Imagen cuantitativa en microscopia 3D con iluminación parcialmente coherente

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Físicas, Departamento de Óptica, leída el 28/01/2021The main research objective of this work is to develop and implement a fast and high resolution technique for reconstructing the 3D refractive index (RI) of weakly absorbing samples. This technique is based on the generalization of optical diffraction tomography (ODT), developed for coherent sample illumination (C-ODT), to the partially coherent case (PC-ODT). In contrast to C-ODT, which involves a specially designed holographic microscope, the proposed PC-ODT technique is implemented in conventional wide-field microscope. The incorporation of the required refocusing module is easy and can be done by a final user unfamiliar with optics (i.e. clinicians and biologists). The low coherent illumination provided by high numerical aperture condenser results in high resolution and reduced speckle noise typical for laser sources used in C-ODT...El principal objetivo de esta tesis es desarrollar e implementar una técnica rápida y de alta resolución para la reconstrucción del índice de refracción (IR) 3D de muestras de absorción débil, basada en la generalización de la tomografía de difracción óptica (TDO), desarrollada para iluminación coherente (TDO-C), al caso parcialmente coherente (TDOPC), lo que evita el ruido coherente y otros artefactos de la iluminación coherente (láser). Al contrario que la TDO-C, que requiere microscopios holográficos (típicamente en configuraciones interferométricas fuera de eje), la técnica TDO-PC propuesta se implementa en un microscopio de campo amplio. La incorporación del módulo de reenfoque óptico es sencillo y puede ser realizado por un usuario no familiarizado con la óptica (como médicos y biólogos). La iluminación poco coherente proporcionada por un condensador de alta apertura numérica logra una alta resolución y reducción del ruido coherente (speckle) típico de las fuentes láser utilizadas en TDO-C...Fac. de Ciencias FísicasTRUEunpu

Label-free quantitative 3D tomographic imaging for partially coherent light microscopy

Crucial benefits provided by partially coherent light microscopy such as improved spatial resolution, optical sectioning and speckle-noise suppression are exploited here to achieve 3D quantitative imaging: reconstruction of the object refractive index (RI). We present a partially coherent optical diffraction tomography technique (PC-ODT) that can be easily implemented in commercially available bright-field microscopes. We show that the high numerical apertures of the objective and condenser lenses, together with optical refocusing, are main issues for achieving fast and successful 3D RI reconstruction of weak objects. In particular, the optical refocusing is performed by a high-speed focus tunable lens mounted in front of the digital camera enabling compatibility with commercial microscopes. The technique is experimentally demonstrated on different examples: diatom cells (biosilica shells), polystyrene micro-spheres and blood cells. The results confirm the straightforward 3D-RI reconstruction of the samples providing valuable quantitative information for their analysis. Thus, the PC-ODT can be considered as an efficient and affordable alternative to coherent ODT which requires specially designed holographic microscopes

Fast label-free optical diffraction tomography compatible with conventional wide-field microscopes

Partially coherent optical diffraction tomography (PC-ODT) is a labelfree quantitative 3D imaging technique based on the refractive index contrast. It provides fast non-interferometric speckle noise-free imaging compatible with conventional wide-field transmission microscopes, but suffers from two principal inconveniences. One of them is the missing cone problem, which is a common drawback for all tomographic modalities with limitedangle inspection, including interferometric coherent ODT. The second one, considered in this paper, is a non-homogeneous contrast for different spatial frequency regions. Analyzing the microscope 3D optical transfer functions for various illuminations over the condenser aperture we have found that the Gaussian illumination shape is a proper one. Numerical simulations and experimental results support this finding. A future line in the development of post-deconvolution processing is also discussed

Optical diffraction tomography with fully and partially coherent illumination in high numerical aperture label-free microscopy

Quantitative label-free imaging is an important tool for the study of living microorganisms that, during the last decade, has attracted wide attention from the optical community. Optical diffraction tomography (ODT) is probably the most relevant technique for quantitative label-free 3D imaging applied in wide-field microscopy in the visible range. The ODT is usually performed using spatially coherent light illumination and specially designed holographic microscopes. Nevertheless, the ODT is also compatible with partially coherent illumination and can be realized in conventional wide-field microscopes by applying refocusing techniques, as it has been recently demonstrated. Here, we compare these two ODT modalities, underlining their pros and cons and discussing the optical setups for their implementation. In particular, we pay special attention to a system that is compatible with a conventional wide-field microscope that can be used for both ODT modalities. It consists of two easily attachable modules: the first for sample illumination engineering based on digital light processing technology; the other for focus scanning by using an electrically driven tunable lens. This hardware allows for a programmable selection of the wavelength and the illumination design, and provides fast data acquisition as well. Its performance is experimentally demonstrated in the case of ODT with partially coherent illumination providing speckle-free 3D quantitative imaging

Partially coherent optical diffraction tomography for label-free quantitative microscopy

In this work we present a tomographic technique for label-free quantitative microscopy that exploits the benefits of the partially coherent illumination. In contrast to conventional holographic techniques based on the coherent optical diffraction tomography (C-ODT), our approach uses an inexpensive non-interferometric setup comprising an electrically tunable lens and low spatially coherent illumination. This setup provides the refocusing needed for axially scan the sample and eventually producing a 3D stack of intensity images. Moreover, the partially coherent illumination avoids speckle noise, which plagues the C-ODT imaging methods. This technique has been developed for optically weak and almost non-absorbing objects that meet the conditions of the first Born approximation. We experimentally demonstrate the capability of the proposed technique by recovering the 3D refractive index distribution of biological samples such as blood cells and diatoms. The results are similar to those obtained by the C-ODT but using a simplified reconstruction process and a refocusing module that can be easily included in commercial widefield microscopes

Label-free bioanalysis of Leishmania infantum using refractive index tomography with partially coherent illumination

In this work, we report the use of refractive index (RI) tomography for quantitative analysis of unstained DH82 cell line infected with Leishmania infantum. The cell RI is reconstructed by using a modality of optical diffraction tomography technique that employs partially coherent illumination, thus enabling inherent compatibility with conventional wide-field microscopes. The experimental results demonstrate that the cell dry mass concentration (DMC) obtained from the RI allows for reliable detection and quantitative characterization of the infection and its temporal evolution. The RI provides important insight for studying morphological changes, particularly membrane blebbing linked to an apoptosis (cell death) process induced by the disease. Moreover, the results evidence that infected DH82 cells exhibit a higher DMC than healthy samples. These findings open up promising perspectives for clinical diagnosis of Leishmania

Ampliación y actualización de las prácticas del laboratorio de Instrumentación Biomédica buscando un aprendizaje y evaluación centrados en el estudiante

El objetivo del proyecto es el diseño de las prácticas de laboratorio para la asignatura Instrumentación Biomédica del Master de Física Biomédica. Se pretende mejorar el aprendizaje potenciando la participación activa y autonomía de los estudiantes. La falta de financiación económica se ha traducido en obtener como resultado del proyecto la redacción de guiones de prácticas que posibiliten el trabajo individual del alumno fuera del laboratorio dado el alto número de alumnos por puesto para llevar a cabo las prácticas. Estos guiones se han complementado con scripts en MatLab para analizar y procesar los datos obtenidos en el laboratorio así como datos obtenidos en diversas bases. Esta propuesta dió información sobre el progreso de los estudiantes en el aprendizaje cuando se utilizó el sistema de rúbricas tambien resultado de este proyecto

Treatment with tocilizumab or corticosteroids for COVID-19 patients with hyperinflammatory state: a multicentre cohort study (SAM-COVID-19)

Objectives: The objective of this study was to estimate the association between tocilizumab or corticosteroids and the risk of intubation or death in patients with coronavirus disease 19 (COVID-19) with a hyperinflammatory state according to clinical and laboratory parameters. Methods: A cohort study was performed in 60 Spanish hospitals including 778 patients with COVID-19 and clinical and laboratory data indicative of a hyperinflammatory state. Treatment was mainly with tocilizumab, an intermediate-high dose of corticosteroids (IHDC), a pulse dose of corticosteroids (PDC), combination therapy, or no treatment. Primary outcome was intubation or death; follow-up was 21 days. Propensity score-adjusted estimations using Cox regression (logistic regression if needed) were calculated. Propensity scores were used as confounders, matching variables and for the inverse probability of treatment weights (IPTWs). Results: In all, 88, 117, 78 and 151 patients treated with tocilizumab, IHDC, PDC, and combination therapy, respectively, were compared with 344 untreated patients. The primary endpoint occurred in 10 (11.4%), 27 (23.1%), 12 (15.4%), 40 (25.6%) and 69 (21.1%), respectively. The IPTW-based hazard ratios (odds ratio for combination therapy) for the primary endpoint were 0.32 (95%CI 0.22-0.47; p < 0.001) for tocilizumab, 0.82 (0.71-1.30; p 0.82) for IHDC, 0.61 (0.43-0.86; p 0.006) for PDC, and 1.17 (0.86-1.58; p 0.30) for combination therapy. Other applications of the propensity score provided similar results, but were not significant for PDC. Tocilizumab was also associated with lower hazard of death alone in IPTW analysis (0.07; 0.02-0.17; p < 0.001). Conclusions: Tocilizumab might be useful in COVID-19 patients with a hyperinflammatory state and should be prioritized for randomized trials in this situatio

EDUCACIÓN AMBIENTAL Y SOCIEDAD. SABERES LOCALES PARA EL DESARROLLO Y LA SUSTENTABILIDAD

Este texto contribuye al análisis científico de varias áreas del conocimiento como la filosofía social, la patología, la educación para el cuidado del medio ambiente y la sustentabilidad que inciden en diversas unidades de aprendizaje de la Licenciatura en Educación para la Salud y de la Maestría en Sociología de la SaludLas comunidades indígenas de la sierra norte de Oaxaca México, habitan un territorio extenso de biodiversidad. Sin que sea una área protegida y sustentable, la propia naturaleza de la región ofrece a sus visitantes la riqueza de la vegetación caracterizada por sus especies endémicas que componen un paisaje de suma belleza