Precision Positioning of Microrobots for Multi-Object Spectrographs

How does one study the evolution of the Milky Way, or the expansion of the Universe, or explore the mysteries of Dark energy? To investigate these complex topics, astronomers require data, and a great deal of it, in the form of the spectra of stars, galaxies, and quasars. In recent years remarkable efforts have been made to develop telescopes and instruments that can measure more light spectra in parallel than ever before. One solution for building these instruments, called Multi-Object Spectrographs, requires the use of several hundreds of small precise automated robots in the telescope's focal plane. Each robot can position an optical fiber with micrometer precision along the focal surface in order to capture and transmit the light of a celestial object to a spectrograph, which then measures the spectrum with great precision.

In the future, new instruments will be able to measure over 10000 celestial objects simultaneously, which would consequently require the implementation of as many optical fiber positioners. To assure the development and successful operation of the instrument, every single fiber positioner requires design and validation testing. Tools for such quality assurance have yet to be developed. This thesis proposes solutions to verify, calibrate, control, and operate optical fiber positioners with two actuated rotation axes, serially linked. A metrology system is presented which is capable of accurately measuring position and alignment of the optical fiber, and a detailed model describing the correct behavior of the positioner is introduced. Based on the model, an automated measurement procedure enables calibration and validation of the positioners. The introduced measurement procedure identifies not only flaws in performance, but also various error sources, which help to detect possible design and manufacturing problems. Finally, a method is proposed on how to operate these positioners in the focal plane. Taken together, the results of this thesis can inform the present and future manufacturer of Multi-Object Spectrographs.

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

Distributed sensing using polarization sensitive optical low-coherence reflectometry and fiber gratings

Dragan Coric

Optical low coherence reflectometry (OLCR) is non-destructive interferometric technique that allows measuring amplitude and phase of the light reflected from the device under test. OLCR is a powerful tool for the characterization of the various optical devices such as fiber gratings and optical waveguides. This thesis work had explored possibilities to combine the OLCR technique with fiber gratings to perform distributed strain and temperature measurements, and to develop novel sensing techniques as an alternative to classical spectral methods. Special attention is devoted to polarization sensitive measurements and techniques, and OLCR techniques that use only amplitude information, as an alternative to more complicated phase sensitive measurements. Using a polarization sensitive system two methods for the measurement of local birefringence of fiber Bragg gratings (FBG) using OLCR were developed. The first technique uses oscillations in the OLCR amplitude signal to directly obtain the beat length and the birefringence. The second method is based on the measurement of the OLCR phase and inverse scattering algorithm. Both methods were compared with birefringence obtained from spectral measurements, and very good agreement was obtained. The indirect method, based on local Bragg grating determination requires two independent measurements and mathematical reconstruction, but provides a very high spatial resolution of ≈ 25 µm. The grating length limits the minimum measurable birefringence in the direct method, but a good sensitivity of 4×10-6 was obtained, which corresponds to a Bragg wavelength shift of 4 pm. The spatial resolution is in the millimeter range in this case. The novel methods were successfully applied for the distributed measurement of birefringence of FBG under diametric load. New types of tunable devices and sensors – fiber Bragg gratings written in high attenuation fibers (HAF) were developed. Active tuning by heating was achieved by optical pumping of a pure single mode fiber without any mechanical or electrical parts, or deposited light absorption coatings. Tuning can be controlled by the applied pump power, the position of the grating, the HAF attenuation level, and the pumping configuration. The proposed design assures a high repeatability and long lifetime. Using OLCR these devices were successfully applied to measure the liquid level with a spatial resolution of 100 micrometers. Liquid level measurements that use both phase and amplitude, or only amplitude were demonstrated. The same principles could be easily applied to design other fiber grating devices (long-period, tilted etc), and sensors based on hot-wire anemometry, like flow or vacuum sensors.

EPFL2007

Mid-infrared surface plasmon resonance sensing applied to refractive index measurements of liquids

Sylvain Herminjard

There has been a very strong development of the sensors based on surface plasmon resonance during the last thirty years, mostly for biological and biomedical applications. If the first experiments in this field were carried out at the beginning of the 20t century, it would have been necessary to wait for the end of the sixties to see the first devices performing accurate measurements of the refractive indices of gas and liquid samples. Indeed, the surface plasmon resonance phenomenon allows to measure refractive index variations with a high sensitivity that is hardly obtained with other optical measurement techniques. The basic principle consists of exciting the free electrons of a metallic layer with light, which induces the formation of a surface wave called surface plasmon. This resonant phenomenon depends on the wavelength of the excitation light and the refractive indices of the metallic layer and the material located close to the metallic layer. A variation of the refractive index of the latter induces a variation of the resonance condition, which allows to realize optical sensors designed to measure determined samples. An appropriate calibration allows to set a relationship between the variation of the concentration of the sample and the measured refractive index value. Among the various parameters that describe the performances of the sensor, one of the most important is the sensitivity. It is possible to demonstrate that the excitation wavelength has an impact on the sensitivity of the system : the latter increases if the measured sample has an absorption peak at a wavelength close to the excitation wavelength. The main goal of this thesis is to explore this sensitivity enhancement technique based on the absorption properties of the probed medium. A proof-of-principle is experimentally demonstrated with an optical setup constituted of laser sources working in the visible range of the electromagnetic spectrum. However, many materials have absorption peaks located in the mid-infrared range due to their fundamental molecular vibrations. Therefore, the realization of a plasmonic sensor working in the mid-infrared could allow to measure these materials with an increased sensitivity. To the best of our knowledge, surface plasmon resonance based sensors have never been developed above the near-infrared. This thesis presents for the first time the design and realization of a plasmonic sensor working in the mid-infrared range able to measure refractive index variations of gas and liquids samples. Even if the optical sources, components and detectors designed for the mid-infrared are clearly different from the ones designed for the visible range, it has been possible to successfully realize two systems working in the mid-infrared range during this project. The first is able to perform gas measurements. As the excitation wavelength depends on the measured sample, it was necessary to select a laser source that had a wavelength located close to the absorption peak of the gas under measurement. For this purpose, we designed a plasmonic sensor working at a 4.4 µm wavelength able to measure carbon dioxide (CO2) absorbing at 4.3 µm. It has been demonstrated that the experimental sensitivity of this system is five times higher than the theoretical sensitivity obtained with a system working in the visible range at a 633 nm wavelength. The second has been conceived to measure liquid samples in order to measure variations of concentration of CO2 dissolved in water. If measurements of refractive index variations (without using the sensitivity enhancement technique) could be performed successfully with variations of concentration of alcohol in water, further work is necessary to get decisive results for CO2 dissolved in water. This project was carried out in collaboration with an industrial partner in order to take into account some industrial constraints during the development of the sensor. Therefore, particular care has been taken in order to design a system that is compact, mechanically robust, without any mechanical moving parts, cheap and easily reproducible. The technological development presented in this thesis finds many applications mainly in the chemical, biological and biomedical fields. Indeed, many proteins show absorption peaks in the mid-infrared. It could be possible to measure their concentration or the modification of their properties – like their composition or structure – with an increased sensitivity. The interest of surface plasmons is that the measurement is not a transmission-based technique, but rather a "reflection-based technique", as the light does not go through the gas or liquid sample. This measurement technique is also adapted for high volume samples that are present in industrial production processes like water or soft drink quality control.

EPFL2010