307 research outputs found

    Robust deep learning for computational imaging through random optics

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    Light scattering is a pervasive phenomenon that poses outstanding challenges in both coherent and incoherent imaging systems. The output of a coherent light scattered from a complex medium exhibits a seemingly random speckle pattern that scrambles the useful information of the object. To date, there is no simple solution for inverting such complex scattering. Advancing the solution of inverse scattering problems could provide important insights into applications across many areas, such as deep tissue imaging, non-line-of-sight imaging, and imaging in degraded environment. On the other hand, in incoherent systems, the randomness of scattering medium could be exploited to build lightweight, compact, and low-cost lensless imaging systems that are applicable in miniaturized biomedical and scientific imaging. The imaging capabilities of such computational imaging systems, however, are largely limited by the ill-posed or ill-conditioned inverse problems, which typically causes imaging artifacts and degradation of the image resolution. Therefore, mitigating this issue by developing modern algorithms is essential for pushing the limits of such lensless computational imaging systems. In this thesis, I focus on the problem of imaging through random optics and present two novel deep-learning (DL) based methodologies to overcome the challenges in coherent and incoherent systems: 1) no simple solution for inverse scattering problem and lack of robustness to scattering variations; and 2) ill-posed problem for diffuser-based lensless imaging. In the first part, I demonstrate the novel use of a deep neural network (DNN) to solve the inverse scattering problem in a coherent imaging system. I propose a `one-to-all' deep learning technique that encapsulates a wide range of statistical variations for the model to be resilient to speckle decorrelations. I show for the first time, to the best of my knowledge, that the trained CNN is able to generalize and make high-quality object prediction through an entirely different set of diffusers of the same macroscopic parameter. I then push the limit of robustness against a broader class of perturbations including scatterer change, displacements, and system defocus up to 10X depth of field. In the second part, I consider the utility of the random light scattering to build a diffuser-based computational lensless imaging system and present a generally applicable novel DL framework to achieve fast and noise-robust color image reconstruction. I developed a diffuser-based computational funduscope that reconstructs important clinical features of a model eye. Experimentally, I demonstrated fundus image reconstruction over a large field of view (FOV) and robustness to refractive error using a constant point-spread-function. Next, I present a physics simulator-trained, adaptive DL framework to achieve fast and noise-robust color imaging. The physics simulator incorporates optical system modeling, the simulation of mixed Poisson-Gaussian noise, and color filter array induced artifacts in color sensors. The learning framework includes an adaptive multi-channel L2-regularized inversion module and a channel-attention enhancement network module. Both simulation and experiments show consistently better reconstruction accuracy and robustness to various noise levels under different light conditions compared with traditional L2-regularized reconstructions. Overall, this thesis investigated two major classes of problems in imaging through random optics. In the first part of the thesis, my work explored a novel DL-based approach for solving the inverse scattering problem and paves the way to a scalable and robust deep learning approach to imaging through scattering media. In the second part of the thesis, my work developed a broadly applicable adaptive learning-based framework for ill-conditioned image reconstruction and a physics-based simulation model for computational color imaging

    Using super resolution microscopy to investigate the role of actin in adenosine receptor organisation

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    Membrane receptors are key to how cells interact with other cells and their environment. G Protein-Coupled Receptors (GPCRs) are a major drug target, with approximately a third of all FDA approved drugs acting on a GPCR [1]. The organisation of GPCRs in the cell membrane can play a key role in determining signalling responses and associated pharmacological parameters. There is significant evidence that the cortical actin skeleton can contribute to this organisation via the picket fence model. The direct contribution of actin architecture and dynamics to organisation of specific receptors requires further study. Therefore, this thesis applies a range of super-resolution microscopy techniques to investigate the role of cortical actin in the organisation of the human adenosine-A2A (A2AR) and -A2B receptors (A2BR). Using A549 cells transiently transfected with N-terminally SNAP-tagged receptor constructs, clustering analysis using dSTORM (direct stochastic optical reconstruction microscopy) indicates effects of actin disruption on A2AR clustering but not A2BR, while assessment of dynamic behaviour via single particle tracking (SPT) indicates differential effects on the motion patterns of each receptor. This was further supported by 3D-SIM (structured illumination microscopy) imaging of actin and receptors together. Assessment of actin using SRRF (super resolved radial fluctuations) processing showed a change in actin architecture after receptor stimulation. Workflows for imaging and analysing finer actin filaments using 3D-SIM expansion microscopy (ExM) were also developed, with incorporation of the A2R interacting protein α-actinin-1 serving both as investigation of a potential actin link and as a demonstration of two colour ExM. Initial experiments using SRRF processing indicated super-resolution imaging of actin was possible on a timescale which allowed concurrent single particle tracking of receptors, opening potential for correlated analysis. These findings indicate a role for actin in mediating A2AR and A2BR membrane organisation, with potential for different regulatory contributions between receptors and across organisational scales. [A thesis submitted to the University of Birmingham and the University of Nottingham for the dual degree of Doctor of Philosophy, August 2022.

    Transcriptional dynamics of the Sonic hedgehog gene

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    Enhancers are capable of driving gene expression over linearly vast distances, allowing precise patterns of spatiotemporal gene expression. They are able to do this independent of orientation to the promoter, and a single gene often has multiple enhancers. There is still limited understanding of how developmental enhancers drive transcription. It must be a highly regulated process, previous evidence has shown that alterations in expression levels can result in developmental malformations. Furthermore, there is debate surrounding the mechanisms of how enhancers interact with their distal promoters. The models currently most popular in the field are looping and transcriptional hubs. Sonic hedgehog (Shh) gene expression is a good model to further our understanding of both developmental transcriptional regulation and distal enhancer-promoter interactions. Shh expression is regulated by many tissue and spatial specific enhancers with, in some instances, single enhancers driving expression in single embryonic domains. The enhancers are all located within a single TAD, at a range of distances from the Shh promoter. Over the years, my lab has taken a special interest in the limb enhancer, ZRS. The ZRS drives transcription in the distal posterior mesenchyme of the developing limb bud in a domain called the ZPA. We have been able to identify a network of activator and repressor binding sites within the ZRS that restricts transcription in the absence of histological boundaries providing an interesting model for me to explore mechanisms for how a developmental enhancer drives transcription. Throughout this thesis I will address three main aims. Firstly, I will establish transcriptional characteristics at the wild-type Shh locus. Before I start exploring how an enhancer drives transcription using different mouse models, I first need a strong understanding of what transcription looks like in wild-type animals. I addressed this using nascent RNA-FISH. Using nascent RNA-FISH I have been able to determine the bursting frequency of Shh in the ZPA. Furthermore, this technique has allowed me to ascertain if an active enhancer can be transcribed through. Meanwhile, the use of RNAscope has allowed me to establish the overall pattern of expression across the ZPA. Determining whether there is a clear-cut boundary or a gradient type pattern. Secondly, I will decipher the role of discrete functional elements of the ZRS in transcription. Previous work from members of my lab has identified different binding sites located throughout the ZRS. For example, there are four known Hox binding sites. Mutations in these sites are known to cause down-regulation of Shh. I used mutants for these sites to determine the action of HOXD proteins on the ZRS and how this impacts transcription. Furthermore, I have investigated how pioneer factor binding of the ZRS influences transcriptional characteristics. This was done by investigating how the mutation of a Lim homeodomain binding site impacted transcriptional characteristics of Shh. To contrast, I then explored how up-regulatory mutations of the ZRS effect transcription by using mice where a ZRS repressor site, the WMS, was disrupted. This work revealed that HOXD proteins, Limb homeodomain proteins and proteins binding the WMS all have different influences on Shh transcription, revealing a range of different roles. Finally, I will explore long-range regulation of distal enhancer-promoter interactions. There have been multiple models proposed to explain how enhancers interact with their target promoters. Two of these models for explaining long range regulation are the looping model and the transcriptional hub model. These two models can be differentiated by the action of a single enhancer on multiple promoters, the looping model predicts promoter choice while the transcriptional hub predicts simultaneous promoter activation. To test these models, I looked at the ability of Shh enhancers to drive transcription of multiple promoters in different contexts. Firstly, I used a mouse line carrying a LacZ reporter integrated within the Shh TAD. This provided a second internal promoter where expression is driven by Shh enhancers in their cognate tissues. Secondly, I performed experiments examining activation of two endogenous genes in adjacent TADs, Shh and Mnx1

    Discos Keplerianos y flujos en estrellas binarias post-AGB

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    Tesis inĂ©dita de la Universidad Complutense de Madrid, Facultad de Ciencias FĂ­sicas, leĂ­da el 17-02-2023The evolution of intermediate mass stars (0:8 < M < 8M ) in their last stages is fast. In these phases the star evolves from the asymptotic giant branch (AGB) over the post-AGB star phase towards the central star of planetary nebula (CSPN) phase. At the end of the AGB phase, the star exhibits high mass-loss rates, up to 10- 4 M a-1.The star begins the post-AGB stage after having expelled most of its stellar envelope. The nebula around a post-AGB star is known as a pre-planetary nebula (pPN), and most of them typically exhibit fast bipolar out ows. We focus on a certain class of post-AGB stars that are part of a binary system. These binary systems present orbital periods between 100 and 3 000 days and separations smaller than 5AU, avoiding (or rapidly going through) the common envelope phase. Their remarkable near-infrared excess in the spectral energy distribution (SED) is an observational probe for the presence of disks around the binary system. This excess at IR wavelengths is indicative of the presence of hot dust produced by the inner regions of these disks. They are formed during the binary interaction. They are stable structures and there are several evidences that prove it, such as the detection of Keplerian dynamics or the presence of highly processed dust grains...La evoluciĂłn de las estrellas de masa intermedia (0:8 < M < 8M ) en las Ășltimas fases de su vida es muy rĂĄpida. En estas fases la estrella evoluciona desde la Rama AsintĂłtica de las Gigantes (AGB, por sus siglas en inglĂ©s) sobre la fase de estrella post-AGB hacia la etapa de estrella central de una nebulosa planetaria (CSPN por sus siglas en inglĂ©s). Al final de la etapa AGB, la estrella muestra grandes tasas de pĂ©rdida de masa, hasta 10-4M a- 1. La estrella empieza la fase post-AGB despuĂ©s de haber expulsado la mayor parte de su manto. La nebulosa que hay alrededor de una estrella post-AGB es conocida como nebulosa pre-Planetaria (pPNe) y la mayorĂ­a de ellas muestra usualmente flujos bipolares de alta velocidad. Nos centramos en una cierta clase de estrellas post-AGB que forman parte de un sistema binario. Estos sistemas binarios presentan periodos orbitales entre 100 y 3 000 dĂ­as y una separaciĂłn menor que 5AU, esquivando (o atravesando rĂĄpidamente) la fase de envoltura comĂșn. Estas fuentes muestran indicios de la presencia de discos alrededor del sistema binario, como son su notable exceso en el infrarrojo cercano (NIR) presente en su distribuciĂłn espectral de energĂ­a (SED). Este exceso a longitudes de onda infrarrojas es indicativo de la presencia de polvo caliente en las regiones mĂĄs internas del disco. Los discos se forman debido a interacciones del sistema binario. Hay varios indicios de que se trata de estructuras estables, como es la detecciĂłn de dinĂĄmica Kepleriana o la presencia de granos de polvo muy procesados...Fac. de Ciencias FĂ­sicasTRUEunpu

    Computational methods for 3D imaging of neural activity in light-field microscopy

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    Light Field Microscopy (LFM) is a 3D imaging technique that captures spatial and angular information from light in a single snapshot. LFM is an appealing technique for applications in biological imaging due to its relatively simple implementation and fast 3D imaging speed. For instance, LFM can help to understand how neurons process information, as shown for functional neuronal calcium imaging. However, traditional volume reconstruction approaches for LFM suffer from low lateral resolution, high computational cost, and reconstruction artifacts near the native object plane. Therefore, in this thesis, we propose computational methods to improve the reconstruction performance of 3D imaging for LFM with applications to imaging neural activity. First, we study the image formation process and propose methods for discretization and simplification of the LF system. Typical approaches for discretization are performed by computing the discrete impulse response at different input locations defined by a sampling grid. Unlike conventional methods, we propose an approach that uses shift-invariant subspaces to generalize the discretization framework used in LFM. Our approach allows the selection of diverse sampling kernels and sampling intervals. Furthermore, the typical discretization method is a particular case of our formulation. Moreover, we propose a description of the system based on filter banks that fit the physics of the system. The periodic-shift invariant property per depth guarantees that the system can be accurately described by using filter banks. This description leads to a novel method to reduce the computational time using singular value decomposition (SVD). Our simplification method capitalizes on the inherent low-rank behaviour of the system. Furthermore, we propose rearranging our filter-bank model into a linear convolution neural network (CNN) that allows more convenient implementation using existing deep-learning software. Then, we study the problem of 3D reconstruction from single light-field images. We propose the shift-invariant-subspace assumption as a prior for volume reconstruction under ideal conditions. We experimentally show that artifact-free reconstruction (aliasing-free) is achievable under these settings. Furthermore, the tools developed to study the forward model are exploited to design a reconstruction algorithm based on ADMM that allows artifact-free 3D reconstruction for real data. Contrary to traditional approaches, our method includes additional priors for reconstruction without dramatically increasing the computational complexity. We extensively evaluate our approach on synthetic and real data and show that our approach performs better than conventional model-based strategies in computational time, image quality, and artifact reduction. Finally, we exploit deep-learning techniques for reconstruction. Specifically, we propose to use two-photon imaging to enhance the performance of LFM when imaging neurons in brain tissues. The architecture of our network is derived from a sparsity-based algorithm for reconstruction named Iterative Shrinkage and Thresholding Algorithm (ISTA). Furthermore, we propose a semi-supervised training based on Generative Adversarial Neural Networks (GANs) that exploits the knowledge of the forward model to achieve remarkable reconstruction quality. We propose efficient architectures to compute the forward model using linear CNNs. This description allows fast computation of the forward model and complements our reconstruction approach. Our method is tested under adverse conditions: lack of training data, background noise, and non-transparent samples. We experimentally show that our method performs better than model-based reconstruction strategies and typical neural networks for imaging neuronal activity in mammalian brain tissue. Our approach enjoys both the robustness of the model-based methods and the reconstruction speed of deep learning.Open Acces

    Keplerian disks and outflows in binary post-AGB stars

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    There is a class of binary post-AGB stars that are surrounded by Keplerian disks and that often present outflows resulting from gas escaping from the disk. We present maps and complex models of 12CO and 13CO J=2-1 emission lines for four objects: AC Herculis, 89 Herculis, IRAS 19125+0343, and R Scuti. Our maps and models allow us to study their morphology, kinematics, and mass distribution. Our maps and modeling of AC Her reveal that 95% of the total nebular mass is located in the disk. So this source is a disk-dominated source (like the Red Rectangle, IW Carinae, IRAS 08544-4431). On the contrary, our maps and modeling of 89 Herculis, IRAS 19125+0343, and R Scuti suggest that the outflow is the dominant component of the nebula, resulting in a new subclass nebulae around binary post-AGB stars: the outflow-dominated ones. Besides CO, the molecular content of this kind of sources was barely known. We also present the first and very deep single-dish radio molecular survey in the 1.3, 2, 3, 7, and 13 mm bands. Our results allow us to classify our sources as O- or C-rich. We also conclude that these sources present in general a low molecular richness, especially those that are disk-dominated, compared to circumstellar envelopes around AGB stars and other post-AGB stars. This thesis presents a comprehensive study at millimetre wavelengths. On the one hand, we perform a detailed kinetic study of these objects through NOEMA interferometric observations and complex models. On the other hand, we study the chemistry of these sources, thanks to our sensitive single-dish observations. The union of these different methods yields a comprehensive study of the molecular gas present in these sources. Hopefully, this Ph.D. thesis will become a reference for future studies of molecular gas in nebulae around binary post-AGB stars.Comment: Doctoral thesis presented by Iv\'an Gallardo Cava; Dissertation Directors: Valent\'in Bujarrabal Fern\'andez and Javier Alcolea Jim\'enez; Tribunal: A. I. G\'omez de Castro, M. A. Guerrero Roncel, R. Corradi, A. Castro Carrizo, and J. Zamorano Calvo; Ph.D. thesis defended on 17 February 2023, Madrid, Spain; Observatorio Astron\'omico Nacional (OAN-IGN) and Universidad Complutense de Madrid (UCM

    Data-driven microscopy: placing high-fidelity data in a population-wide context

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    Mikroskopi Àr idag ett fundamentalt verktyg inom forskning, dÀr det tillÄter oss att skÄda in och utforska vÄra prover i hög detalj. Mycket utav utvecklingen av nya mikroskopimetoder har strÀvat efter att öka den detaljnivÄ vi kan uppnÄ. Samtidigt har utvecklingen inom hÄrdvara, med tillgÄng till bÀttre och mer kraftfulla instrument, lett till utveckligen av metoder dÀr fokuset Àr att studera en hel population av celler. Till skillnad frÄn nÀr vi studerar ett fÄtal celler i hög detalj, tillÄter det oss att sÀtta perspektiv pÄ det vi ser. Det ger oss en förmÄga att sÀga vad det normala beteendet som man kan förvÀnta sig Àr, och vilka celler som sticker ut i en population. Med andra ord, vad som Àr intressant.Samtidigt finns det ett stort intresse av att veta hur varje individuell cell beter sig. Varje cell Àr, precis som oss mÀnniskor, unik. De har olika historia, olika Älder och befinner sig i olika tillstÄnd. Precis som vÄra celler i kroppen Àr unika, Àr Àven de cellerna som kan orsaka sjukdom unika. För att förstÄ varför vissa personer Àr mer kÀnsliga mot sjukdom, och hur en infektion svarar pÄ vÄra behandlingar behövs en förstÄelse och an förmÄga att studera celler pÄ individuell nivÄ, samtidigt som vi bibehÄller ett perspektiv utifrÄn populations-nivÄ.Denna brist pÄ perspektiv har lÀnge varit ett problem inom mikroskopi. Den vanliga lösningen pÄ detta problem Àr att vi, som mÀnniskor, kan tolka en bild och peka pÄ vad det Àr som Àr intressant eller inte. Vi Àr, trots allt, extremt duktiga pÄ att tolka visuell information. Men detta Àr inte en helt felfri lösning. Som mÀnniskor kan vi vara relativt okonsekventa, vi tolkar oftast utifrÄn hur vi vill att datan ser ut. Med andra ord, vi saknar förmÄgan att vara objektiva i vÄr metodik för att samla in bilder i hög detalj.Min avhandling har till stor del handlat om att utveckla ett verktyg som tillÄter oss att sÀtta perspektiv pÄ det vi studerar med mikroskopi. Detta har lett till Arbete 1, dÀr vi presenterar en allmÀn strategi (data-styrd mikroskopi) för hur vi kan arbeta med mikroskopi för att samla in data pÄ en hel population, samtidigt som vi kan samla in data med hög detalj pÄ relevanta fynd i populationen. Vi presenterar Àven hÀr en teknisk lösning, och utför metoden i tre olika scenarion: ett för att studera en population av celler mer allmÀnt, ett för att fÄnga det ögonblick som bakterier infekterar mÀnskliga celler, och ett dÀr vi studerar och fÄngar in data pÄ relevanta (frÄn ett populations-kontext) cancerceller och följer dem över tid. Denna metod tillÄter oss att samla in data i hög detalj pÄ ett objektivt sÀtt, och att sÀtta perspektiv pÄ det vi studerar.I Arbete 2 har vi vidare utvecklat pÄ vÄr metod, dÀr vi försöker lösa problemet att hitta en och samma cell i flera olika mikroskop. Eftersom vi, genom mikroskopi, jobbar pÄ en sÄ ofantligt liten skala, Àr det oftast vÀldigt svÄrt att orientera sig och hitta rÀtt inom ett prov. Det Àr lite som att spela PÄ spÄret och gissa vart man Àr, fast utan alla ledtrÄdar man fÄr pÄ varje nivÄ. Eftersom vi har tillgÄng till data pÄ en hel population, sÄ utgick vi frÄn att det borde finnas samband mellan celler och deras grannar i ett prov som Àr unika för just dem. Genom att anvÀnda sig av dessa unika samband kom vi fram med en lösning dÀr vi snabbt kan kalibrera ett prov pÄ ett nytt mikroskop. Det öppnar dörrarna för oss forskare att ÄteranvÀnda prov, att lÀttare justera provet med nya markörer (för det vi vill visualisera inom cellerna), och att kunna tolka ett prov med data insamlat frÄn flera system.COVID-19 pandemin var en stor omstÀllning för samhÀllet och vÄrden. LikvÀl var det en stor omstÀllning för mÄnga forskningslabb, dÀr en kapplöpning startade för att sÄ snabbt som möjligt förstÄ sig pÄ hur viruset fungerar och hur vÄrt immunförsvar svarar pÄ dess infektion. Det var i detta kontext som mitt tredje arbete utfördes. Genom den erfarenhet jag samlat pÄ mig inom mikroskopi och att analysera bilder pÄ stora dataset, bidrog jag med hjÀlp för att studera hur framtagna antikroppar kan förhindra bindningen av virus-lika partiklar till celler. Antikroppar Àr ett protein som immunförsvaret producerar i respons mot en patogen. En bÀttre förstÄelse kring hur antikroppar verkar, och vad skillnaden mellan en bra och en dÄlig antikropp Àr kan leda till framtagningen av bÀttre vaccin-program och behandlingar inom sjukvÄrden.I Arbete 4 medverkade jag i ett arbete dÀr bakterien Streptococcus pyogenes var i fokus. S. pyogenes enda vÀrd Àr mÀnniskor, och ansvarar för över 600 miljoner infektionsfall per Är globalt. PÄ bakteriens yta dominerar ett protein, M-proteinet, ett multi-funktionellt protein som bakterien (bland annat) anvÀnder sig för att binda till ytor och förhindra immunförsvarets förmÄga att göra sig av med bakterien. I arbetet upptÀckte vi att fibronektin binder till bakterien (specifikt M-proteinet) olika mycket beroende pÄ mÀngden antikroppar som finns i miljön. Fibronektin Àr ett protein som vi mÀnniskor producerar, och bidrar (bland annat) till att skapa den miljön som celler befinner sig i. MÀngden fibronektin varierar beroende pÄ var i kroppen man kollar. Till exempel, i saliv har du en relativt lÄg mÀngd fibronektin jÀmfört med i blodet. Detta ledde till hypotesen att bakterien Àr special-anpassad för olika miljöer i dess förmÄga att undkomma immunförsvaret. En bÀttre förstÄelse kring hur bakterien Àr anpassad till vÄra olika miljöer och dess infektionsförlopp kan leda till bÀttre och mer anpassade behandlingar inom sjukvÄrden

    Towards a circular economy: fabrication and characterization of biodegradable plates from sugarcane waste

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    Bagasse pulp is a promising material to produce biodegradable plates. Bagasse is the fibrous residue that remains after sugarcane stalks are crushed to extract their juice. It is a renewable resource and is widely available in many countries, making it an attractive alternative to traditional plastic plates. Recent research has shown that biodegradable plates made from Bagasse pulp have several advantages over traditional plastic plates. For example, they are more environmentally friendly because they are made from renewable resources and can be composted after use. Additionally, they are safer for human health because they do not contain harmful chemicals that can leach into food. The production process for Bagasse pulp plates is also relatively simple and cost-effective. Bagasse is first collected and then processed to remove impurities and extract the pulp. The pulp is then molded into the desired shape and dried to form a sturdy plate. Overall, biodegradable plates made from Bagasse pulp are a promising alternative to traditional plastic plates. They are environmentally friendly, safe for human health, and cost-effective to produce. As such, they have the potential to play an important role in reducing plastic waste and promoting sustainable practices. Over the years, the world was not paying strict attention to the impact of rapid growth in plastic use. As a result, uncontrollable volumes of plastic garbage have been released into the environment. Half of all plastic garbage generated worldwide is made up of packaging materials. The purpose of this article is to offer an alternative by creating bioplastic goods that can be produced in various shapes and sizes across various sectors, including food packaging, single-use tableware, and crafts. Products made from bagasse help address the issue of plastic pollution. To find the optimum option for creating bagasse-based biodegradable dinnerware in Egypt and throughout the world, researchers tested various scenarios. The findings show that bagasse pulp may replace plastics in biodegradable packaging. As a result of this value-added utilization of natural fibers, less waste and less of it ends up in landfills. The practical significance of this study is to help advance low-carbon economic solutions and to produce secure bioplastic materials that can replace Styrofoam in tableware and food packaging production

    Coded Aperture Imaging: novel approaches to high-energy high-resolution laboratory imaging

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    Rapid advancement is being made in laser driven x-ray and particle sources, pushing the boundaries in temporal duration, spatial and spectral distribution, and maximum energy. These advancements need to be complimented with development of imaging capabilities, in order to fully characterise and utilise the new source potential. Here, coded apertures are used to investigate novel approaches to high-energy high-resolution aperture based imaging. Firstly, coded aperture theory is applied to high-energy x-ray sources such as those generated using laser wakefield techniques. The coded aperture is compared to a single pinhole aperture, to discuss whether the prior assumption of highly attenuating substrates is required when using coded apertures. The coded aperture with scatter and partial attenuation included, dubbed a `CASPA', is demonstrated with a 511 keV source simulation, showing that the fully attenuating 18~mm thick tungsten substrate for a single pinhole can be replaced with a 250 um thick tungsten CASPA. Furthermore, the thin CASPA is not mechanism specific, and the physical processes behind the scatter and partial attenuation is found to be inconsequential as long as the combined result yields adequate hologram contrast for image decoding to occur. Secondly, an investigation is conducted into imaging with spectral and spatial information for applications such as laser-solid interaction hotspots. Combing coded apertures with Ross pair filters, a banded spectrally-resolving coded aperture is discussed, dubbed a `BaSCA', using multiple non-redundant array designs on a single aperture and single non-spectrally resolving detector. Finally, the application of a CASPA for imaging high-resolution high-energy neutron sources from inertial confinement fusion experiments is discussed. Using the National Ignition Facility at Lawrence Livermore National Laboratory as an example, a CASPA is designed for the 14.1 MeV neutrons, and reconstruction techniques discussed. In comparison to the currently implemented 20 cm thick gold grand array, it is suggested here that a 10 mm tungsten CASPA would suffice - potentially reducing manufacturing costs, increasing ease of implementation and field of view

    Remote refocusing light-sheet fluorescence microscopy for high-speed 2D and 3D imaging of calcium dynamics in cardiomyocytes

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    The high prevalence and poor prognosis of heart failure are two key drivers for research into cardiac electrophysiology and regeneration. Dyssynchrony in calcium release and loss of structural organization within individual cardiomyocytes (CM) has been linked to reduced contractile strength and arrhythmia. Correlating calcium dynamics and cell microstructure requires multidimensional imaging with high spatiotemporal resolution. In light-sheet fluorescence microscopy (LSFM), selective plane illumination enables fast optically sectioned imaging with lower phototoxicity, making it suitable for imaging subcellular dynamics. In this work, a custom remote refocusing LSFM system is applied to studying calcium dynamics in isolated CM, cardiac cell cultures and tissue slices. The spatial resolution of the LSFM system was modelled and experimentally characterized. Simulation of the illumination path in Zemax was used to estimate the light-sheet beam waist and confocal parameter. Automated MATLAB-based image analysis was used to quantify the optical sectioning and the 3D point spread function using Gaussian fitting of bead image intensity distributions. The results demonstrated improved and more uniform axial resolution and optical sectioning with the tighter focused beam used for axially swept light-sheet microscopy. High-speed dual-channel LSFM was used for 2D imaging of calcium dynamics in correlation with the t-tubule structure in left and right ventricle cardiomyocytes at 395 fps. The high spatio-temporal resolution enabled the characterization of calcium sparks. The use of para-nitro-blebbistatin (NBleb), a non-phototoxic, low fluorescence contraction uncoupler, allowed 2D-mapping of the spatial dyssynchrony of calcium transient development across the cell. Finally, aberration-free remote refocusing was used for high-speed volumetric imaging of calcium dynamics in human induced pluripotent stem-cell derived cardiomyocytes (hiPSC-CM) and their co-culture with adult-CM. 3D-imaging at up to 8 Hz demonstrated the synchronization of calcium transients in co-culture, with increased coupling with longer co-culture duration, uninhibited by motion uncoupling with NBleb.Open Acces
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