as inputs. This structure is therefore an interesting experimental motif to test sequence-dependent mechanical properties of the DNA molecule. The purpose of this thesis is to explore the experimental methods to determine the shape and free energy of formation of DNA minicircles. The shapes have been be determined from cryo-electron micrographs. Efficiency of formation has been investigated by a novel method called annealing-cyclization. Cryo-electron microscopy allows observation of very small molecules (here 17 or 11 nm diameter) in vitrified water, in order to keep the 3D shape of the molecules as close as possible to the shape they had in solution. In Chapter 1, I used stereo cryo-electron micrographs to determine and compare the three-dimensional shape of 95 individual DNA minicircles of 158 base pairs, which were identical in sequence except within a 18 bp block which contained either a TATA box sequence or a CAP site. I defined the notion of shape-distance which I used to estimate the error of reconstruction, and I detected clusters of shapes using an appropriate sorting algorithm. However the cluster did not seem to be associated with the variable sequence (TATA or CAP). I then analyzed (in Chapter 2) two-dimensional shapes of shorter DNA minicircles (94 bp) determined by negative staining electron microscopy, designed with either two nicks (breaks in one of the strands) or two gaps (missing nucleotides) at diametrically opposite sites of the minicircles. I observed that the gapped minicircles have an elongated shape with respect to the nicked minicircles, and I used this result together with the results of atomic level molecular dynamics simulations to conclude that the gap flexibility, perhaps together with base unpairing at the gap site, is responsible for this elongated minicircle shape. Finally, I proposed a ligase-free assay to measure the minicircle formation efficiency, which can give a high yield of nicked minicircles. The method avoids the use of ligase and the associated concerns about the effect of ligase concentration on the measurements. I determined an equation from a chemical model of the reaction that fits the experimental data, and I defined the Ja factor which gives a measure of the cyclization yield independent of DNA initial concentration. This method seems to confirm that minicircles with two gaps cyclize more efficiently that minicircles with two nicks, probably because of the gap flexibility. As a perspective, the conclusions of this thesis could be used for the design of minicircle constructs whose shape would be sensitive enough to sequence mutation in order to be detected by electron microscopy. Such shapes could be then compared, thanks to the shape-distance tool defined herein, to shapes computed with known or putative DNA models.

EPFL2008

Developing luminescent Brownian probes for near-field investigations of the intracellular environment

Flavio Maurizio Mor

Life sciences have a constantly growing need for novel methodological approaches suitable to investigate the intracellular environment with increased temporal and spatial resolutions. Recently, optical near-field probes, such as laser-irradiated pointed metal, uncoated/metal-coated tapered optical fibers, as well as nano-emitters, such as single molecules or nanoparticles, have attracted increasing attention as key components of high-resolution microscopes. In parallel, super-resolution fluorescence microscopy, operating in far-field regime and overcoming the light diffraction limit, has markedly improved imaging resolution. Although both near- and far-field optical approaches drastically improve image resolution, they still represent numerous drawbacks, such as, technical difficulties concerning probe preparation (near-field) or potential damaging through very high light intensities (far-field). The aforementioned shortcomings of high-resolution optical microscopy can be overcome to a great extent by photonic force microscopy (PFM). PFM employs a strongly focused near-infrared (NIR) laser light to hold a dielectric or metallic particle as a local probe. Such an optical trap enables one to ‘cage’ a mesoscopic particle and track its three-dimensional (3D) thermal fluctuations in the surrounding environment, e.g. a viscous liquid. Therefore, besides offering near-field 3D imaging, PFM reports also on other important quantities concerning local mechanical properties, including force and viscosity as well as dynamical properties of the surrounding medium. This additional information is derived from the careful analysis of the jittery motion (so-called Brownian motion) of the trapped single-particle probe that collides with thermally-activated surrounding molecules. In this thesis, we first study in detail the Brownian motion of a single spherical particle that is confined within the harmonic potential of the optical trap. To this end, we optimize the NIR light path and electronic noise floor of our custom-built PFM set-up for detecting and quantifying resonances in the Brownian motion. These resonances arise from the coupling between the hydrodynamic memory and strong strength of the optical trap. Due to the high sensitivity of the short-time dynamics, the size of the particle can be measured by simultaneous fitting of the velocity autocorrelation function and power spectral density of its thermal fluctuations. In order to choose the best-suitable spherical probe for a given experiment in PFM, computational modeling based on a Matlab toolbox is performed. The generalized Lorenz-Mie theory is computed using the T -matrix method for various experimental conditions, including changes in the size and refractive index of the sphere, as well as different laser polarization states. The axial equilibrium position is examined to predict its location compared with the position of the laser focus. Optical forces acting on the sphere are investigated in 3D to highlight, in particular, the limits of the perfectly harmonic potential assumption. These limits are quantified and discussed. Subsequently, we identify different types of inorganic nano/micro-sized particles that can be trapped by the NIR laser of the PFM and used simultaneously as mechanical and near-field light sources. To this end, we take advantage of the visible light emission from single probes confined in the optical trap and excited by the NIR laser light. The emphasis is on particles revealing nonlinear optical properties. In particular, nonlinear optical field enhancement resulting from excitation of surface plasmon resonance (SPR) on trapped gold particles of 200 nm is demonstrated by measuring the emission of that second-harmonic generation (SHG). Furthermore, we show, under optical trapping conditions, SHG emission from single potassium niobate (KNbO3) nano/micro-sized particles. We also report on the multicolor upconversion luminescence (UCL), at the single particle level, for randomly shaped crystals of sodium yttrium fluoride codoped with ytterbium and erbium, NaYF4:Yb,Er (UCC), when they are held in the optical trap. In parallel, 3D Brownian fluctuations of a single trapped particle are quantified. Moreover, luminescence resonance energy transfer (LRET) between a KNbO3 particle or an UCC and molecules of an organic dye (rose bengal) adsorbed on their surface is highlighted and analyzed. Finally, randomly shaped UCCs and hexagonal nanoplates are characterized in detail at the single particle level. The hexagonal β phase in the polycrystalline and monocrystalline crystals is identified, in both randomly and hexagonally shaped particles by electron microscopy. UCL emission from the trapped crystal is quantified in terms of power and found to be dependent on the laser power density, size and shape of the particle as well as its orientation within the trap. Most importantly, the different colors in the UCL spectrum do not vary with the same trend for different orientations of the randomly or hexagonally shaped crystal upon trapping. This suggests the existence of crystalline anisotropy-dependent UCL. Thereby, we should be able to design the best-suited particle probe for LRET experiments with subwavelength resolution.