All-optical interrogation of neural circuits during behaviour

This thesis explores the fundamental question of how patterns of neural activity encode information and guide behaviour. To address this, one needs three things: a way to record neural activity so that one can correlate neuronal responses with environmental variables; a flexible and specific way to influence neural activity so that one can modulate the variables that may underlie how information is encoded; a robust behavioural paradigm that allows one to assess how modulation of both environmental and neural variables modify behaviour. Techniques combining all three would be transformative for investigating which features of neural activity, and which neurons, most influence behavioural output. Previous electrical and optogenetic microstimulation studies have told us much about the impact of spatially or genetically defined groups of neurons, however they lack the flexibility to probe the contribution of specific, functionally defined subsets. In this thesis I leverage a combination of existing technologies to approach this goal. I combine two-photon calcium imaging with two-photon optogenetics and digital holography to generate an “all-optical” method for simultaneous reading and writing of neural activity in vivo with high spatio-temporal resolution. Calcium imaging allows for cellular resolution recordings from neural populations. Two-photon optogenetics allows for targeted activation of individual cells. Digital holography, using spatial light modulators (SLMs), allows for simultaneous photostimulation of tens to hundreds of neurons in arbitrary spatial locations. Taken together, I demonstrate that this method allows one to map the functional signature of neurons in superficial mouse barrel cortex and to target photostimulation to functionally-defined subsets of cells. I develop a suite of software that allows for quick, intuitive execution of such experiments and I combine this with a behavioural paradigm testing the effect of targeted perturbations on behaviour. In doing so, I demonstrate that animals are able to reliably detect the targeted activation of tens of neurons, with some sensitive to as few as five cortical cells. I demonstrate that such learning can be specific to targeted cells, and that the lower bound of perception shifts with training. The temporal structure of such perturbations had little impact on behaviour, however different groups of neurons drive behaviour to different extents. In order to probe which characteristics underly such variation, I tested whether the sensory response strength or correlation structure of targeted ensembles influenced their behavioural salience. Whilst these final experiments were inconclusive, they demonstrate their feasibility and provide us with some key actionable improvements that could further strengthen the all-optical approach. This thesis therefore represents a significant step forward towards the goal of combining high resolution readout and perturbation of neural activity with behaviour in order to investigate which features of the neural code are behaviourally relevant

Camera based Display Image Quality Assessment

This thesis presents the outcomes of research carried out by the PhD candidate Ping Zhao during 2012 to 2015 in Gjøvik University College. The underlying research was a part of the HyPerCept project, in the program of Strategic Projects for University Colleges, which was funded by The Research Council of Norway. The research was engaged under the supervision of Professor Jon Yngve Hardeberg and co-supervision of Associate Professor Marius Pedersen, from The Norwegian Colour and Visual Computing Laboratory, in the Faculty of Computer Science and Media Technology of Gjøvik University College; as well as the co-supervision of Associate Professor Jean-Baptiste Thomas, from The Laboratoire Electronique, Informatique et Image, in the Faculty of Computer Science of Universit´e de Bourgogne. The main goal of this research was to develop a fast and an inexpensive camera based display image quality assessment framework. Due to the limited time frame, we decided to focus only on projection displays with static images displayed on them. However, the proposed methods were not limited to projection displays, and they were expected to work with other types of displays, such as desktop monitors, laptop screens, smart phone screens, etc., with limited modifications. The primary contributions from this research can be summarized as follows: 1. We proposed a camera based display image quality assessment framework, which was originally designed for projection displays but it can be used for other types of displays with limited modifications. 2. We proposed a method to calibrate the camera in order to eliminate unwanted vignetting artifact, which is mainly introduced by the camera lens. 3. We proposed a method to optimize the camera’s exposure with respect to the measured luminance of incident light, so that after the calibration all camera sensors share a common linear response region. 4. We proposed a marker-less and view-independent method to register one captured image with its original at a sub-pixel level, so that we can incorporate existing full reference image quality metrics without modifying them. 5. We identified spatial uniformity, contrast and sharpness as the most important image quality attributes for projection displays, and we used the proposed framework to evaluate the prediction performance of the state-of-the-art image quality metrics regarding these attributes. The proposed image quality assessment framework is the core contribution of this research. Comparing to conventional image quality assessment approaches, which were largely based on the measurements of colorimeter or spectroradiometer, using camera as the acquisition device has the advantages of quickly recording all displayed pixels in one shot, relatively inexpensive to purchase the instrument. Therefore, the consumption of time and resources for image quality assessment can be largely reduced. We proposed a method to calibrate the camera in order to eliminate unwanted vignetting artifact primarily introduced by the camera lens. We used a hazy sky as a closely uniform light source, and the vignetting mask was generated with respect to the median sensor responses over i only a few rotated shots of the same spot on the sky. We also proposed a method to quickly determine whether all camera sensors were sharing a common linear response region. In order to incorporate existing full reference image quality metrics without modifying them, an accurate registration of pairs of pixels between one captured image and its original is required. We proposed a marker-less and view-independent image registration method to solve this problem. The experimental results proved that the proposed method worked well in the viewing conditions with a low ambient light. We further identified spatial uniformity, contrast and sharpness as the most important image quality attributes for projection displays. Subsequently, we used the developed framework to objectively evaluate the prediction performance of the state-of-art image quality metrics regarding these attributes in a robust manner. In this process, the metrics were benchmarked with respect to the correlations between the prediction results and the perceptual ratings collected from subjective experiments. The analysis of the experimental results indicated that our proposed methods were effective and efficient. Subjective experiment is an essential component for image quality assessment; however it can be time and resource consuming, especially in the cases that additional image distortion levels are required to extend the existing subjective experimental results. For this reason, we investigated the possibility of extending subjective experiments with baseline adjustment method, and we found that the method could work well if appropriate strategies were applied. The underlying strategies referred to the best distortion levels to be included in the baseline, as well as the number of them

Case series of breast fillers and how things may go wrong: radiology point of view

INTRODUCTION: Breast augmentation is a procedure opted by women to overcome sagging breast due to breastfeeding or aging as well as small breast size. Recent years have shown the emergence of a variety of injectable materials on market as breast fillers. These injectable breast fillers have swiftly gained popularity among women, considering the minimal invasiveness of the procedure, nullifying the need for terrifying surgery. Little do they know that the procedure may pose detrimental complications, while visualization of breast parenchyma infiltrated by these fillers is also deemed substandard; posing diagnostic challenges. We present a case series of three patients with prior history of hyaluronic acid and collagen breast injections. REPORT: The first patient is a 37-year-old lady who presented to casualty with worsening shortness of breath, non-productive cough, central chest pain; associated with fever and chills for 2-weeks duration. The second patient is a 34-year-old lady who complained of cough, fever and haemoptysis; associated with shortness of breath for 1-week duration. CT in these cases revealed non thrombotic wedge-shaped peripheral air-space densities. The third patient is a 37‐year‐old female with right breast pain, swelling and redness for 2- weeks duration. Previous collagen breast injection performed 1 year ago had impeded sonographic visualization of the breast parenchyma. MRI breasts showed multiple non- enhancing round and oval shaped lesions exhibiting fat intensity. CONCLUSION: Radiologists should be familiar with the potential risks and hazards as well as limitations of imaging posed by breast fillers such that MRI is required as problem-solving tool

Studying Large Multi-Protein Complexes Using Single Molecule Localization Microscopy

Biology would not be where it is today without fluorescence microscopy. It is arguably one of the most commonly used tools in the biologists toolbox and it has helped scientists study the localization of cellular proteins and other small things for decades, but it is not without its limitations. Due to the diffraction limit, conventional fluorescence microscopy is limited to micrometer-range structures. Science has long relied upon electron microscopy and X-ray crystallography to study phenomena that occur below this limit. However, many of lifes processes occur between these two spatial domains. Super-resolution microscopy, the next stage of evolution of fluorescence microscopy, has the potential to bridge this gap between micro and nano. It combines superior resolutions of down to a few nanometers with the ability to view objects in their natural environments. It is the ideal tool for studying the large, multi-protein complexes that carry out most of lifes functions, but are too complex and fragile to put on an electron microscope or into a synchrotron. A form of super-resolution microscopy called SMLM Microscopy shows especially high promise in this regard. With its ability to detect individual molecules, it combines the high resolution needed for structural studies with the quantitative readout required for obtaining data on the stoichiometry of multi-protein complexes. This thesis describes new tools which expand the toolbox of SMLM with the specific aim of studying multi-protein complexes. First, the development of a novel fluorescent tagging system that is a mix of genetic tagging and immuno-staining. The system, termed BC2, consists of a short, genetically encodable peptide that is targeted by a nanobody (BC2 nanobody). The system brings several advantages. The small tag is not disruptive to the protein it is attached to and the small nanobody can get into tight spaces, making it an excellent tag for dense multi-protein structures. Next, several new variants of some commonly used green-to-red fluorescent proteins. The novel variants, which can be converted with a combination of blue and infrared light are especially useful for live-cell imaging. The developed fluorescent proteins can also be combined with photo-activatable fluorescent proteins to enable imaging of several targets with the same color protein. Finally, an application of the latter technique to study the multi-protein kinetochore complex and gain first glimpses into its spatial organization and the stoichiometry of its subunits