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

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

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

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

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

Fast and robust wave optics-based reconstruction protocol for Fourier lightfield microscopy

Fourier lightfield microscopy (FLMic) is a powerful technique to record 3D images of thick dynamic samples. Belonging FLMic to the general class of computational imaging techniques, its efficiency is determined by several factors, like the optical system, the calibration process, the reconstruction algorithm, or the computation architecture. In the case of FLMic the calibration and the reconstruction algorithm should be fully adapted to the singular features of the technique. To this end, and concerning the reconstruction, we discard the use of experimental PSFs, and propose the use of a synthetic one, which is calculated on the basis of paraxial optics and taking into account the equal influence of diffraction and pixelation. Using this quite simple PSF, performing the adequate calibration and finally implementing the algorithm in GPU, we demonstrate here the possibility of obtaining 3D images with good results in terms of resolution and strong improvement in terms of computation time. In summary, and aiming to accelerate the widespread of FLMic among microscopy users and researchers, we are proposing a fast protocol fully adapted to FLMic and that is very flexible and robust against any slight misalignment or against the change of any optical element

Resolution limit in opto-digital systems revisited

The resolution limit achievable with an optical system is a fundamental piece of information when characterizing its performance, mainly in case of microscopy imaging. Usually this information is given in the form of a distance, often expressed in microns, or in the form of a cutoff spatial frequency, often expressed in line pairs per mm. In modern imaging systems, where the final image is collected by pixelated digital cameras, the resolution limit is determined by the performance of both, the optical systems and the digital sensor. Usually, one of these factors is considered to be prevalent over the other for estimating the spatial resolution, leading to the global performance of the imaging system ruled by either the classical Abbe resolution limit, based on physical diffraction, or by the Nyquist resolution limit, based on the digital sensor features. This estimation fails significantly to predict the global performance of opto-digital imaging systems, like 3D microscopes, where none of the factors is negligible. In that case, which indeed is the most common, neither the Abbe formula nor the Nyquist formula provide by themselves a reliable prediction for the resolution limit. This is a serious drawback since systems designers often use those formulae as design input parameters. Aiming to overcome this lack, a simple mathematical expression obtained by finely articulating the Abbe and Nyquist formulas, to easily predict the spatial resolution limit of opto-digital imaging systems, is proposed here. The derived expression is tested experimentally, and shows to be valid in a broad range of opto-digital combinations

Three-dimensional imaging through patterned type-1 microscopy

We report a scanning non-confocal fluorescence microscopy scheme that provides images with optical sectioning and with a lateral resolution that surpasses by a factor of two the diffraction resolution limit. This technique is based on the type-1 microscopy concept combined with patterned illumination. The method does not require the application of phase-shifting or post-processing algorithms and provides artifact-free superresolved 3D images. We have validated the theory by means of experimental data