The conventional epi-uorescent wide-eld microscope features a uniform illumination of an extended sample region. A problem arises with this setup since light from outof-focus uorophores is also detected. The result is poor quality in the nal image, as out-of-focus structures appear blurred; furthermore, their emission light contributes to the background and leads to a reduction in image contrast. Removing out-of-focus light yields an optically sectioned image: a thin slice of a thick sample that only contains in-focus information. Taking a stack of such sectioned images allows for a three-dimensional (3D) view of the specimen. Structured illumination microscopy for sectioning (sSIM) is a method to obtain optically sectioned data, similar to that obtained from the widely used confocal microscope. However, sSIM suffers from a limited acquisition rate, as at least three individual raw images are needed to reconstruct one sectioned slice. The technique of polarised illumination coded structured illumination microscopy (picoSIM) combines optical sectioning with high temporal resolution. In picoSIM the individual light patterns needed to acquire the raw sSIM images are encoded in the polarisation of the illumination light. This enables the simultaneous acquisition of the data needed for the sSIM reconstruction, allowing optical sectioning with high acquisition rates. This thesis describes the theory of picoSIM and presents experimental results.

Argumentation theory conceptualises the human practice of debating. Implemented as computational argumentation it enables a computer to perform a virtual debate. Using existing knowledge from research into argumentation theory, this thesis investigates the potential of computational argumentation within biology. As a form of non-monotonic reasoning, argumentation can be used to tackle inconsistent and incomplete information - two common problems for the users of biological data. Exploration of argumentation shall be conducted by examining these issues within one biological subdomain: in situ gene expression information for the developmental mouse. Due to the complex and often contradictory nature of biology, occasionally it is not apparent whether or not a particular gene is involved in the development of a particular tissue. Expert biological knowledge is recorded, and used to generate arguments relating to this matter. These arguments are presented to the user in order to help him/her decide whether or not the gene is expressed. In order to do this, the notion of argumentation schemes has been borrowed from philosophy, and combined with ideas and technologies from arti cial intelligence. The resulting conceptualisation is implemented and evaluated in order to understand the issues related to applying computational argumentation within biology. Ultimately, this work concludes with a discussion of Argudas - a real world tool developed for the biological community, and based on the knowledge gained during this work.

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Many biological structures exist on a scale smaller than can be resolved by conventional fluorescence microscopy, which has limited the study of cellular processes. For this reason, there has been a large amount of research over the past decade dedicated to the development of super resolution microscopy techniques, which allow optical imaging of structures below the so-called resolution limit. In particular there has been much interest in developing super resolution microscopy techniques capable of non-destructive imaging of living samples. A novel super resolution microscopy technique, lifetime image reconstruction super resolution (LIR-SR), is presented here; this uses continuous wave stimulated emission depletion (CW STED) to shorten the fluorescence lifetimes of fluorophores within a labelled sample. Differential lifetime shortening across the area of a scanning laser beam pair in the microscope results in spatial variation in the distribution of detected fluorescence on a nanosecond timescale, which can be subsequently used to reconstruct a super resolution image. Detailed theory of LIR-SR is explained, as well as the microscope hardware and computational methods used for its implementation. The technique is then tested on structures of known size and shape to gauge performance, and future directions for the technique are discussed. Spectroscopic studies of CW STED are also undertaken to better understand the underlying photophysics of the process. The effects of solvent viscosity on CW STED are investigated and analysed, and a mathematical model of CW STED is presented. Further investigations are then undertaken which address a wide range of factors which could affect CW STED, including out-of-focus fluorescence and involvement of the triplet state, and possible refinements to the model of CW STED are suggested in light of the experimental results.

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Quantitative structure-activity relationships (QSAR) rationalize interrelation between molecular structure and biological response in terms of either physicochemical parameters, as in linear free energy relationships (LFER), or via purely empirical parameters, as is the case for De Novo schemes. In LFER the leading process is often the partitioning of a compound between two solvent phases, taken to represent the transfer of a drug molecule across a biological membrane. This study has investigated the partitioning behaviour of three series of hydroxybenzoate esters, viz. o-, m- and predominantly p-esters, the latter being preservatives in pharmaceutical formulations. The thermodynamic parameters AH, AG and AS for the transfer process were derived in an attempt to establish a QSAR. on a fundamental thermodynamic basis. Such parameters have identifiable physicochemical meaning and lend themselves more readily to interpretation. This facilitates application to alternative systems. A new Gibbs function factor analysis was developed and utilized to obtain thermodynamic contributions for parent and incremental methylene group portions of thestudy molecules. The empirical Collander equation for interrelation of various solute/solvent systems was also rationalized on a thermodynamic basis. Further extension of the Gibbs function factor analysis allowed scaling of "solvent" systems including chromatographic packings, solvents and liposomes. The scheme indicated capacity for optimized selection of bulk solvent systems to mimic biological membranes. A novel analytical procedure for direct measurement of biological response was developed. The bioassay appeared capable of discrimination i) between the closely related structural homologues, ii) between gram-negative and gram-positive bacteria, and further, iii) between certain cell batches of the same bacteria type. Also, the bioassay demonstrated a Collander interrelation between the two bacteria types. Flow microcalorimetry was the technique employed to measure thermal response of respiring E. coli and Staph, aur. bacteria. The modification of biological response with drug concentration was quantitated and a log dose max term was derived for each homologue. The results indicated potential for a predictive, additive structure-activity scheme based on assessment of biological response (BR) direct rather than through f(BR) via physicochemical or empirical parameters.

The ability to observe at tiny length scales has enabled key advances across the physical and life sciences. Much of what we know about the structure of cells and tissues comes from experiments on the micron length scale, enabled by new microscopy techniques. Modern manufacturing is increasingly concerned with materials that are structured on the nanometre scale, and devices which have ever-smaller features. Manipulating and measuring microscopic objects is a problem common to fields as diverse as microfabrication and cell biology, and it is these challenges that my doctoral studies have addressed. Tiny sizes mean tiny forces; so small that the light from a laser can be used to propel objects. Optical tweezers, a technique pioneered some two and a half decades ago, exploit light’s momentum to trap and manipulate objects. Now an established tool, single particles can be trapped and tracked to measure forces on a molecular scale, and this work is responsible for much of our current knowledge of motor proteins. This thesis describes advances in the holographic technology used to control multiple optical traps (and hence many trapped particles), and improved methods for monitoring the positions and forces involved. The speed with which multiple holographic optical traps can be moved has traditionally been limited by the time taken to calculate holograms, but by using consumer graphics cards and high speed Spatial Light Modulators (SLMs) I have implemented holographic systems fast enough to react to the Brownian motion of trapped particles. Brownian motion can, to some extent, be suppressed by this approach, and it also allows the trap's stiffness to be engineered to balance sensitivity against tight constraint of position. Feedback control using an SLM, rather than the other beam steering technologies that have been employed, is able to react to motion in three dimensions. This requires 3D position measurement, which is provided by the stereo microscopy technique described in Chapter 2. By illuminating and viewing the sample from two different angles it is possible to reconstruct the depth of objects. This is accomplished through a single high numerical aperture microscope objective, the same lens used to focus the trapping laser. In conjunction with a fast CMOS camera, it is possible to track particles with an accuracy of 2-3nm at several thousand frames per second. This allows measurement of forces and displacements within the control loop, that can be fed back to influence the position of the optical traps. This force information can also be relayed to the operator using a force-feedback joystick as detailed in Chapter 7. Interface design is an important part of making technology accessible to scientists from other disciplines; to this end I have also developed a multi-touch tablet application to control optical tweezers. By creating simple, reliable systems and coupling them to an intuitive interface, I have endeavoured to produce developments which are of use to the non specialist as well as to experts in optical tweezers-a number of which are now available commercially (Section 8.7). These technologies form the basis of a toolkit for working with multi-part probes in optical tweezers, and they should bear fruit in the coming years as a new form of scanning-probe microscopy emerges.

The DIYbio (Do-It-Yourself biology) group was established with the aim of turning biology and biotechnology into a creative practice accessible to everyone. The group is composed of graduate and post-graduate students and drop-out graduate students, but also disenfranchised researchers and professionals who see in the initiative the possibility of reviving their passion for science. Inspired by the analogy of the personal computer as a 'spokes-technology' for a free, egalitarian and decentralized society, that of the free and open-source software movement, and inspired by the image of the Victorian amateur and his home laboratory, DIYbio members organize regionally in what they call 'community laboratories,' or they practice in the comfort of their homes. Based on a series of interviews with DIYbio members, participants' observations of DIYbio's transient practices and a literary analysis of DIYbio members' use of social media, this thesis traces what I provisionally call 'the making of a personal biology.' Starting from the narrative formation the network, it then moves from the foundation of the DIYbio network in 2008 to the establishment of the first 'community laboratories', tracing the contingent orchestration of a diverse set of people, sites, tools and events, into a four-year community building effort. Due to its recent emergence in the field of Science and Technology Studies, only a limited number of research initiatives engage with the DIYbio network. Such works, mainly in the form of dissertations chapters and short articles, are analytically rich but limited in their observations, and often focus only on specific aspects of the network (Aguiton, 2010; Roosth, 2010; Delfanti, 2011; Meyer, 2012). This thesis recognizes the emergence of the DIYbio network as a cultural phenomenon in itself, and addresses the gap in the literature by tracing how DNA became hackable and biology became personal. Following Donna Haraway's effort to critically address the politics of technoscience as a practice of 'turning tropes into worlds' (1997: 59), the overarching topic of this research is how the trope of the biohacker became a world, and what type of world it became. The aim of this research is, therefore, to explore how members of the DIYbio network and biohackers define themselves, construct their identities and organize their work. This research also aims to situate the discourses and practices of DIYbio members in a context where governments and industries are intensifying their effort to make the coming century of biology into a reality.