New advances in high-resolution optical microscopy

La microscopio ía tiene como nalidad observar muestras que no pueden distinguirse a simple vista por el ojo humano dado que el tamaño de ellas es menor que su l mite de resoluci on. Un microscopio produce una imagen ampliada de la muestra a analizar. Debido a que la microscopía es un instrumento b asico para la ciencia de la vida y de los materiales, en esta Tesis se desarrolla un estudio exhaustivo de la microscopía óptica. Los microscopios opticos se pueden con gurar de diferentes formas para producir im agenes con diferentes caracter sticas. El microscopio de campo amplio, el m as simple de todos, presenta algunas limitaciones que debe ser superadas para obtener im agenes de mayor calidad. Entre estas limitaciones encontramos: el barrido axial mec anico para proporcionar toda la informaci on de la estructura de la muestra, la presencia de aberraci ón esf érica debido a los desajustes del í ndice de refracci ón entre el medio de inmersi on del objetivo, el cubreobjetos y la muestra, la limitaci ón de la resoluci ón espacial impuesta por la difracci ón y la incapacidad de obtener im ágenes cuantitativas de fase. Tales limitaciones se analizan en esta Tesis y se proponen algunas soluciones con el fi n de proporcionar mejores im ágenes micros ópicas. En particular, nosotros hemos obtenido: (1) un r ápido barrido axial de muestras gruesas, en tiempo real y sin ning ún movimiento mecánico, (2) un microscopio invariante a la aberraci ón esf érica, (3) im agenes con alta resoluci on lateral y seccionado optico y (4) im agenes cuantitativas de fase precisas y sin deteriorar el l mite de resoluci on. Todos estos resultados se han demostrado tanto te oricamente como experimentalmente.Microscopy is the science which aim is to view objects that can not be distinguished with the naked eye because the size of those objects are not within its resolution range. A microscope produces an enlarged image of a sample under research. Since microscopy is an essential tool for live and material sciences, this Thesis is devoted to study thoroughly optical microscopy. Optical microscopes can be performed in di erent ways to provide resulting images with di erent features. The simplest optical microscope is the wide eld microscope. However it presents some limitations that needs to be overcome to obtain high-quality images. Among these limitations we nd: axial mechanical scanning to provide the whole structure of a sample, the presence of spherical aberration due to the refractive-index mismatches between the immersion medium of the microscope objective, the coverglass and the specimen, the limitation of spatial resolution imposed by di raction and the inability of obtaining quantitative phase images. Such limitations are analyzed in this Thesis and some solutions are proposed in order to provide better microscopic images. In particular, we have achieved: (1) a fast-axial scanning of thick samples in real time and without any mechanical movement, (2) an SA-invariant imaging system, (3) images with high lateral resolution and optical sectioning and (4) accurate quantitative phase images and without deteriorating the resolution limit imposed by di raction. All these ndings have been veri ed both theoretically and experimentall

Digital holographic microscopy for diabetes screening

A digital holographic microscope operating in telecentric mode could be used to diagnose diabetes and evaluate long-term glycemic control in patients with diabetes

Shaded-Mask Filtering for Extended Depth-of-Field Microscopy

This paper proposes a new spatial filtering approach for increasing the depth-of-field (DOF) of imaging systems, which is very useful for obtaining sharp images for a wide range of axial positions of the object. Many different techniques have been reported to increase the depth of field. However the main advantage in our method is its simplicity, since we propose the use of purely absorbing beam-shaping elements, which allows a high focal depth with a minimum modification of the optical architecture. In the filter design, we have used the analogy between the axial behavior of a system with spherical aberration and the transverse impulse response of a 1D defocused system. This allowed us the design of a ring-shaded filter. Finally, experimental verification of the theoretical statements is also provided

Off-axis Digital Holographic Microscopy: practical design parameters for operating at diffraction limit

The utilization of microscope objectives (MOs) in digital holographic microscopy (DHM) has associated effects that are not present in conventional optical microscopy. The remaining phase curvature, which can ruin the quantitative phase imaging, is the most evident and analyzed. As phase imaging is considered, this interest has made possible the development of different methods of overcoming its undesired consequences. Additionally to the effects in phase imaging, there exist a set of less obvious conditions that have to be accounted for as MOs are utilized in DHM to achieve diffraction-limit operation. These conditions have to be considered even in the case in which only amplitude or intensity imaging is of interest. In this paper, a thorough analysis of the physical parameters that control the appropriate utilization of MOs in DHM is presented. A regular DHM system is theoretically modeled on the basis of the imaging theory. The Fourier spectrum of the recorded hologram is analyzed to evaluate the performance of the DHM. A set of the criteria that consider the microscope features and the recording parameters to achieve DHM operation at the diffraction limit is derived. Numerical modeling and experimental results are shown to validate our findings