Emilio Sánchez-Ortiga

Digital holographic microscopy with pure-optical spherical phase compensation

Telecentric architecture is proposed for circumventing, by the pure-optical method, the residual parabolic phase distortion inherent to standard configuration of digital holographic microscopy. This optical circumvention produces several important advantages. One is that there is no need for computer compensation of the parabolic phase during the phase map recovering procedure. The other is that in off-axis configuration, the spatial frequency useful domain is enlarged. The validity of the method is demonstrated by performing quantitative measurement of depth differences with high axial resolution.

Accurate single-shot quantitative phase imaging of biological specimens with telecentric digital holographic microscopy

Abstract. The advantages of using a telecentric imaging system in digital holographic microscopy (DHM) to study biological specimens are highlighted. To this end, the performances of nontelecentric DHM and telecentric DHM are evaluated from the quantitative phase imaging (QPI) point of view. The evaluated stability of the microscope allows single-shot QPI in DHM by using telecentric imaging systems. Quantitative phase maps of a section of the head of the drosophila melanogaster fly and of red blood cells are obtained via single-shot DHM with no numerical postprocessing. With these maps we show that the use of telecentric DHM provides larger field of view for a given magnification and permits more accurate QPI measurements with less number of computational operations.

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.

Subtractive imaging in confocal scanning microscopy using a CCD camera as a detector

We report a scheme for the detector system of confocal microscopes in which the pinhole and a large-area detector are substituted by a CCD camera. The numerical integration of the intensities acquired by the active pixels emulates the signal passing through the pinhole. We demonstrate the imaging capability and the optical sectioning of the system. Subtractive-imaging confocal microscopy can be implemented in a simple manner, providing superresolution and improving optical sectioning.

Shift-variant digital holographic microscopy: inaccuracies in quantitative phase imaging

Inaccuracies introduced in quantitative phase digital holographic microscopy by the use of nontelecentric imaging systems are analyzed. Computer modeling of the experimental result shows that even negligible errors in the radius and center of curvature of the numerical compensation needed to get rid of the remaining quadratic phase factor introduce errors in the phase measurements; these errors depend on the position of the object in the field-of-view. However, when a telecentric imaging system is utilized for the recording of the holograms, the numerical modeling and experimental results show the shift-invariant behavior of the quantitative-phase digital holographic microscope.

Multispectral integral imaging acquisition and processing using a monochrome camera and a liquid crystal tunable filter

This paper presents an acquisition system and a procedure to capture 3D scenes in different spectral bands. The acquisition system is formed by a monochrome camera, and a Liquid Crystal Tunable Filter (LCTF) that allows to acquire images at different spectral bands in the [480, 680]nm wavelength interval. The Synthetic Aperture Integral Imaging acquisition technique is used to obtain the elemental images for each wavelength. These elemental images are used to computationally obtain the reconstruction planes of the 3D scene at different depth planes. The 3D profile of the acquired scene is also obtained using a minimization of the variance of the contribution of the elemental images at each image pixel. Experimental results show the viability to recover the 3D multispectral information of the scene. Integration of 3D and multispectral information could have important benefits in different areas, including skin cancer detection, remote sensing and pattern recognition, among others.

Reduction of spherical-aberration impact in microscopy by wavefront coding

In modern high-NA optical scanning instruments, like scanning microscopes, the refractive-index mismatch between the sample and the immersion medium introduces a significant amount of spherical aberration when imaging deep inside the specimen, spreading out the impulse response. Since such aberration depends on the focalization depth, it is not possible to achieve a static global compensation for the whole 3D sample in scanning microscopy. Therefore a depth-variant impulse response is generated. Consequently, the design of pupil elements that increase the tolerance to this aberration is of great interest. In this paper we report a hybrid technique that provides a focal spot that remains almost invariant in the depth-scanning processing of thick samples. This invariance allows the application of 3D deconvolution techniques to that provide an improved recovery of the specimen structure when imaging thick samples.

Non-Homogeneity of Lateral Resolution in Integral Imaging

We evaluate the lateral resolution in reconstructed integral images. Our analysis takes into account both the diffraction effects in the image capture stage and the lack of homogeneity and isotropy in the reconstruction stage. We have used Monte Carlo simulation in order to assign a value for the resolution limit to any reconstruction plane. We have modelled the resolution behavior. Although in general the resolution limit increases proportionally to the distance to the lens array, there are some periodically distributed singularity planes. The phenomenon is supported by experiments.

Resolution enhancement in integral microscopy by physical interpolation.

Integral-imaging technology has demonstrated its capability for computing depth images from the microimages recorded after a single shot. This capability has been shown in macroscopic imaging and also in microscopy. Despite the possibility of refocusing different planes from one snap-shot is crucial for the study of some biological processes, the main drawback in integral imaging is the substantial reduction of the spatial resolution. In this contribution we report a technique, which permits to increase the two-dimensional spatial resolution of the computed depth images in integral microscopy by a factor of √2. This is made by a double-shot approach, carried out by means of a rotating glass plate, which shifts the microimages in the sensor plane. We experimentally validate the resolution enhancement as well as we show the benefit of applying the technique to biological specimens.

Fast Axial-Scanning Widefield Microscopy With Constant Magnification and Resolution

In this paper, we propose the use of electrically-addressable lens devices for performing fast non-mechanical axial scanning when imaging three-dimensional samples. This non-mechanical method can be implemented in any commercial microscope. The approach is based on the insertion of the tunable lens at the aperture stop of the microscope objective. By tuning the voltage, a stack of depth images of 3D specimens can be captured in real time and with constant magnification and resolution. The main advantage of our technique is the possibility of performing fast axial scanning free of mechanical vibrations.