Genetic dissection of the glutamatergic neuron system in cerebral cortex.

Diverse types of glutamatergic pyramidal neurons mediate the myriad processing streams and output channels of the cerebral cortex1,2, yet all derive from neural progenitors of the embryonic dorsal telencephalon3,4. Here we establish genetic strategies and tools for dissecting and fate-mapping subpopulations of pyramidal neurons on the basis of their developmental and molecular programs. We leverage key transcription factors and effector genes to systematically target temporal patterning programs in progenitors and differentiation programs in postmitotic neurons. We generated over a dozen temporally inducible mouse Cre and Flp knock-in driver lines to enable the combinatorial targeting of major progenitor types and projection classes. Combinatorial strategies confer viral access to subsets of pyramidal neurons defined by developmental origin, marker expression, anatomical location and projection targets. These strategies establish an experimental framework for understanding the hierarchical organization and developmental trajectory of subpopulations of pyramidal neurons that assemble cortical processing networks and output channels

New optical techniques and hardware for studying live cell dynamics

This thesis was previously held under moratorium from 12/02/2019 to 21/05/2021.Fluorescence optical microscopy has become an integral technique in the life sciences and has opened the door to investigating live biological specimens non-invasively at sub-cellular spatial resolutions with high specificity and temporal resolutions. One of the limiting factors of optical microscopy is that the spatial resolution is dictated by the diffraction limit of light.;This work shows the first use of LEDs to carry out widefield axial super-resolution standing wave microscopy with high temporal resolution. The technique was used to image red blood cell membrane dynamics in real time with no increase in photobleaching or toxicity rates compared to standard widefield imaging. This work also presents 3D computational reconstructions of the data allowing for easier visualisation and the possibility of carrying out further quantitative analysis.;Following on from Chapter 2, is an investigation into the development and application of multi-wavelength standing wave microscopy on live specimens in both emission and excitation modalities. These techniques are henceforth referred to in this thesis as TartanSW. This investigation found that using multiple excitation wavelengths allowed for a reduction in the nodal contribution of the images resulting in obtaining 32.3 % more spatial information about the structure of the specimen. It is also shown that by taking the difference images between each excitation channel the standing wave antinodal planes could be reduced in thickness enabling axial resolutions on the order of 55 nm when imaging live cell experiments.;The multi-emission technique was shown that it could be applied to be applied to imaging biological specimens using both widefield and confocal microscopy. However, the widefield data was not in line with the expected theoretical structure. There is the possibility of using plane ordering though to infer the directionality of a specimen structure and extract height maps though further work to develop computational tools to enable this will have to be implemented.;Finally, this thesis describes the work carried out making use of a new high-brightness 340 nm LED to develop a fast switching 340/380 nm illuminator and demonstrate its application for ratiometric Fura-2 Ca2+ imaging of live cell specimens with sub-5 nM precision that supports full frame video-rate temporal resolutions.Fluorescence optical microscopy has become an integral technique in the life sciences and has opened the door to investigating live biological specimens non-invasively at sub-cellular spatial resolutions with high specificity and temporal resolutions. One of the limiting factors of optical microscopy is that the spatial resolution is dictated by the diffraction limit of light.;This work shows the first use of LEDs to carry out widefield axial super-resolution standing wave microscopy with high temporal resolution. The technique was used to image red blood cell membrane dynamics in real time with no increase in photobleaching or toxicity rates compared to standard widefield imaging. This work also presents 3D computational reconstructions of the data allowing for easier visualisation and the possibility of carrying out further quantitative analysis.;Following on from Chapter 2, is an investigation into the development and application of multi-wavelength standing wave microscopy on live specimens in both emission and excitation modalities. These techniques are henceforth referred to in this thesis as TartanSW. This investigation found that using multiple excitation wavelengths allowed for a reduction in the nodal contribution of the images resulting in obtaining 32.3 % more spatial information about the structure of the specimen. It is also shown that by taking the difference images between each excitation channel the standing wave antinodal planes could be reduced in thickness enabling axial resolutions on the order of 55 nm when imaging live cell experiments.;The multi-emission technique was shown that it could be applied to be applied to imaging biological specimens using both widefield and confocal microscopy. However, the widefield data was not in line with the expected theoretical structure. There is the possibility of using plane ordering though to infer the directionality of a specimen structure and extract height maps though further work to develop computational tools to enable this will have to be implemented.;Finally, this thesis describes the work carried out making use of a new high-brightness 340 nm LED to develop a fast switching 340/380 nm illuminator and demonstrate its application for ratiometric Fura-2 Ca2+ imaging of live cell specimens with sub-5 nM precision that supports full frame video-rate temporal resolutions

Strain softening and strain localisation in irreversible deformation of snow

The aim of this work was to visualise heterogeneous deformation in snow under controlled laboratory conditions. Heterogeneous deformation was observed for both homogenous and heterogeneous loading conditions. Understanding deformation of snow is important in many scientific fields including vehicle traction, avalanche forecasting, and winter sports. This thesis investigates the deformation behaviour of snow on the centimetre scale under moderate strain rates (0.005 to 0.1 s-1) when subject to one-dimensional compression or to indentation. In order to allow controlled and repeatable snow deformation experiments, a new type of artificial snow was developed. This snow type was examined by low temperature scanning electron microscopy and by traditional avalanche observer’s methodology. Penetrometer experiments were conducted on the artificial snow and on natural seasonal snow in Scotland. The two snow types were found to be similar: results obtained on artificial snow are thus applicable to natural snow. A reproducible technique of manufacture and a thorough characterisation of the artificial snow are presented. One-dimensional compression experiments were conducted on the artificial snow. The experiments were in confined compression in a specially constructed apparatus, designed to provide for back-lit photography. Images were taken at 0.25 second intervals and analysed using digital image correlation, thus providing 2D strain fields. With careful control of photographic parameters, it is demonstrated that process of applying tracer substances to the snow is not necessary, thus allowing an unprecedented resolution. Spontaneously-forming strain localisations were observed for the first time, indicating strain softening behaviour. Damage was observed to propagate through the specimen as a moving front, resembling a wave. The force required to propagate the front remained nearly constant until the whole specimen was compacted, at which point a new front formed and the process repeated. The experimental method was extended to 2D indention experiments with a range of sizes and shapes of indenter. Complex deformation fields were observed, extending up to 6 times the width of the indenter on each side. Observed deformation included tensile tearing as well as compression and shear. The maximum local strain achieved in the indentation experiments was similar to that achieved by the first compaction front in one-dimensional compression. The work here presented has implications for snow deformation generally: strain localisation introduces a characteristic length, which may prevent scaling of models or results. The indentation results are particularly relevant to snow penetrometry, where indentation experiments are used to try and extract microstructural information from buried snow layers for the purpose of avalanche prediction. The common assumption that the penetrometer interacts only with snow very close to its tip may need to be reconsidered

Understanding novel gap-bridged remote laser welded (RLW) joints for automotive high-rate and temperature applications

This paper investigates the microstructure, high-rate and temperature dependent tensile behaviour of fillet edge joints produced by novel ‘gap-bridged’ remote laser welding (RLW) using an automotive grade aluminium alloy AA6014, commercially known as AC-170PX, extensively used for automotive skin panel applications. Three part-to-part gap-bridged RLW fillet edge welds, produced with different gaps (0.2 mm, 0.4 mm and 0.6 mm) were examined for joint geometry and microstructure. Relatively larger columnar grains resulting from directional solidification were observed in the fusion zone and microhardness was reduced by ~15- 20% due to precipitates disappearance. Moderate (0.1 m/s) to high speed rate (10 m/s) tensile tests performed at room temperature (~23°C) were used to determine high-rate tensile performance. Although the strain rate dependency was found to be low, an increase in tensile extension was obtained. Additionally, the joint tensile performance was evaluated over a range of temperatures between -50°C and 300°C. Using digital image correlation (DIC), fracture strains were obtained in the range from 0.21 to 0.25 for all gap and speed conditions. Fusion zone based finite element simulations were performed using the Johnson-Cook material failure model to predict joint strength. Additionally, the suitability of gap-bridged RLW joints for automotive applications was determined by comparison with two industrial joining methods, self-piercing riveting (SPR) and resistance spot welding (RSW)

Noise reduction results of the ACASIAS Active Lining Panel

Advanced concepts for aero-structures with multifunctional capabilities are investigated within the EU-project ACASIAS. In work package 3 of ACASIAS, components of an active noise reduction system are structurally integrated into a curved sandwich panel by means of 3D printed inserts. This so-called smart lining is intended for application in aircraft as a modular and lightweight interior noise treatment in propeller-driven aircraft. The broad application scenario of smart linings ranges from retro-fitting of current regional aircraft such as ATR 42, ATR 72, DHC-8 Q400 to the application in new short-range aircraft with energy efficient counter rotating open rotor (CROR) engines or with distributed electric propellers. A key feature of the smart lining with integrated active components is its modularity, facilitating a flexible application in the aircraft cabin. This requires a fully self-contained sensing mechanism based on structurally integrated accelerometers. Using the normal surface vibration data from the integrated sensors, the smart lining is able to predict the sound field in front of it. The so-called virtual microphone method with remote sensors and observer filter allows to get rid of real microphones and wiring in the aircraft cabin. This makes retro-fitting easier because it reduces wiring effort and costs which is beneficial for future aircraft as well. However, the use of virtual instead of real microphones might deteriorate the performance or even the stability of the active noise reduction system because it relies on accurate plant models. Laboratory experiments in a sound transmission loss facility are conducted to assess the behavior of the smart lining with virtual microphones and compare it to a smart lining with real microphones. The sensitivity of the smart lining to environmental changes and the noise reduction performance and control system stability are investigated in this study

Single-molecule techniques in biophysics : a review of the progress in methods and applications

Single-molecule biophysics has transformed our understanding of the fundamental molecular processes involved in living biological systems, but also of the fascinating physics of life. Far more exotic than a collection of exemplars of soft matter behaviour, active biological matter lives far from thermal equilibrium, and typically covers multiple length scales from the nanometre level of single molecules up several orders of magnitude to longer length scales in emergent structures of cells, tissues and organisms. Biological molecules are often characterized by an underlying instability, in that multiple metastable free energy states exist which are separated by energy levels of typically just a few multiples of the thermal energy scale of kBT, where kB is the Boltzmann constant and T the absolute temperature, implying complex, dynamic inter-conversion kinetics across this bumpy free energy landscape in the relatively hot, wet environment of real, living biological matter. The key utility of single-molecule biophysics lies in its ability to probe the underlying heterogeneity of free energy states across a population of molecules, which in general is too challenging for conventional ensemble level approaches which measure mean average properties. Parallel developments in both experimental and theoretical techniques have been key to the latest insights and are enabling the development of highly-multiplexed, correlative techniques to tackle previously intractable biological problems. Experimentally, technological developments in the sensitivity and speed of biomolecular detectors, the stability and efficiency of light sources, probes and microfluidics, have enabled and driven the study of heterogeneous behaviours both in vitro and in vivo that were previously undetectable by ensemble methods..