Atomic Force Microscopy for Martian Investigations

The Phoenix Mars Lander includes a Microscopy, Electrochemistry and Conductivity Analyser (MECA) instrument for the study of dust and regolith at the Martian arctic. The microscopy payload comprises an AFM and Optical Microscope (OM) to which samples are delivered by a robot arm. The setup allows imaging of individual dust and soil particles at a higher spatial resolution than any other in-situ instrument. A fully functioning test-bed of the flight microscopy setup within an environmental chamber to simulate Mars conditions was assembled at Imperial College, enabling characterization of the microscopes. Samples are collected on small disks rotated to the vertical position for imaging, with each substrate surface promoting different adhesion mechanisms. The vertical mounting necessitates good adhesion of particles to substrates. Moreover, to achieve safe operation and good AFM scans, a sparse field of particles is required. This work investigates models and experimental setups which consider the adhesion mechanisms of particles, including under Mars conditions. These models incorporate the forces from the AFM cantilever during scanning, particle-substrate adhesion and particle-tip adhesion. The solution offered to the problem of unstable particles is substrates with engineered features, micromachined in silicon, to trap and stabilise particles for AFM and reduce the loading of the sample to a suitable level. Various designs were investigated in a series of tests, and a final design was created for a substrate for AFM during the mission. The substrates were fabricated and incorporated on the sample wheel on Phoenix, now on Mars. The MECA results are discussed, focusing in particular on the characterization, calibration and cataloguing of samples using the Imperial College testbed. The best ways of obtaining data from the setup were investigated. These strategies were used during the Phoenix mission. Finally, the extant microscopy data acquired during surface operations are presented and the overall operations procedures discussed

HIGH-BANDWIDTH IDENTIFICATION AND COMPENSATION OF HYSTERETIC DYNAMICS

Ph.DDOCTOR OF PHILOSOPH

Nanotechnology approaches to combat antimicrobial resistance: novel therapeutics and diagnostics

Antimicrobial resistance (AMR) is a global issue caused by misuse of antibiotics and a lack of new antibiotics coming to market. Combating the development of AMR requires the development of new antimicrobial agents to treat bacterial infections and better diagnostic tools for improved stewardship. This thesis describes novel approaches to address these issues using nanotechnology. The main technique employed in these studies was atomic force microscopy (AFM), used in both conventional imaging mode and in an innovative sensing capacity. From a therapeutic perspective, the mechanism of action of novel antimicrobial structures (protein / DNA) were studied, using real-time imaging on model membranes and live E. coli cells. Nanometre resolution was achieved on both systems, allowing rapid membrane poration and subsequent cell death to be observed for a de novo designed antimicrobial peptide and a pioneering antimicrobial DNA-lipid origami structure. In addition, this thesis describes the first visualisation of the Membrane Attack Complex (MAC) on live bacterial cells showing remarkable similarity with the recently solved cryo-EM structure. In working to develop novel phenotypic diagnostic tools for AMR, we report on a novel antibiotic susceptibility testing (AST) device. This device uses single cell optical interference to provide a rapid (∼45 min) and simple measure of the effect of antimicrobials on suspended bacterial cells. Homebuilt code was developed to analyse datasets, allowing antibiotic sensitivity to be systematically determined for lab and clinical strains of E. coli. This thesis provides insights into a number of potential avenues to pursue in the face of increasing AMR, with future work entailing moving the described from the lab closer to clinical use

High Resolution Atomic Force Microscopy of Functional Biological Molecules

Nanoscale dynamic biological processes are central to the regulation of cellular pro- cesses within the body. The direct visualisation of these processes represents a challenge because of the intrinsic difficulties of imaging at the nanoscale, well below the diffraction limit of light. Here we use the Atomic Force Microscope to ‘feel’ the structure of single biomolecules adsorbed to a flat substrate at sub-nanometre resolution. We have enhanced the performance and resolution of Atomic Force Microscopy (AFM) for imaging DNA plasmids in solution, resolving its secondary structure in the form of the double helix. We are able to observe local deviations from the average structure, and in particular variations in the depth of the grooves in the double-stranded DNA which may be attributed to supercoiling of the DNA. Such local variations of the DNA double helix structure are important in mediating protein-DNA binding specificity and thus in regulating gene expression. We show preliminary data on DNA minicircles, which can be used as a synthetic system to study how supercoiling affects DNA structure and influences DNA-protein binding interactions with implications for many genetic processes. Going from fundamental science to a biomedical application, we have used AFM to study the functional mechanisms of antimicrobial peptides, which are developed in response to the growing problem of antimicrobial resistance. Antimicrobial peptides disrupt microbial phospholipid membranes but direct observation of the mode of action for the disruption is lacking. Here we visualise the mode of action of syn- thetic antimicrobial cationic alpha-helical peptides. Two of these peptides attack membrane via previously unknown mechanism: Amhelin forms pores which are not limited in size but expand from the nano to micrometre scale; Amhelit also forms pores which penetrate a single layer of the lipid bilayer that forms the membrane. We present the first nanoscale visualisation of membrane disruption by the naturally occurring antimicrobial peptide cecropin B. This is complemented by the visualisa- tion of peptides similar in sequence to cecropin B, but with structural modifications which are used to elucidate the structural origins of cecropin B’s mechanism of ac- tion. Improvements in imaging capabilities of the AFM, as tested on DNA, were shown to benefit imaging of the mode of action for antimicrobial peptides, including time-lapse imaging of a novel expanding monolayer state. We have thus used AFM to elucidate mechanisms of action for antimicrobial pep- tides. Relating these mechanisms to the peptide sequences, we can gain insight into how peptide sequence affects structure and function for these antimicrobial agents. This may aid in the development and improvement of novel peptide antibiotics

Thermal characterisation of miniature hotplates used in gas sensing technology

The reliability of micro-electronic devices depends on the device operating temperature and therefore self-heating can have an adverse effect on the performance and reliability of these devices. Hence, thermal measurement is crucial including accurate maximum operating temperature measurements to ensure optimum reliability and good electrical performance. In the research presented in this thesis, the high temperature thermal characterisation of novel micro-electro-mechanical systems (MEMS) infra-red (IR) emitter chips for use in gas sensing technology for stable long-term operation were studied, using both IR and a novel thermo-incandescence microscopy. The IR emitters were fabricated using complementary-metal-oxide semiconductor (CMOS) based processing technology and consisted of a miniature micro-heater, fabricated using tungsten metallisation. There is a commercial drive to include MEMS micro-heaters in portable electronic applications including gas sensors and miniaturised IR spectrometers where low power consumption is required. IR thermal microscopy was used to thermally characterise these miniature MEMS micro-heaters to temperatures approaching 700 °C. The research work has also enabled further development of novel thermal measurement techniques, using carbon microparticle infra-red sensors (MPIRS) with the IR thermal microscopy. These microparticle sensors, for the first time, have been used to make more accurate high temperature (approaching 700 °C) spot measurements on the IR transparent semiconductor membrane of the micro-heater. To substantially extend the temperature measurement range of the IR thermal microscope, and to obtain the thermal profiles at elevated temperatures (> 700 °C), a novel thermal measurement approach has been developed by calibrating emitted incandescence radiation in the optical region as a function of temperature. The calibration was carried out using the known melting point (MP) of metal microparticles. The method has been utilised to obtain the high temperature thermo-optical characterisation of the MEMS micro-heaters to temperatures in excess of 1200 °C. The measured temperature results using thermo-incandescence microscopy were compared with calculated electrical temperature results. The results indicated the thermo-incandescence measurements are in reasonable agreement (± 3.5 %) with the electrical temperature approach. Thus, the measurement technique using optical incandescent radiation extends the range of conventional IR microscopy and shows a great potential for making very high temperature spot measurements on electronic devices. The high power (> 500mW) electrical characterisation of the MEMS micro-heaters were also analysed to assess the reliability. The electrical performance results on the MEMS micro-heaters indicated failures at temperatures greater than 1300 °C and Scanning Electron Microscope (SEM) was used to analyse the failure modes

Roadmap for optical tweezers

Artículo escrito por un elevado número de autores, solo se referencian el que aparece en primer lugar, el nombre del grupo de colaboración, si le hubiere, y los autores pertenecientes a la UAMOptical tweezers are tools made of light that enable contactless pushing, trapping, and manipulation of objects, ranging from atoms to space light sails. Since the pioneering work by Arthur Ashkin in the 1970s, optical tweezers have evolved into sophisticated instruments and have been employed in a broad range of applications in the life sciences, physics, and engineering. These include accurate force and torque measurement at the femtonewton level, microrheology of complex fluids, single micro- and nano-particle spectroscopy, single-cell analysis, and statistical-physics experiments. This roadmap provides insights into current investigations involving optical forces and optical tweezers from their theoretical foundations to designs and setups. It also offers perspectives for applications to a wide range of research fields, from biophysics to space explorationEuropean Commission (Horizon 2020, Project No. 812780

Roadmap for optical tweezers

Optical tweezers are tools made of light that enable contactless pushing, trapping, and manipulation of objects, ranging from atoms to space light sails. Since the pioneering work by Arthur Ashkin in the 1970s, optical tweezers have evolved into sophisticated instruments and have been employed in a broad range of applications in the life sciences, physics, and engineering. These include accurate force and torque measurement at the femtonewton level, microrheology of complex fluids, single micro- and nano-particle spectroscopy, single-cell analysis, and statistical-physics experiments. This roadmap provides insights into current investigations involving optical forces and optical tweezers from their theoretical foundations to designs and setups. It also offers perspectives for applications to a wide range of research fields, from biophysics to space exploration.journal articl

Roadmap for Optical Tweezers 2023

Optical tweezers are tools made of light that enable contactless pushing, trapping, and manipulation of objects ranging from atoms to space light sails. Since the pioneering work by Arthur Ashkin in the 1970s, optical tweezers have evolved into sophisticated instruments and have been employed in a broad range of applications in life sciences, physics, and engineering. These include accurate force and torque measurement at the femtonewton level, microrheology of complex fluids, single micro- and nanoparticle spectroscopy, single-cell analysis, and statistical-physics experiments. This roadmap provides insights into current investigations involving optical forces and optical tweezers from their theoretical foundations to designs and setups. It also offers perspectives for applications to a wide range of research fields, from biophysics to space exploration

Investigation of the phospholipid peripheral region of lactose permease in model membranes

[cat] La interacció entre una proteïna de membrana i els fosfolípids que l’envolten és crucial pel bon plegament i la correcta funció de la proteïna. Aquesta tesi està centrada en la investigació de la interacció entre la Lactosa permeasa (LacY), un paradigma dels transportadors secundaris situat a la membrana interna d’Escherichia coli, i sistemes models que mimetitzen el seu entorn lipídic. Aquest treball representa una contribució al camp a través de l’estudi de la interacció a dos nivells: (i) la interacció entre LacY i els fosfolípids presents a la regió anular propera a la proteïna ha estat estudiada a través de mesures de FRET entre un mutant de LacY amb un únic triptòfan i diversos fosfolípids marcats i (ii) la interacció entre LacY amb els fosfolípids més llunyans o bulk s’ha investigat a través de làmines de lípid i proteïna sobre un suport, les quals s’han analitzat a partir de diversos modes de microscòpia de força atòmica (topografia, espectroscòpia de força i force-volume). En primer lloc, s’ha validat la preferència de LacY pels fosfolípids en fases fluïdes (Lα). A més, s’ha confirmat una composició lipídica entre la regió anulars i el bulk. Així, els fosfolípids bulk, considerats com a fosfolípids en fase Lα, tenen PG com a principal component, mentre que PE és el major component de la regió anular. Això sembla indicar una selectivitat entre LacY i els fosfolípids anulars. En segon lloc, s’ha descrit que la selectivitat de LacY per fosfolípid determinat a la regió anular està relacionada amb (i) càrrega neutra i (ii) curvatura espontània (C0) negativa. A més, D68 s’ha assenyalat com un aminoàcid important per la selectivitat de la proteïna envers els lípids anulars. Finalment, s’ha descrit una interacció recíproca entre LacY i els lípids bulk. Així, la presencia de la proteïna modifica la topografia i la nanomecànica del sistema lipídic, especialment de la fase Lα, i, alhora, la nanomecànica de la pròpia LacY varia segons la matriu lipídica que l’envolta. En conseqüència, la composició lipídica de la bicapa sembla determinar les forces que governen l’estreta interacció de LacY amb la membrana i, per tant, aquesta composició és decisiva per la correcta inserció i activitat de la proteïna.[eng] The interaction between a membrane protein and its surrounding phospholipids is thought to be crucial for the correct folding and function of the protein. This thesis is focused on the investigation of the interplay between Lactose permease (LacY), a paradigm for secondary transporters present in the inner membrane of Escherichia coli and model systems mimicking its natural lipid environment. Since the role of phospholipids in LacY’s activity is currently being refined, this work represents a contribution to the field by studying the interaction at two different levels: (i) the LacY interplay with the phospholipids present at the annular region in the vicinity of the protein was studied through FRET measurements between a single-tryptophan LacY mutant and diverse pyrene-marked phospholipids, and (ii) the LacY interaction with the more distanced bulk phospholipids was studied through supported proteo-lipid sheets that were analysed using topography, force-spectroscopy and force-volume Atomic Force Microscopy modes. First, after validating LacY preference for phospholipid fluid (Lα) phases in the studied two-component model systems, a different composition between bulk and annular regions was confirmed. Hence, bulk lipids, which were assimilated to the phospholipids in Lα phase, were mainly formed by PG, while PE was the main component of the annular region. This points to a direct annular phospholipid-LacY selectivity because it discards a random phospholipid distribution near the protein. Second, the LacY selectivity for precise phospholipid species at the annular region was found to be related to: (i) a neutral charged phosholipid (PE or PC, with preference for the former), and (ii) phospholipids with large negative spontaneous curvature (C0) (DOPE > POPE). In addition, D68 was revealed as an important amino acid for the protein annular lipid selectivity. Third, the interaction between LacY and the bulk lipids was described as reciprocal. Accordingly, the presence of the protein largely modified the topography and the nanomechanics of the lipid system, especially for the Lα phase, whilst the nanomechanics of LacY itself were different depending on the surrounding lipid matrix: more force was needed to pull LacY form the DPPE:POPG (3:1, mol/mol) system than from the POPE:POPG (3:1, mol/mol) one. Therefore, the bilayer lipid composition seems to determine the forces governing the LacY tight interaction with the membrane and can be thus decisive for the protein correct insertion and activit