Single biomolecule studies using optical tweezers

Single biological molecule studies enable to probe and visualize exciting details of the events in physiological in vivo processes. The basic underlying question of this dissertation is to understand biological processes at a single molecule level. In contrast to ensemble techniques, advances in single molecule manipulation (e.g. optical and magnetic tweezers, atomic force microscopy) and / or fluorescence techniques allow to investigate the properties of individual molecules in real time with a possibility to change external conditions (buffers) in situ and modulate inter- and intra-molecular interactions. This thesis reports the application of a single molecule technique, dual beam optical tweezers, for the study of single biomolecules. A range of single molecule systems was investigated such as i)VirE2 protein DNA machinery, ii) DNA-surfactant, EtBr (ethidium bromide), SYBR® Green-DNA interactions and iii) dsDNA denaturation studies. In addition the development of the present experimental setup is described to enable combined force measurement as well as single molecule fluorescence studies. The presented biomolecular results provide new and complementary information on the different biological systems demonstrating the diversity of experiments that can be performed on single DNA molecules using optical tweezers. Chapter one gives a brief introduction to optical tweezers, describes how optical tweezers work, the physics behind it, details of the experimental setup and the method of force calibration required in micromanipulation. Optical tweezers have opened exciting avenues of research, especially in biology. Biologists will be able to investigate the nature of molecular machines one by one, and infer from their behavior those properties common to the population. In chapter 2, we show how optical tweezers were employed to study the change in the mechanical properties of single DNA molecules upon binding of small agents. The first part of this chapter reports on the changes in mechanics of single dsDNA in the presence of cationic and anionic surfactants (used as non-viral vectors in gene therapy). The second part describes the interaction of DNA binding ligands (SYBR® Green, EtBr) with individual DNA strands. Agrobacterium tumefaciens (AT), a Gram-negative bacterium, evolved a complex and unique mechanism to transfer a long single stranded DNA (ssDNA) molecule from its cytoplasm to the eukaryotic host plant cell nucleus. Central to this mechanism, chapter 3 discusses the results of the measurements on VirE2 protein interacting with single stranded DNA (ssDNA). VirE2 protein is a multifunctional protein from AT that coat the transferred-ssDNA (T-DNA), interacts with host factors assisting nuclear import of the complex, forms channels in lipid bilayers and displays a highly cooperative binding to ssDNA. The biological findings are presented in a new generic model which can be used to explain how generation of forces helps bacterial DNA to enter the plant cell based on our single molecule data. Single molecule dsDNA denaturation, relevant in many molecular biological experiments, induced by NaOH and mechanical pulling are studied in chapter 4. Here optical tweezers experiments give access to the ‘melting’ of hydrogen bonds by mechanical forces or alkali denaturation (NaOH) of dsDNA in real time. The mechanical stability and the transition of dsDNA to ssDNA is investigated at different ionic strength as well as in buffers. Fluorescent images of single λ DNA labeled with SYBR® Green were observed up to forces ≥ 65 pN and indicate a B-DNA to S −DNA transition. Chapter 5 describes the implementation of single-molecule fluorescence detection (SMF) in optical tweezers. The design and instrumental capabilities of optical tweezers combined with SMF are discussed in detail. The development of this instrument provides a worldwide unique experimental setup and opens up new possibilities in the studies of complex biological systems. Finally chapter 6 summarizes the results of this thesis and discusses future experimental applications. The appendices provide further details for DNA sample preparation, molecular biology and chemical surface activation recipes, an instruction manual for the setup and the list of currently published papers

Superconducting properties of NbN film, bridge and meanders

The transport properties of superconducting NbN nanostructures in the form of thin film, bridge of width (w) = 50 mu m and three meanders of w = 500, 250 and 100 nm have been investigated by resistance (R) measurements in temperature (T) range = 2 - 300 K and magnetic field (B) range = 0 - 7 Tesla. The nanostructuring was carried out using Focused Ion Beam (FIB) milling. Reduction of sample width results in significant changes in the normal and superconducting state properties. For instance, the observed metallic behavior in the thin film sample is lost and the normal state resistance increases drastically from 2.4 Omega to 418 k Omega for the 100 nm meander. In the superconducting state, the value of critical temperature T-c (upper critical field B-c2 at T = 0 K) reduces gradually with width reduction, it changes from 13.15K(42.8 Tesla) in the case of thin film sample to 5.7K(12.7 Tesla) for the 100 nm meander sample. The superconducting transitions are found to get broader for the bridge sample and the meanders additionally show low-temperature resistive tails. In case of all the samples with reduced width, the transition onsets are found to be rounded at surprisingly high values of T similar to 25K > > T-c. These results are discussed in terms of the possible effects of FIB processing and weak localization in our samples

Superconducting properties of NbN film, bridge and meanders

The transport properties of superconducting NbN nanostructures in the form of thin film, bridge of width (w) = 50 μm and three meanders of w = 500, 250 and 100 nm have been investigated by resistance (R) measurements in temperature (T) range = 2 -300 K and magnetic field (B) range = 0 - 7 Tesla. The nanostructuring was carried out using Focused Ion Beam (FIB) milling. Reduction of sample width results in significant changes in the normal and superconducting state properties. For instance, the observed metallic behavior in the thin film sample is lost and the normal state resistance increases drastically from 2.4 Ω to 418 kΩ for the 100 nm meander. In the superconducting state, the value of critical temperature Tc (upper critical field Bc2 at T = 0 K) reduces gradually with width reduction, it changes from 13.15 K (42.8 Tesla) in the case of thin film sample to 5.7 K (12.7 Tesla) for the 100 nm meander sample. The superconducting transitions are found to get broader for the bridge sample and the meanders additionally show low-temperature resistive tails. In case of all the samples with reduced width, the transition onsets are found to be rounded at surprisingly high values of T ∼ 25 K >> Tc. These results are discussed in terms of the possible effects of FIB processing and weak localization in our samples

Superconducting properties of NbN film, bridge and meanders

The transport properties of superconducting NbN nanostructures in the form of thin film, bridge of width (w) = 50 μm and three meanders of w = 500, 250 and 100 nm have been investigated by resistance (R) measurements in temperature (T) range = 2 -300 K and magnetic field (B) range = 0 - 7 Tesla. The nanostructuring was carried out using Focused Ion Beam (FIB) milling. Reduction of sample width results in significant changes in the normal and superconducting state properties. For instance, the observed metallic behavior in the thin film sample is lost and the normal state resistance increases drastically from 2.4 Ω to 418 kΩ for the 100 nm meander. In the superconducting state, the value of critical temperature Tc (upper critical field Bc2 at T = 0 K) reduces gradually with width reduction, it changes from 13.15 K (42.8 Tesla) in the case of thin film sample to 5.7 K (12.7 Tesla) for the 100 nm meander sample. The superconducting transitions are found to get broader for the bridge sample and the meanders additionally show low-temperature resistive tails. In case of all the samples with reduced width, the transition onsets are found to be rounded at surprisingly high values of T ∼ 25 K >> Tc. These results are discussed in terms of the possible effects of FIB processing and weak localization in our samples

Ultrafast carrier and phonon dynamics in thin films of bismuth telluride on a flexible substrate

International audienceThin films of topological insulators (TIs) possess exotic nonlinear optical properties such as strong light-matter interaction, broadband spectral sensitivity, thickness-dependent tunable bandgap, higher harmonic generation, etc. Due to the presence of metallic surface states with Dirac fermions and an insulating bulk band in TI, they are projected to be a viable material for studying novel physics, resulting in exciting new properties and technologies. The peculiar electron-phonon interactions at the surface have been linked to various unexpected physical features of topological insulators. Although electron behaviour in topological insulators has been extensively investigated on non-flexible substrates, electron-phonon interactions at TI bulk and surfaces states are less well known on a flexible substrate. Because of its potential uses in wearable devices, communications, sensors, and other fields, there is a significant need for the manufacture of high performance flexible optoelectronic responses employing novel exotic materials. In this paper, we preformed ultrafast pump-probe method to explore TI (Bi2Te3) thin films on a flexible PET (polyethylene terephthalate) substrate. We studied the dynamics of Bi2Te3 thin films' hot carrier relaxation progression and coherent phonon behaviour using transient absorbance measurements. Thickness-dependent low-frequency coherent acoustical phonon oscillations are observed in 10 nm thick films, and the changes vanish for 25 nm thick films, and high-frequency optical phonon oscillations are absent in our work. Subpicosecond range of time constant for the photon excitation, diffusion, carrier thermalization, and relaxation are reported. Longer characteristics time was observed for 25 nm film as compared to 10 nm film, and its variation has been discussed here. The thickness-dependent coherent acoustic phonon oscillating in the terahertz frequency range has been experimentally calculated. The film's terahertz frequency response varies with its thickness, allowing it to be employed in future terahertz applications based on flexible topological insulator thin films