1,714 research outputs found

    Adaptive beam control and analysis in fluorescence microscopy

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    This thesis details three novel advances in instrumentation that are each related to performance improvement in wide-field visible-spectrum imaging systems. In each case our solution concerns the assessment and improvement of optical imaging quality. The three instruments are as follows: The first is a portable transmission microscope which is able to correct for artificially induced aberrations using adaptive optics (AO). The specimens and the method of introducing aberrations into the optical system can be altered to simulate the performance of AO-correction in both astronomical and biological imaging. We present the design and construction of the system alongside before-and-after AO-correction images for simulated astronomical and biological images. The second instrument is a miniature endoscope camera sensor we re-purposed for use as a quantitative beam analysis probe using a custom high dynamic range (HDR) imaging and reconstruction procedure. This allowed us to produce quantitative flux maps of the illumination beam intensity profile within several operational fluorescence microscope systems. The third and final project in this thesis was concerned with an adaptive modification to the light sheet illumination beam used in light sheet microscopy, specifically for a single plane illumination microscope (SPIM), embracing the trade-off between the thickness of the light sheet and its extent across the detection field-of-view. The focal region of the beam was made as small as possible and then matched to the shape of curved features within a biological specimen by using a spatial light modulator (SLM) to alter the light sheet focal length throughout the vertical span of the sheet. We used the HDR beam profiling camera probe mentioned earlier to assess the focal shape and quality of the beam. The resulting illumination beam may in the future be used in a modified SPIM system to produce fluorescence microscope images with enhanced optical sectioning of specific curved features

    Adaptive optics wavefront compensation for solid immersion microscopy in backside imaging

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    Thesis (Ph.D.)--Boston UniversityThis dissertation concerns advances in high-resolution optical microscopy needed to detect faults in next generation semiconductor chips. In this application, images are made through the chips' back side to avoid opaque interconnect metal layers on the frontside. Near infrared wavelengths are required, since the silicon is relatively transparent at these wavelengths. A significant challenge in this technique is to resolve features as small as 200nm using wavelengths exceeding 1OOOnm. The highest imaging resolution achievable with refractive optics at infrared wavelengths is demonstrated in this dissertation using an aplanatic solid immersion lens (SIL). This is the only method that has been found to be of sufficient resolution to image the next generation of integrated circuits. While the use of an aplanatic solid immersion lens theoretically allows numerical aperture far in excess of conventional microscopy (NASIL ~ 3.5), it also makes the system performance particularly sensitive to aberrations, especially when the samples have thicknesses that are more than a few micrometers thicker or thinner than designed thickness, or when the refractive index of the SIL is slightly different than that of the sample. In the work described here, practical design considerations of the SILs are examined. A SIL-based confocal scanning microscope system is designed and constructed. The aberrations of the system due to thickness uncertainty and material mismatch are simulated using both analytical model and ray-tracing software, and are measured in the SIL experimental apparatus. The dominant aberration for samples with thickness mismatch is found to be spherical aberration. Wavefront errors are compensated by a microelectromechanical systems deformable mirror (MEMS DM) in the optical system's pupil. The controller is implemented either with closed-loop real time sensor feedback or with predictive open-loop estimation of optical aberrations. Different DM control algorithms and aberration compensation techniques are studied and compared. The experimental results agree well with simulation and it has been demonstrated through models and experiments in this work that the stringent sample thickness tolerances previously needed for high numerical aperture SIL microcopy can be relaxed considerably through aberration compensation. Near-diffraction-limited imaging performance has been achieved in most cases that correspond to practical implementation of the technique

    Fast Objective Coupled Planar Illumination Microscopy

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    Among optical imaging techniques light sheet fluorescence microscopy stands out as one of the most attractive for capturing high-speed biological dynamics unfolding in three dimensions. The technique is potentially millions of times faster than point-scanning techniques such as two-photon microscopy. This potential is especially poignant for neuroscience applications due to the fact that interactions between neurons transpire over mere milliseconds within tissue volumes spanning hundreds of cubic microns. However current-generation light sheet microscopes are limited by volume scanning rate and/or camera frame rate. We begin by reviewing the optical principles underlying light sheet fluorescence microscopy and the origin of these rate bottlenecks. We present an analysis leading us to the conclusion that Objective Coupled Planar Illumination (OCPI) microscopy is a particularly promising technique for recording the activity of large populations of neurons at high sampling rate. We then present speed-optimized OCPI microscopy, the first fast light sheet technique to avoid compromising image quality or photon efficiency. We enact two strategies to develop the fast OCPI microscope. First, we devise a set of optimizations that increase the rate of the volume scanning system to 40 Hz for volumes up to 700 microns thick. Second, we introduce Multi-Camera Image Sharing (MCIS), a technique to scale imaging rate by incorporating additional cameras. MCIS can be applied not only to OCPI but to any widefield imaging technique, circumventing the limitations imposed by the camera. Detailed design drawings are included to aid in dissemination to other research groups. We also demonstrate fast calcium imaging of the larval zebrafish brain and find a heartbeat-induced motion artifact. We recommend a new preprocessing step to remove the artifact through filtering. This step requires a minimal sampling rate of 15 Hz, and we expect it to become a standard procedure in zebrafish imaging pipelines. In the last chapter we describe essential computational considerations for controlling a fast OCPI microscope and processing the data that it generates. We introduce a new image processing pipeline developed to maximize computational efficiency when analyzing these multi-terabyte datasets, including a novel calcium imaging deconvolution algorithm. Finally we provide a demonstration of how combined innovations in microscope hardware and software enable inference of predictive relationships between neurons, a promising complement to more conventional correlation-based analyses

    Appearance Enhancement for Visually Impaired with Projector Camera Feedback

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    Visually impaired is a common problem for human life in the world wide. The projector-based AR technique has ability to change appearance of real object, and it can help to improve visibility for visually impaired. We propose a new framework for the appearance enhancement with the projector camera system that employed model predictive controller. This framework enables arbitrary image processing such as photo-retouch software in the real world and it helps to improve visibility for visually impaired. In this article, we show the appearance enhancement result of Peli's method and Wolffshon's method for the low vision, Jefferson's method for color vision deficiencies. Through experiment results, the potential of our method to enhance the appearance for visually impaired was confirmed as same as appearance enhancement for the digital image and television viewing

    Light controlled motility of Escherichia coli. Characterization and applications

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    Characterization of wild type E. coli motility in response to light stimuli. Gene editing of bacteria to implement specifc functions (e.g. photokinesis). The engineered strain has been used to demonstrate that density modulation of photokinetic bacteria can be obtained by projecting spatially structured light on the sample. Additionally these bacteria have been also used as propelling units in microfabricated structures

    Novel optics-based approaches for cardiac electrophysiology: a review

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    Optical techniques for recording and manipulating cellular electrophysiology have advanced rapidly in just a few decades. These developments allow for the analysis of cardiac cellular dynamics at multiple scales while largely overcoming the drawbacks associated with the use of electrodes. The recent advent of optogenetics opens up new possibilities for regional and tissue-level electrophysiological control and hold promise for future novel clinical applications. This article, which emerged from the international NOTICE workshop in 20181, reviews the state-of-the-art optical techniques used for cardiac electrophysiological research and the underlying biophysics. The design and performance of optical reporters and optogenetic actuators are reviewed along with limitations of current probes. The physics of light interaction with cardiac tissue is detailed and associated challenges with the use of optical sensors and actuators are presented. Case studies include the use of fluorescence recovery after photobleaching and super-resolution microscopy to explore the micro-structure of cardiac cells and a review of two photon and light sheet technologies applied to cardiac tissue. The emergence of cardiac optogenetics is reviewed and the current work exploring the potential clinical use of optogenetics is also described. Approaches which combine optogenetic manipulation and optical voltage measurement are discussed, in terms of platforms that allow real-time manipulation of whole heart electrophysiology in open and closed-loop systems to study optimal ways to terminate spiral arrhythmias. The design and operation of optics-based approaches that allow high-throughput cardiac electrophysiological assays is presented. Finally, emerging techniques of photo-acoustic imaging and stress sensors are described along with strategies for future development and establishment of these techniques in mainstream electrophysiological research

    Publications of the Jet Propulsion Laboratory, 1988

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    This bibliography describes and indexes by primary author the externally distributed technical reporting, released during calendar year 1988, that resulted from scientific and engineering work performed, or managed, by the Jet Propulsion Laboratory. Three classes of publications are included: JPL publications in which the information is complete for a specific accomplishment; articles from the quarterly Telecommunications and Data Acquisition (TDA) Progress Report; and articles published in the open literature

    Molecular Photoswitches for STED-inspired Laser Lithography

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    Tools for interfacing, extracting, and analyzing neural signals using wide-field fluorescence imaging and optogenetics in awake behaving mice

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    Imaging of multiple cells has rapidly multiplied the rate of data acquisition as well as our knowledge of the complex dynamics within the mammalian brain. The process of data acquisition has been dramatically enhanced with highly affordable, sensitive image sensors enable high-throughput detection of neural activity in intact animals. Genetically encoded calcium sensors deliver a substantial boost in signal strength and in combination with equally critical advances in the size, speed, and sensitivity of image sensors available in scientific cameras enables high-throughput detection of neural activity in behaving animals using traditional wide-field fluorescence microscopy. However, the tremendous increase in data flow presents challenges to processing, analysis, and storage of captured video, and prompts a reexamination of traditional routines used to process data in neuroscience and now demand improvements in both our hardware and software applications for processing, analyzing, and storing captured video. This project demonstrates the ease with which a dependable and affordable wide-field fluorescence imaging system can be assembled and integrated with behavior control and monitoring system such as found in a typical neuroscience laboratory. An Open-source MATLAB toolbox is employed to efficiently analyze and visualize large imaging data sets in a manner that is both interactive and fully automated. This software package provides a library of image pre-processing routines optimized for batch-processing of continuous functional fluorescence video, and additionally automates a fast unsupervised ROI detection and signal extraction routine. Further, an extension of this toolbox that uses GPU programming to process streaming video, enabling the identification, segmentation and extraction of neural activity signals on-line is described in which specific algorithms improve signal specificity and image quality at the single cell level in a behaving animal. This project describes the strategic ingredients for transforming a large bulk flow of raw continuous video into proportionally informative images and knowledge
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