Aberration compensation for objective phase curvature in phase holographic microscopy: comment

In a recent Letter by Seo et al. [Opt. Lett. 37, 4976 (2012)], the numerical correction of the quadratic phase distortion introduced by the microscope objective in digital holographic microscopy (DHM) has been presented. In this comment, we would like to draw to the attention of the authors and the readers in general that this approach could not be the optimal solution for maintaining the accuracy of the quantitative phase via DHM. We recall that the use of telecentric imaging systems in DHM simplifies the numerical processing of the phase images and produces more accurate measurements

Técnicas de microscopía para la mejora en resolución, obtención de seccionado óptico y medida cuantitativa de fase

La microscopía óptica convencional alcanzó el límite en resolución que predice la difracción por medio del empleo de sofisticados objetivos de microscopio, que consiguen la captura de la información espacial en el espacio de la muestra con un ángulo de captura de prácticamente 70º en aire, esto es, aperturas numéricas (NA) de 0.9 (y mayores si existe un medio de inmersión). Existe la posibilidad de sobrepasar dicho límite o, mejor dicho, encontrar otro límite en resolución siempre que el microscopio que se emplee no pueda entenderse como un microscopio convencional. Por otro lado, los microscopios convencionales carecen de capacidad de seccionado óptico ya que, cuando se tiene una muestra tridimensional, no sólo se obtiene la imagen de la zona enfocada dentro de la misma sino que se recibe luz de todos los planos que se encuentren fuera del plano de foco. Este efecto perturba la calidad de las imágenes bidimensionales obtenidas con el microscopio reduciendo notablemente el contraste y, además, impide la realización de imágenes tridimensionales a partir de la compilación de pilas de secciones ópticas obtenidas para distintos planos de foco. Nuevamente, es posible obtener la mencionada capacidad de seccionado óptico por medio del empleo de algunos microscopios ópticos no convencionales Además en los últimos años se han desarrollado una serie de técnicas alter-nativas a la microscopia convencional que son capaces de proporcionar in-formación de fase de las muestras, esto es, información de las variaciones de índice de refracción en su interior o del espesor de las mismas. Como veremos, dicha información de fase puede ser relevante en el estudio y la caracterización de distintos tipos de muestras. A pesar de que para alcanzar las prestaciones arriba mencionadas se debe aumentar la complejidad tecnológica con respecto a los microscopios convencionales, los beneficios que ello reporta justifica con creces dicha complejidad. Los objetivos de esta Memoria son múltiples. En primer lugar, se pretende aunar en un mismo marco teórico distintas técnicas pertenecientes a la microscopía óptica no convencional, en concreto, CLSM, SIM y DHM. Asimismo se mostrarán algunas de las limitaciones prácticas de las mismas. Para cada una de estas técnicas se presentarán propuestas de implementaciones alternativas así como las ventajas que representa el empleo de estos nuevos sistemas. Para llevar a cabo los objetivos arriba señalados, se comenzará tratando desde un punto de vista teórico la microscopía convencional en el Capítulo 2. Con ello, no sólo se presentará el tipo de cálculos que se requieren para la comprensión de las distintas técnicas de microscopía aquí recogidas, sino que también se podrán entender las limitaciones de la microscopía convencional de una manera estricta. En el Capítulo 3 se hará, en primer lugar, una introducción teórica acerca de la microscopía confocal de barrido. En éste realiza-remos una propuesta de un sistema de adquisición de imágenes confocales alternativo que, además, permitirá mejorar la calidad de las imágenes obtenidas. En el Capítulo 4 introduciremos la técnica SIM de manera rigurosa, pre-sentando los cálculos necesarios para la obtención de imágenes mediante este tipo de sistema. En este capítulo, distinguiremos entre los distintos sistemas que se pueden implementar y propondremos un novedoso sistema para gene-rar iluminación estructurada en el que la frecuencia del patrón de iluminación se pueda variar de una manera muy sencilla. Además, propondremos un algo-ritmo de reconstrucción alternativo para la obtención de imágenes. En el Capítulo 5 nos centraremos en la microscopía holográfica digital. Para llegar a entender el funcionamiento de este tipo de técnica, haremos una pequeña introducción teórica sobre holografía clásica y holografía digital. Esto nos permitirá entender las limitaciones de la técnica, a partir de lo cual realizaremos tanto una propuesta para mejorar sus prestaciones como un desarrollo teórico con el fin de optimizar los parámetros de captura. Por último, en el Capítulo 6, aunaremos las técnicas SIM y DHM en lo que llamaremos microscopía holográfica digital por iluminación estructurada (SI-DHM) en una propuesta novedosa para mejorar la resolución de un DHM convencional. Asimismo, pre-sentaremos un algoritmo mediante el cual se facilita la implementación práctica de esta técnica

Digital holographic microscopy as a screening technology for diabetes

Label-free quantitative phase imaging (QPI) is the hallmark of digital holographic microscopy (DHM). One of the most interesting medical applications of QPI-DHM is that it can be used to analyze illnesses in which the refractive index or/and the morphology of cells/tissues are distorted, from the acquisition of a single image. In this contribution, we obtain the phase maps of red blood cells (RBCs) samples of patients suffering from diabetes mellitus type 1 (DM1) by using a DHM. Our experimental results show that the measured phase values are significantly different between control non-diabetic and diabetic patients. The high correlation coefficient between the phase and the glycated hemoglobin (HbA1C) values determined by the gold standard method to screen diabetes and the clear separation between the two groups indicate that DHM may potentially be used to evaluate long-term glycemic control in diabetic patients as well as to diagnose diabetes

Fourier-domain lightfield microscopy: a new paradigm in 3D microscopy

Recently, integral (also known as lightfield or plenoptic) imaging concept has been applied successfully to microscopy. The main advantage of lightfield microscopy when compared with conventional 3D imaging techniques is that it offers the possibility of capturing the 3D information of the sample after a single shot. However, integral microscopy is now facing many challenges, like improving the resolution and depth of field of the reconstructed specimens or the development and optimization of specially-adapted reconstruction algorithms. This contribution is devoted to review a new paradigm in lightfield microscopy, namely, the Fourier-domain integral microscope (FiMic), that improves the capabilities of the technique, and to present recent advances and applications of this new architecture

Three-dimensional real-time darkfield imaging through Fourier lightfield microscopy

We report a protocol that takes advantage of the Fourier lightfield microscopy concept for providing 3D darkfield images of volumetric samples in a single-shot. This microscope takes advantage of the Fourier lightfield configuration, in which a lens array is placed at the Fourier plane of the microscope objective, providing a direct multiplexing of the spatio-angular information of the sample. Using the proper illumination beam, the system collects the light scattered by the sample while the background light is blocked out. This produces a set of orthographic perspective images with shifted spatial-frequency components that can be recombined to produce a 3D darkfield image. Applying the adequate reconstruction algorithm high-contrast darkfield optical sections are calculated in real time. The presented method is applied for fast volumetric reconstructions of unstained 3D samples

Optical sectioning microscopy through single-shot Lightfield protocol

Optical sectioning microscopy is usually performed by means of a scanning, multi-shot procedure in combination with non-uniform illumination. In this paper, we change the paradigm and report a method that is based in the light field concept, and that provides optical sectioning for 3D microscopy images after a single-shot capture. To do this we fi rst capture multiple orthographic perspectives of the sample by means of Fourier-domain integral microscopy (FiMic). The second stage of our protocol is the application of a novel refocusing algorithm that is able to produce optical sectioning in real time, and with no resolution worsening, in the case of sparse f luorescent samples.We provide the theoretical derivation of the algorithm, and demonstrate its utility by applying it to simulations and to experimental data

La perte de sûreté dans les lignes de remorquage par des problèmes de fatigue dans les tressages de fibres synthétiques

En las operaciones de remolque de buques existen situaciones críticas debido a que el control del movimiento del buque pasa por la integridad de la línea de remolque. En los puertos del Mediterráneo la composición de la línea de remolque viene dada por un cable de acero y un trenzado, de polipropileno habitualmente, más pequeño que actúa como amortiguador. En el momento del diseño de los diferentes elementos se utiliza el coeficiente de seguridad adecuado, pero dado a su uso dinámico y como demuestra nuestro estudio, con el tiempo el material se fatiga con la consiguiente pérdida de seguridad.During tow operations of ships, safety and control of movements of ships depend on towlines integrity. In the Mediterranean Sea, common towline elements are steel cables and ropes, usually of polypropilene, that perform as a shock absorber, due to its less stiffness. A static safety coefficient is defined in the design of the towline elements. In our study, we demonstrate that dynamical use of towline weakens the rope material and suffers fatigue. Hence a reduction of towline safety is reported.Dans les opérations de remorquage de bateaux i y a des situations critiques à cause de que le contrôle du mouvement du bateau passe par l'intégrité de la ligne de remorquage. Dans les ports de la Méditerranée la composition de la ligne de remorquage est un câble en acier et en tressage de polypropylène, habituellement plus petit qui agit comme amortisseur. Au moment du dessin de différents éléments le coefficient adéquat de sûreté est utilisé, mais étant donné son usage dynamique et comme notre étude démontre, avec le temps le matériel se fatigue avec la perte résultante de sûreté

Stable and simple quantitative phase-contrast imaging by Fresnel biprism

Digital holographic (DH) microscopy has grown into a powerful nondestructive technique for the real-time study of living cells including dynamic membrane changes and cell fluctuations in nanometer and sub-nanometer scales. The conventional DH microscopy configurations require a separately generated coherent reference wave that results in a low phase stability and a necessity to precisely adjust the intensity ratio between two overlapping beams. In this work, we present a compact, simple, and very stable common-path DH microscope, employing a self-referencing configuration. The microscope is implemented by a diode laser as the source and a Fresnel biprism for splitting and recombining the beams simultaneously. In the overlapping area, linear interference fringes with high contrast are produced. The frequency of the interference pattern could be easily adjusted by displacement of the biprism along the optical axis without a decrease in fringe contrast. To evaluate the validity of the method, the spatial noise and temporal stability of the setup are compared with the common off-axis DH microscope based on a Mach-Zehnder interferometer. It is shown that the proposed technique has low mechanical noise as well as superb temporal stability with sub-nanometer precision without any external vibration isolation. The higher temporal stability improves the capabilities of the microscope for studying micro-object fluctuations, particularly in the case of biological specimens. Experimental results are presented using red blood cells and silica microspheres to demonstrate the system performance

Over-expression of vascular endothelial growth factor in pituitary adenomas is associated with extrasellar growth and recurrence

Some pituitary adenomas (PA) demonstrate aggressive behavior with local invasion and recurrences. Angiogenesis is regarded as an essential step in the formation of solid tumors. The aim of this study is to find out whether angiogenic factors may have information about the aggressiveness of PA that could be useful in determining the frequency of follow-up and whether adjuvant therapy is necessary. In this retrospective descriptive study, we evaluated vascular endothelial growth factors (VEGF) and VEGF receptor (KDR) mRNA expression by RT-PCR analysis on 46 human PA samples. Clinical data, histological subtype and radiologic characteristics were studied to determine the associations between the variables and the pre-operative behavior of the tumor. In addition, we monitored 12 patients without adjuvant post-operative therapies over 46 months after surgery, determining progression of tumor remnants and its association with these markers. VEGF expression correlates with KDR expression (r = 0.40, p = 0.006). VEGF demonstrates different expression between histological subtypes (p = 0.036). The extension at magnetic resonance imaging showed that VEGF expression was related to suprasellar extension (p = 0.007), being expressed more on tumors with extrasellar growth than intrasellar ones (p = 0.008). Our results demonstrate a 27.5 times increased risk of extrasellar growth when VEGF expression exceeds 0.222 normalized copy number (NCN) (p = 0.002). Likewise, tumors with KDR greater than 0.750 NCN had less recurrence-free survival time (p = 0.032). Our results suggest that the expression of VEGF and its receptor could be a marker for poor outcome after partial tumor resection. These data should be considered in future studies evaluating angiogenic factors as therapeutic targets in patients with PA

Machine Learning-Based View Synthesis in Fourier Lightfield Microscopy

Current interest in Fourier lightfield microscopy is increasing, due to its ability to acquire 3D images of thick dynamic samples. This technique is based on simultaneously capturing, in a single shot, and with a monocular setup, a number of orthographic perspective views of 3D microscopic samples. An essential feature of Fourier lightfield microscopy is that the number of acquired views is low, due to the trade-off relationship existing between the number of views and their corresponding lateral resolution. Therefore, it is important to have a tool for the generation of a high number of synthesized view images, without compromising their lateral resolution. In this context we investigate here the use of a neural radiance field view synthesis method, originally developed for its use with macroscopic scenes acquired with a moving (or an array of static) digital camera(s), for its application to the images acquired with a Fourier lightfield microscope. The results obtained and presented in this paper are analyzed in terms of lateral resolution and of continuous and realistic parallax. We show that, in terms of these requirements, the proposed technique works efficiently in the case of the epi-illumination microscopy mode