Robust particle outline extraction and its application to digital on-line holography

Peer reviewedPostprin

Focus detection in digital holography by cross-sectional images of propagating waves

In digital holography, computing a focused image of an object requires a prior knowledge of the distance of the object from the camera. When this distance is not known, it is necessary to repeat the image reconstruction at a range of distances followed by evaluation of each image with a sharpness metric to determine the in-focus distance of the object. Here, we present a method to find the focus distance by processing the image transverse to the object plane instead of the processing in the image plane as it is usually done. Since the reconstructed hologram image is spatially symmetric around the focus point along the propagation axis, simply finding the symmetry points in the image cross-section specifies the focus location, and no other sharpness metrics are necessary to use. Also with this method, it is possible to find the focus distances of multiple objects simultaneously, including the phase only objects without any staining. We will present the simulations and the experimental results obtained by a digital holographic microscope

Deep convolutional neural networks and digital holographic microscopy for in-focus depth estimation of microscopic objects

Deep artificial neural network learning is an emerging tool in image analysis. We demonstrate its poten- tial in the field of digital holographic microscopy by addressing the challenging problem of determining the in-focus reconstruction depth of an arbitrary epithelial cell cluster encoded in a digital hologram. A deep convolutional neural network learns the in-focus depths from half a million hologram amplitude images. The trained network correctly determines the in-focus depth of new holograms with high probability, with- out performing numerical propagation. To our knowledge, this is the first application of deep learning in the field of digital holographic microscopy

Mathematical Principles of Object 3D Reconstruction by Shape-from-Focus Methods

Shape-from-Focus (SFF) methods have been developed for about twenty years. They able to obtain the shape of 3D objects from a series of partially focused images. The plane to which the microscope or camera is focused intersects the 3D object in a contour line. Due to wave properties of light and due to finite resolution of the output device, the image can be considered as sharp not only on this contour line, but also in a certain interval of height-the zone of sharpness. SSFs are able to identify these focused parts to compose a fully focused 2D image and to reconstruct a 3D profile of the surface to be observed

High Quality 3D Shape Reconstruction via Digital Refocusing and Pupil Apodization in Multi-wavelength Holographic Interferometry.

Multi-wavelength holographic interferometry (MWHI) has good potential for evolving into a high quality 3D shape reconstruction technique. There are several remaining challenges, including 1) depth-of-field limitation, leading to axial dimension inaccuracy of out-of-focus objects; and 2) smearing from shiny smooth objects to their dark dull neighbors, generating fake measurements within the dark area. This research is motivated by the goal of developing an advanced optical metrology system that provides accurate 3D profiles for target object or objects of axial dimension larger than the depth-of-field, and for objects with dramatically different surface conditions. The idea of employing digital refocusing in MWHI has been proposed as a solution to the depth-of-field limitation. One the one hand, traditional single wavelength refocusing formula is revised to reduce sensitivity to wavelength error. Investigation over real example demonstrates promising accuracy and repeatability of reconstructed 3D profiles. On the other hand, a phase contrast based focus detection criterion is developed especially for MWHI, which overcomes the problem of phase unwrapping. The combination for these two innovations gives birth to a systematic strategy of acquiring high quality 3D profiles. Following the first phase contrast based focus detection step, interferometric distance measurement by MWHI is implemented as a next step to conduct relative focus detection with high accuracy. This strategy results in ±100mm 3D profile with micron level axial accuracy, which is not available in traditional extended focus image (EFI) solutions. Pupil apodization has been implemented to address the second challenge of smearing. The process of reflective rough surface inspection has been mathematically modeled, which explains the origin of stray light and the necessity of replacing hard-edged pupil with one of gradually attenuating transmission (apodization). Metrics to optimize pupil types and parameters have been chosen especially for MWHI. A Gaussian apodized pupil has been installed and tested. A reduction of smearing in measurement result has been experimentally demonstrated.Ph.D.Mechanical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/91461/1/xulium_1.pd

Design and implementation of a digital holographic microscope with fast autofocusing

Holography is a method for three-dimensional (3D) imaging of objects by applying interferometric analysis. A recorded hologram is required to be reconstructed in order to image an object. However one needs to know the appropriate reconstruction distance prior to the hologram reconstruction, otherwise the reconstruction is out-of-focus. If the focus distance of the object is not known priori, then it must be estimated using an autofocusing technique. Traditional autofocusing techniques used in image processing literature can also be applied to digital holography. In this thesis, eleven common sharpness functions developed for standard photography and microscopy are applied to digital holograms, and the estimation of the focus distances of holograms is investigated. The magnitude of a recorded hologram is quantitatively evaluated for its sharpness while it is reconstructed on an interval, and the reconstruction distance which yields the best quantitative result is chosen as the true focus distance of the hologram. However autofocusing of highresolution digital holograms is very demanding in means of computational power. In this thesis, a scaling technique is proposed for increasing the speed of autofocusing in digital holographic applications, where the speed of a reconstruction is improved on the order of square of the scale-ratio. Experimental results show that this technique offers a noticeable improvement in the speed of autofocusing while preserving accuracy greatly. However estimation of the true focus point with very high amounts of scaling becomes unreliable because the scaling method detriments the sharpness curves produced by the sharpness functions. In order to measure the reliability of autofocusing with the scaling technique, fifty computer generated holograms of gray-scale human portrait, landscape and micro-structure images are created. Afterwards, autofocusing is applied to the scaleddown versions of these holograms as the scale-ratio is increased, and the autofocusing performance is statistically measured as a function of the scale-ratio. The simulation results are in agreement with the experimental results, and they show that it is possible to apply the scaling technique without losing significant reliability in autofocusing

Long-Term Quantitative Microscopy: From Microbial Population Dynamics to Growth of Plant Roots

Quantitative optical measurements at the micron scale have been crucial to the study of multiple biological processes, including bacterial chemotaxis, eukaryotic gene expression and y development. Extending measurements to long time scales allows complete observation of processes that are otherwise studied piecemeal, such as development and evolution. This thesis describes the development of two types of microscope for making long term, quantitative measurements, and the tools for image analysis. The rst device is a digital holographic microscope for measuring microbial population dynamics. It allows three dimensional localization of hundreds of cells within a mm3 sized volume, at micron resolution and an acquisition period of minutes. The technique is simple and inexpensive, which enabled us to construct ten replicate devices for parallel measurements. Each device incorporates precise and programmable control of light and temperature for the microbial ecosystem. Experiments were performed with the green algae Chlamydomonas reinhardtii and the ciliate Tetrahymena reinhardtii, both together and in isolation, and continued for as long as 90 days. The population dynamics exhibited a striking degree of repeatability, despite the presence of added noise in the illumination, spatial gradients of cell density, convection currents and phenotypic changes of both species. The second device is a thin light sheet fluorescence microscope for tracking nuclei in growing roots of the flowering plant Arabidopsis thaliana. The device incorporates a chamber designed to maintain optical quality while providing conditions for root growth. Optical feedback to a translation stage is used to maintain the root tip in the fi eld of view as the root grows by centimeters over several days. Data from a three day experiment is presented to demonstrate the technique. Over 1,000 nuclei were tracked simultaneously, and hundreds of cell divisions were automatically identif ed. The device was also used to image the regeneration of a root tip after surgical excision. The data corroborate earlier investigations at a more detailed level than was previously possible