Graphene setting the stage: tracking DNA hybridization with nanoscale resolution

In this study we use nanophotonic effects of graphene to study DNA hybridization: the z−4 nanoscale distance-dependence of the fluorescence lifetime for fluorophores located in the vicinity of graphene is for the first time used to track a DNA hybridization reaction with nanoscale resolution in real time. First, a nanostaircase with ≈2 nm steps from 0 to a total height of 48 nm is used as a nanoruler to confirm the distance dependence law. We find that the axial sensitivity is suited to determine the nanoscale surface roughness of these samples. The proof-of-concept DNA experiments in aqueous medium involve the hybridization of fluorescently labelled DNA beacons attached to CVD grown graphene with complementary (target) DNA added in solution. We track the conformational changes of the beacons statistically by determining the fluorescence lifetimes of the labelling dye and converting them into nanoscale distances from the graphene. In this way, we are able to monitor the vertical displacement of the label during DNA-beacon unfolding with an axial resolution reaching down to 1 nm. The measured distance increase during the DNA hybridization reaction of about 10 nm matches the length of the target DNA strand. Furthermore, the width of the fluorescence lifetime distributions could be used to estimate the molecular tilt angle of the hybridized ds-DNA configuration. The achieved nanoscale sensitivity opens innovation opportunities in material engineering, genetics, biochemistry and medicine.INL received support for this project from the CCDR-N via the project 'Nanotechnology based functional solutions' (Grant No. NORTE-01-0145-FEDER-000019) and from the Portuguese Foundation for Science and Technology (FCT) via the project 'ON4SupremeSens' PTDC/NAN-OPT/29417/2017. Edite Figueiras received a Marie Curie fellowship via the EU-EC COFUND program 'NanoTRAINforGrowth' (Grant No. 600375). U Minho research was partially supported by the FCT in the framework of the Strategic Funding UID/FIS/04650/2013

Optical projection tomography technique for image texture and mass transport studies in hydrogels based on gellan gum

The microstructure and permeability are crucial factors for the development of hydrogels for tissue engineering, since they influence cell nutrition, penetration and proliferation. The currently available imaging methods able to characterize hydrogels have many limitations. They often require sample drying and other destructive processing, which can change hydrogel structure, or they have limited imaging penetration depth. In this work, we show for the first time an alternative non-destructive method, based on optical projection tomography (OPT) imaging, to characterize hydrated hydrogels without the need of sample processing. As proof of concept we used gellan gum (GG) hydrogels obtained by several crosslinking methods. Transmission mode OPT was used to analyse image microtextures and emission mode OPT to study mass transport. Differences in hydrogels structure related to different types of crosslinking and between modified and native GG were found through the acquired Haralickâ s image texture features followed by multiple discriminant analysis (MDA). In mass transport studies, the mobility of FITC-dextran (MW 20, 150, 2000 kDa) was analysed through the macroscopic hydrogel. The FITC-dextran velocities were found to be inversely proportional to the size of the dextran as expected. Furthermore, the threshold size in which the transport is affected by the hydrogel mesh was found to be 150 kDa (Stokesâ radii between 69 à and 95 à ). On the other hand, the mass transport study allowed us to define an index of homogeneity to assess the crosslinking distribution, structure inside the hydrogel and repeatability of hydrogel production. As a conclusion,  we showed that  the set of OPT imaging based material characterization methods presented here are useful for screening many characteristics of hydrogel compositions in relatively short time in an inexpensive manner, providing  tools for improving the process of designing hydrogels for tissue engineering and drugs/cells delivery applications.Portuguese Foundation for Science and Technology (FCT) - Grant No. SFRH/BPD/100590/201

Doppler flowmetry: line imaging techniques

A imagiologia da perfusão por Laser Doppler tem-se transformado no principal método óptico para a medição da perfusão sanguínea. Este trabalho descreve o projecto e o desenvolvimento de um modelo de bancada de um sistema de Imagiologia Laser Doppler para a monitorização do fluxo sanguíneo numa pequena porção de pele. O sistema é composto por um sensor de linha, um gerador de linha laser, um motor sem escovas, uma mesa de posicionamento, dois conjuntos (ópticos e electromecânicos) de interruptores de fim de curso, um módulo de aquisição de dados (National Instruments (NI) USB-6008) e um sistema electrónico de suporte (alimentação, interface, etc). Estes componentes são controlados por duas toolboxes do Matlab: a Image Acquisition Toolbox (IAT), versão 2.9, e a Data Acquisition Toolbox (DAT), versão 1.8. O DAT permite o controlo da NI USB-6008 que é usada para controlar o funcionamento do motor, do gerador de linha laser e para a leitura dos interruptores ópticos de fim de curso. A IAT é usada para controlar o sensor de linha

Métodos de instrumentação para fluxometria laser: aplicações à microcirculação sanguínea

Tese de doutoramento em Física, especialidade de Física Tecnológica, apresentada à Faculdade de Ciências e Tecnologia da Universidade de CoimbraDifferent diseases affect in different ways the behavior of the microcirculatory layers of the skin. Since there are clinical interests in the knowledge of their interrelationship, it is required the discrimination of the blood perfusion signal (information about the velocity and concentration of scatterers) arose from the super cial and the deep skin layers. Therefore, we were conducted to studies with the aim to improve the laser Doppler owmetry (LDF) technique in what concerns the sample depth discrimination. These studies resulted in the development of a owmeter prototype that enables us to study, simultaneously, the influence of the wavelength and of the distance between emitting and receiving fibres in the skin blood perfusion measurements. The developed technology was tested in vitro and validated through Monte Carlo simulations. They have shown that the average depth of the scattered photons increases with the wavelength and with the distance between the emitting and the receiving fibres. In vivo validation tests showed clear differences when comparing perfusion results obtained with different wavelengths and with different fibres distances. Perfusion signals obtained with the commercial flowmeter were analyzed in time and frequency domains. It was demonstrated that the obtained perfusion in healthy subjects in different regions of the skin, have different properties, and the perfusion obtained in patients with systemic sclerosis have a different regularity, when compared with the obtained perfusion in healthy subjects or patients with Raynaud's phenomenon. The need to follow the variations of the microcirculatory blood ow in the rat brain, has led researchers to use the invasive LDF optical bres probes commercially available. However, due to their large external diameter (ca. 450 m), the invasive LDF probes can damage the tissue to be monitored. Studies conducted with the aim of reducing the dimensions of the LDF probes, led to the construction of a self-mixing based microprobe using a single thin optical bre. These probes have an outer diameter of 260 m. The developed micro-probes showed a good linearity when tested for different scatterers velocities. In vivo measurements showed good indicators that lead us to conclude that the evaluated micro-probes can be used to monitor the microcirculatory blood ow in real time, in the rat brain. The obtained results with the two prototypes (non-invasive and invasive) are encouraging, and there are good indicators for their applicability in clinical and research environments.Diferentes doenças afectam de modo diferente o comportamento das camadas da rede microcirculatória da pele. Havendo interesse clínico no conhecimento do seu interrelacionamento, é necessária a discriminação do sinal de perfusão sanguínea (informação acerca da velocidade e concentração dos dispersores) com origem nas camadas da pele, super cial e profundas. Assim fomos conduzidos à realização de estudos com o objectivo de aperfeiçoar a técnica de uxometria laser Doppler (FLD) no que diz respeito à discrimina ção da profundidade amostrada. Estes estudos culminaram com o desenvolvimento de um uxómetro que permite o estudo, em simultâneo, da in uência do comprimento de onda e da distância entre as bras emissora e receptora na monitorização da perfusão sanguínea na pele. A tecnologia desenvolvida foi testada in vitro e validada através de simulações Monte Carlo. As simulações comprovaram que a profundidade média dos fot ões dispersos aumenta com o comprimento de onda e com a distância entre bras. Nos testes de validação in vivo foram veri cadas diferenças evidentes quando se compararam fl uxos obtidos com diferentes comprimentos de onda e diferentes distâncias entre fibras. Concomitantemente ao desenvolvimento do protótipo referido, foram realizadas análises, no domínio do tempo e no domínio das frequências, de sinais de perfusão obtidos com fl uxómetros comerciais. Nestes estudos demonstrou-se que a perfusão obtida, em participantes saudáveis, em diferentes regiões de pele, têm propriedades diferentes, e que a perfusão obtida em pacientes com esclerose sistémica tem uma regularidade diferente da dos sinais obtidos em indivíduos saudáveis ou com fenómeno de Raynaud. A necessidade de se observarem as variações do uxo capilar sanguíneo no cérebro de ratos, levou os investigadores a recorrer às sondas de bra óptica invasivas, para FLD, disponíveis no mercado. No entanto, devido ao seu elevado diâmetro externo (ca. 450 m), o tecido cerebral a ser monitorizado ca muito dani cado. Estudos realizados com o objectivo de reduzir as dimensões destas sondas culminaram com a construção de micro-sondas de uma única bra óptica, baseadas na técnica de detecção self-mixing, com um diâmetro externo de 260 m. As micro-sondas desenvolvidas apresentaram uma boa linearidade quando testadas com diferentes velocidades de dispersores. Nas medições efectuadas in vivo houve boas indicações de que as micro-sondas avaliadas podiam ser utilizadas para seguir o uxo microcirculatório, em tempo real, no cérebro dos ratos. Os resultados obtidos com os protótipos (não-invasivo e invasivo) são animadores e prenunciam a sua utilização, tanto a nível clínico como em investigação

Signal and Image Processing of Physiological Data: Methods for Diagnosis and Treatment Purposes

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Finite element mapping for efficient image reconstruction in rotational electrical impedance tomography

Electrical impedance tomography (EIT) is a label free harmless imaging method capable of imaging differences in electrical conductivity of a sample. In EIT, a low frequency current is injected into the sample, voltage differences on sample surface are measured, and from these measurements, interior conductivity distribution is reconstructed. To increase the accuracy of reconstruction, rotational EIT (rEIT) has been proposed where independent measurements are taken from multiple rotational positions around the sample. However, the benefit of conventional electrode configurations are limited to small number of rotational positions. We have presented an approach called Limited Angle Full Revolution rEIT (LAFR-rEIT) that uses a small number of electrodes and large number of rotational measurement position measurements over 360°. The results are comparable to previous rotational EIT implementations, and furthermore, the limited EIT boundary access provides space for simultaneous attachment of other measurement modalities. On the other hand, the increased number of measurement positions cause an increase in computational complexity, and optimization is required until 3D applications are feasible. This work presents modifications into finite element mesh presentation of the imaging domain and outlines an optimization, that enables sufficiently light rotation for 3D LAFR-rEIT computations.submittedVersionPeer reviewe

Rotational electrical impedance tomography using electrodes with limited surface coverage provides window for multimodal sensing

Electrical impedance tomography (EIT) is an imaging method that could become a valuable tool in multimodal applications. One challenge in simultaneous multimodal imaging is that typically the EIT electrodes cover a large portion of the object surface. This paper investigates the feasibility of rotational EIT (rEIT) in applications where electrodes cover only a limited angle of the surface of the object. In the studied rEIT, the object is rotated a full 360◦ during a set of measurements to increase the information content of the data. We call this approach Limited Angle Full Revolution rEIT (LAFR-rEIT). We test LAFR-rEIT setups in two-dimensional geometries with computational and experimental data. We use up to 256 rotational measurement positions, which requires a new way to solve the forward and inverse problem of rEIT. For this, we provide a modification, available for EIDORS, in the supplementary material. The computational results demonstrate that LAFR-rEIT with eight electrodes produce the same image quality as conventional 16-electrode rEIT, when data from an adequate number of rotational measurement positions are used. Both computational and experimental results indicate that the novel LAFR-rEIT provides good EIT with setups with limited surface coverage and a small number of electrodes.publishedVersionPeer reviewe

Gaussian Light Model in Brightfield Optical Projection Tomography

This study focuses on improving the reconstruction process of the brightfield optical projection tomography (OPT). OPT is often described as the optical equivalent of X-ray computed tomography, but based on visible light. The detection optics used to collect light in OPT focus on a certain distance and induce blurring in those features out of focus. However, the conventionally used inverse Radon transform assumes an absolute focus throughout the propagation axis. In this study, we model the focusing properties of the detection by coupling Gaussian beam model (GBM) with the Radon transform. The GBM enables the construction of a projection operator that includes modeling of the blurring caused by the light beam. We also introduce the concept of a stretched GBM (SGBM) in which the Gaussian beam is scaled in order to avoid the modeling errors related to the determination of the focal plane. Furthermore, a thresholding approach is used to compress memory usage. We tested the GBM and SGBM approaches using simulated and experimental data in mono- and multifocal modes. When compared with the traditionally used filtered backprojection algorithm, the iteratively computed reconstructions, including the Gaussian models GBM and SGBM, provided smoother images with higher contrast.publishedVersionPeer reviewe