Midfield microscope: Exploring the extraordinary

In this thesis the development of the midfield microscope is presented. This is a microscope in which the extraordinary transmission (EOT) through sub-wavelength hole-arrays is applied. Before trying to combine microscopy and EOT, we look at them separately. In chapter 1 an overview is given of the current microscope techniques. The main research questions posed are which qualities, like resolution, the midfield microscope will possess and more importantly whether it will be an addition to the current variety of microscopes. EOT has been subject of research ever since its discovery. A literature study is given in chapter 2, in which both experimental and theoretical results are given. Physical explanations are developed, like surface plasmons and coupled diffracted evanescent waves. For applications, like microscopy or lithography, these explanations are of less importance, as long as the device is working. Because of the lack of a unified theory to predict the transmitted spectra and intensity distribution, we analyze them ourselves. The influence of various parameters, like period or incidence angle, is measured on home-made arrays and is described in chapter 3. We confirmed experimentally that the simple surface plasmon model predicts the transmission spectrum only up to a certain level; experimental verification is an absolute must. Polarization measurements show that the polarization has no influence on the spectrum in 2D, which supports the idea of the array being an active device. The limited angular spread is supported by far-field measurements of the transmission for both Köhler and collimated illumination. Now that we measured the spectra, we should know whether the wavelengths corresponding to the peaks result in an interesting transmission pattern. A theoretical calculation, which is limited to the case of a (two-dimensional) slit-array, is done in chapter 4. The transmission pattern having the best contrast and highly localized spots is not the one corresponding to the peak intensity, but to an interference pattern because the wavelength smaller than the period. Fortunately the intensity is still enhanced (at that wavelength more light is transmitted through a periodic than through a random hole-array, chapter 3). Through an analytical approach was found that for a wavelength smaller than the period the contribution to the pattern is mostly due to the transmission (and not so much the surface waves). The theoretical predictions gave already enough information to formulate the working of the midfield microscope. In chapter 5 it is shown that the microscope is comparable with a confocal one, but instead of two pinholes the hole-array is used in the illumination pathway and a CCD in the imaging pathway. Based on the predicted size of lobes in the intensity pattern the midfield resolution is similar to the confocal one, because the point spread functions have a similar size. The possible use of increased resolution by means of structured illumination in possible combination with nonlinear techniques is discussed. In chapter 6 fluorescence measurements are described. Fluorescent molecules are used as probes for the local intensity. The measurement with continuous fluorescence results in an intensity distribution, similar to the one predicted earlier. This is the first time fluorescence is used to measure the transmitted intensity pattern through a hole-array. Moreover, it is a proof-of-principle that the midfield microscope is able to image a sample, in this case continuously labeled and placed in the transmitted "midfield" intensity distribution. Chapter 7 answers the research questions, conclude this thesis and gives recommendations for further research.Applied Science

A Proof-of-Concept of an Integrated VR and AI Application to Develop Classroom Management Competencies in Teachers in Training

We designed an interactive virtual reality (VR) application to provide a controlled and yet unpredictable environment for the development of classroom management skills. The simulated environment allows teachers in training to interact with virtual students in realistic and meaningful ways. The VR application allows rich verbal interaction by using artificial intelligence (AI). Initial findings suggest it is a successful proof of concept. In this paper, we focus on the technical implementation. Predictions on educational effectiveness and the educational challenges of pre-service teacher education are discussed. Future developments include rigorous testing and incorporating non-verbal communication based on a multi-dimensional interpersonal behavior model.BN/AfdelingsbureauEducation and Student AffairsNew Media CentreScience & Engineering Educatio

DNA binding proteins explore multiple local configurations during docking via rapid rebinding

Finding the target site and associating in a specific orientation are essential tasks for DNA-binding proteins. In order to make the target search process as efficient as possible, proteins should not only rapidly diffuse to the target site but also dynamically explore multiple local configurations before diffusing away. Protein flipping is an example of this second process that has been observed previously, but the underlying mechanism of flipping remains unclear. Here, we probed the mechanism of protein flipping at the single molecule level, using HIV-1 reverse transcriptase (RT) as a model system. In order to test the effects of long-range attractive forces on flipping efficiency, we varied the salt concentration and macromolecular crowding conditions. As expected, increased salt concentrations weaken the binding of RT to DNA while increased crowding strengthens the binding. Moreover, when we analyzed the flipping kinetics, i.e. the rate and probability of flipping, at each condition we found that flipping was more efficient when RT bound more strongly. Our data are consistent with a view that DNA bound proteins undergo multiple rapid re-binding events, or short hops, that allow the protein to explore other configurations without completely dissociating from the DNA.BN/Cees Dekker LabBN/Technici en AnalistenBN/Elio Abbondanzieri La

Hysteresis in DNA compaction by Dps is described by an ising model

In all organisms, DNA molecules are tightly compacted into a dynamic 3D nucleoprotein complex. In bacteria, this compaction is governed by the family of nucleoid-associated proteins (NAPs). Under conditions of stress and starvation, an NAP called Dps (DNA binding protein from starved cells) becomes highly up-regulated and can massively reorganize the bacterial chromosome. Although static structures of Dps-DNA complexes have been documented, little is known about the dynamics of their assembly. Here, we use fluorescence microscopy and magnetic-tweezers measurements to resolve the process of DNA compaction by Dps. Real-time in vitro studies demonstrated a highly cooperative process of Dps binding characterized by an abrupt collapse of the DNA extension, even under applied tension. Surprisingly, we also discovered a reproducible hysteresis in the process of compaction and decompaction of the Dps-DNA complex. This hysteresis is extremely stable over hour-long timescales despite the rapid binding and dissociation rates of Dps. A modified Ising model is successfully applied to fit these kinetic features. We find that long-lived hysteresis arises naturally as a consequence of protein cooperativity in large complexes and provides a useful mechanism for cells to adopt unique epigenetic states.Title of the "Accepted Author Manuscript": An Ising model describes hysteresis in the process of DNA compaction by DpsBN/Elio Abbondanzieri LabBN/Nynke Dekker LabBN/Technici en AnalistenBN/Anne Meyer La

A novel concept for a mid-field microscope

When light passes through a hole smaller than the wavelength of the light, the transmission is very low and the light is diffracted. This however changes if holes are arranged in a periodic array on metal. In that case the light couples to surface plasmons; this results in enhanced transmission, spectral selection and a small angular diffraction. We develop a novel microscopic method based on a periodic hole-array, which will be used as a multiple-apertures near-field source for illuminating a biological sample while the light is collected in far-field. The measurement speed is high, due to the use of an array instead of a single source. The main advantage of this microscope originates from the low diffraction of light through a relatively thick sample with enhanced transmission. It results in the ability to measure the samples interior and 3D reconstruction can be made by semi-confocal techniques. This overcomes the major limitation of near-field methods for which only a shallow layer of the surface (~20 nm) is detectable. For our measurements we use glass coated devices. The holes are processed with a focused ion beam. The photonplasmon coupling process is characterized as a function of the wavelength. Our experiments aim on gaining a better understanding of the transmission process. We tested the dependence of the transmitted spectrum on angle of incidence was tested as well as far-field spectral imaging measurements of the transmission in both Koehler and collimated light illumination. The results as well as the description of the microscope that we are constructing are presented.Imaging Science and TechnologyApplied Science

Measuring the wavelength-dependent divergence of transmission through sub-wavelength hole-arrays by spectral imaging

We present a study on the far-field patterns of light transmitted through sub-wavelength metallic hole-arrays. Spectral imaging measurements are used here on hole arrays for the first time. It provides both spatial and spectral information of the transmission in far-field. The visibility of the images, measured in two illumination modes: Köhler and collimated, is calculated for different planes in and out of focus. The transmission under collimated illumination reveals that 75% of the beam if non-divergent. The results are in agreement with the low divergence measured by Lezec [Science 297, 820 (2002)].Imaging Science and TechnologyApplied Science

Toward the development of a Three-Dimensional Mid–Field Microscope

Recently, an extraordinary transmission of light through small holes (<200 nm) in a thin metallic film has been described. This phenomenon has been shown to be the result of the photon-plasmon interaction in thin films where a periodic structure (such as a set of holes) is embedded in the film. One of the extraordinary results is that the beam that passes through a hole has a very small diffraction in extreme contrast to the wide angle predicted by diffraction theory. Based on this effect, we propose here a new type of microscopy that we term mid-field microscopy. It combines an illumination of the sample through a metallic hole-array with far-field collection optics, a scanning mechanism and a CCD. When compared to other high resolution methods, what we suggest here is relatively simple because it is based on a thin metallic film with an array of nano-sized holes. Such a method can be widely used in high-resolution microscopy and provide a novel simple-to-use tool in many life-sciences laboratories. When compared to near-field scanning optical microscopy (NSOM), the suggested mid-field method provides a significant improvement. This is chiefly for three reasons: 1. The penetration depth of the microscope increases from a few nanometers to a few micrometers, hence the name mid-field microscope. 2. It allows one to measure an image faster because the image is measured through many holes in parallel rather then through a single fiber tip used in conventional near-field microscopy, and 3. It enables one to perform three-dimensional reconstruction of images due to a semi-confocal effect. We describe the physical basics of the photon-plasmon interaction that allows the coupling of light to the surface plasmons and determines the main spectral characteristics of the device. This mechanism can be ascribed due to the super-periodicity of the electron oscillations on the metallic surface engendered by the grating-like structure of the holearray.Imaging Science and TechnologyApplied Science

Measuring the near-field of extra-ordinary transmission through a periodic hole-array

The knowledge of the near-field of extra-ordinary transmission through hole-arrays is mostly theoretical; there is less experimental validation of the theory. We study the near-field properties by measuring fluorescent molecules that are immersed in a solution and their Brownian motion. The measurements are performed by filling the space above the hole-array with fluorescent solution and exciting these molecules through the hole-array. By measuring both the fluorescence and the direct exciting light, it is possible to learn about the near-field properties.Imaging Science and TechnologyApplied Science

High-Speed Super-Resolution Imaging Using Protein-Assisted DNA-PAINT

Super-resolution imaging allows for the visualization of cellular structures on a nanoscale level. DNA-PAINT (DNA point accumulation in nanoscale topology) is a super-resolution method that depends on the binding and unbinding of DNA imager strands. The current DNA-PAINT technique suffers from slow acquisition due to the low binding rate of the imager strands. Here we report on a method where imager strands are loaded into a protein, Argonaute (Ago), which allows for faster binding. Ago preorders the DNA imager strand into a helical conformation, allowing for 10 times faster target binding. Using a 2D DNA origami structure, we demonstrate that Ago-assisted DNA-PAINT (Ago-PAINT) can speed up the current DNA-PAINT technique by an order of magnitude, while maintaining the high spatial resolution. We envision this tool to be useful for super-resolution imaging and other techniques that rely on nucleic acid interactions.BN/Chirlmin Joo LabBN/Cees Dekker LabBN/Technici en Analiste

Structured illumination microscopy using extraordinary transmission through sub-wavelength hole-arrays

Imaging Science and TechnologyApplied Science