Set up of a light sheet fluorescence microscope for cellular studies

Light-sheet fluorescence microscopy (LSFM) has been present in cell biology laboratories for quite some time, mainly as custom-made systems, with imaging applications ranging from single cells (in the μm scale) to small organisms (mm). Such microscopes distinguish themselves for having very low phototoxicity levels and high spatial and temporal resolution, properties that render it ideal for 3D characterization of cell motility in migration and traction force studies. Cellular motion has proven to be essential in biological processes such as tumor metastasis and tissue development. Experimental setups make extensive use of microdevices (bioMEMS) that are providing higher degrees of empirical complexity. The following report details the process of setting-up a functional LSFM device for imaging cell motion in microfluidic devices. It begins with a brief summary of fluorescence imaging and current techniques, important to understand why single-plane illumination microscopy (SPIM) was chosen among other light-sheet methods. Then, the whole SPIM set-up process is described, containing explanations about the physical and material properties of the hardware used, the intricacies of the control system, and important procedures. These procedures include: calibration of the microscope, sample preparation in microdevices, and image acquisition from the software provided. Real fluorescence images acquired serve as evidence of the functionality of the instrument. The current limitations are highlighted, and pointers on how to improve or enhance the device are given. The report contains many diagrams, tables and pictures to aid in the understanding of important concepts. In the Annex, a comprehensive table listing the project costs by category is attached. This table includes links to the manufacturers and providers. The aim of this writing is to serve as an exhaustive guideline and be of reproducible use for researchers aiming to build SPIM systems for similar applications.Ingeniería Biomédic

Manufacturing Metrology

Metrology is the science of measurement, which can be divided into three overlapping activities: (1) the definition of units of measurement, (2) the realization of units of measurement, and (3) the traceability of measurement units. Manufacturing metrology originally implicates the measurement of components and inputs for a manufacturing process to assure they are within specification requirements. It can also be extended to indicate the performance measurement of manufacturing equipment. This Special Issue covers papers revealing novel measurement methodologies and instrumentations for manufacturing metrology from the conventional industry to the frontier of the advanced hi-tech industry. Twenty-five papers are included in this Special Issue. These published papers can be categorized into four main groups, as follows: Length measurement: covering new designs, from micro/nanogap measurement with laser triangulation sensors and laser interferometers to very-long-distance, newly developed mode-locked femtosecond lasers. Surface profile and form measurements: covering technologies with new confocal sensors and imagine sensors: in situ and on-machine measurements. Angle measurements: these include a new 2D precision level design, a review of angle measurement with mode-locked femtosecond lasers, and multi-axis machine tool squareness measurement. Other laboratory systems: these include a water cooling temperature control system and a computer-aided inspection framework for CMM performance evaluation

In-line quality control for Zero Defect Manufacturing: design, development and uncertainty analysis of vision-based instruments for dimensional measurements at different scales

Lo scopo di questo progetto di dottorato industriale finanziato attraverso una borsa di studio della Regione Marche è stato quello di sviluppare ricerca con potenziale impatto su un settore industriale, promuovere il coinvolgimento delle fabbriche e delle imprese locali nella ricerca e innovazione svolta in collaborazione con l'università e produrre ricerca in linea con le esigenze dell'ambiente industriale, non solo a livello regionale. Quindi, attraverso la collaborazione con una torneria locale (Zannini SpA) e una piccola azienda high-tech focalizzata sull'introduzione dell'innovazione meccatronica nel settore della tornitura (Z4Tec srl), e anche grazie a una collaborazione internazionale con l'Università di Anversa, abbiamo progettato e sviluppato nuovi strumenti per il controllo qualità in linea, basati su tecnologie senza contatto, in particolare tecnologie elettro-ottiche. Portando anche l'attenzione sull'importanza di prendere in considerazione l'incertezza, poiché è fondamentale nel processo decisionale basato sui dati che sono alla base di una strategia di Zero Defect Manufacturing. Infatti, la scarsa qualità delle misure può pregiudicare la qualità dei dati. In particolare, questo lavoro presenta due strumenti di misura che sono stati progettati e sviluppati con lo scopo di effettuare controllo qualità in linea di produzione e l’incertezza di misura di ogni strumento è stata analizzata in confronto ad altri strumenti presenti sul mercato. Nella parte finale di questo lavoro si è valutata l’incertezza di un profilometro a triangolazione di linea laser. Pertanto, la ricerca condotta in questa tesi può essere organizzata in due obiettivi principali: lo sviluppo di nuovi sistemi di misura dimensionale basati sulla visione da implementare in linea di produzione e l'analisi dell'incertezza di questi strumenti di misura. Per il primo obiettivo ci siamo concentrati su due tipi di misure dimensionali imposte dall'industria manifatturiera: macroscopiche (misure in mm) e microscopiche (misure in µm). Per le misure macroscopiche l'obiettivo era il controllo in linea della qualità dimensionale di pezzi torniti attraverso la profilometria ottica telecentrica. Il campione da ispezionare è stato posto tra l'illuminatore e l'obiettivo per ottenere la proiezione dell'ombra del campione. Le misure sono state eseguite mediante analisi grafica dell'immagine. Abbiamo discusso le disposizioni meccaniche mirate a ottimizzare le immagini acquisite e i problemi che eventuali disallineamenti meccanici dei componenti potrebbero introdurre nella qualità delle immagini. Per le misure microscopiche abbiamo progettato un sistema di misurazione della rugosità superficiale basato sulla visione retroilluminata, con l'obiettivo di determinare le condizioni ottimali di imaging utilizzando la modulation transfer function e l'uso di una electrically tunable lens. Un campione tornito (un cilindro) è posto di fronte a una telecamera ed è retroilluminato da una sorgente di luce collimata; tale configurazione ottica fornisce l'immagine del bordo del campione. Per testare la sensibilità del sistema di misura è stata utilizzata una serie di campioni di acciaio torniti con diverse rugosità superficiali. Per il secondo obiettivo, le tecniche di valutazione dell'incertezza di misura utilizzate in questo lavoro sono state un'analisi dell'incertezza statistica di tipo A e un'analisi Gage R&R. Nel caso del profilometro telecentrico, l'analisi è stata eseguita in confronto con altri dispositivi presenti sul mercato con un'analisi di tipo A e una Gage R&R. L'incertezza di misura del profilometro si è rivelata sufficiente per ottenere risultati nell'intervallo di tolleranza richiesto. Per il sistema di visione retroilluminato, il confronto dei risultati è stato effettuato con altri strumenti allo stato dell'arte, con un'analisi di Tipo A. Il confronto ha mostrato che le prestazioni dello strumento retroilluminato dipendono dai valori di rugosità superficiale considerati; mentre a valori maggiori di rugosità l'offset aumenta, per valori inferiori di rugosità i risultati sono compatibili con quelli dello strumento di riferimento (a stilo). Infine, sono state valutate la ripetibilità e la riproducibilità di un profilometro a triangolazione di linea laser, attraverso uno studio Gage R&R. Ogni punto di misura è stato ispezionato da tre operatori e l'insieme dei dati è stato elaborato con un'analisi dell'incertezza di Tipo A. Successivamente, uno studio Gage R&R ha contribuito a indagare la ripetibilità, la riproducibilità e la variabilità del sistema. Questa analisi ha dimostrato un'incertezza accettabile.The purpose of this industrial PhD project financed through a scholarship from the Regione Marche was to develop research with potential impact on an industrial sector, to promote the involvement of local factories and companies in research and innovation performed jointly with the university and to produce research in line with the needs of the industrial environment, not only at regional level. Hence, through collaborating with a local turning factory (Zannini SpA) and a small high-tech company focused on introducing mechatronic innovation in the turning sector (Z4Tec srl), and also thanks to an international collaboration with the University of Antwerp, we designed and developed new instruments for in-line quality control, based on non-contact technologies, specifically electro-optical technologies. While also bringing attention to the importance of taking uncertainty into consideration, since it is pivotal in data-based decision making which are at the base of a Zero Defect Manufacturing strategy. This means that poor quality of measurements can prejudice the quality of the data. In particular, this work presents two measurement instruments that were designed and developed for the purpose of in-line quality control and the uncertainty of each of the two instruments was evaluated and analyzed in comparison with instruments already present on the market. In the last part of this work, the uncertainty of a hand-held laser-line triangulation profilometer is estimated. Hence, the research conducted in this thesis can be organized in two main objectives: the development of new vision-based dimensional measurement systems to be implemented in production line and the uncertainty analysis of these measurement instruments. For the first objective we focused on two types of dimensional measurements imposed by the manufacturing industry: macroscopic (measuring dimensions in mm) and microscopic (measuring roughness in µm). For macroscopic measurements the target was the in-production dimensional quality control of turned parts through telecentric optical profilometry. The sample to be inspected was placed between illuminator and objective in order to obtain the projection of the shadow of the sample over a white background. Dimensional measurements were then performed by means of image processing over the image obtained. We discussed the mechanical arrangements targeted to optimize images acquired as well as the main issues that eventual mechanical misalignments of components might introduce in the quality of images. For microscopic measurements we designed a backlit vision-based surface roughness measurement system with a focus on smart behaviors such as determining the optimal imaging conditions using the modulation transfer function and the use of an electrically tunable lens. A turned sample (a cylinder) is placed in front of a camera and it is backlit by a collimated source of light; such optical configuration provides the image of the edge of the sample. A set of turned steel samples with different surface roughness was used to test the sensitivity of the measurement system. For the second objective, the measurement uncertainty evaluation techniques used in this work were a Type A statistical uncertainty analysis and a Gage R&R analysis. In the case of the telecentric profilometer, the analysis was performed in comparison with other on-the-market devices with a Type A analysis and a Gage R&R analysis. The measurement uncertainty of the profilometer proved to be sufficient to obtain results within the tolerance interval required. For the backlit vision system, the comparison of the results was made with other state-of-the-art instruments, with a Type A analysis. The comparison showed that the performance of the backlit instrument depends on the values of surface roughness considered; while at larger values of roughness the offset increases, the results are compatible with the ones of the reference instrument (stylus-based) at lower values of roughness. Lastly, the repeatability and reproducibility of a laser-line triangulation profilometer were assessed, through a Gage R&R study. Each measuring point was inspected by three different operators and the data set has been, at first, processed by a Type A uncertainty analysis. Then, a Gage R&R study helped investigate repeatability, reproducibility and the system variability. This analysis showed that the presented laser-line triangulation system has an acceptable uncertainty

Advanced Stereoscopy towards On-Machine Surface Metrology and Inspection

With the goal of inventing an integral on-machine integral 3D machine vision inspection system, which monitors the parts quality and extract required patterns or structures during the manufacturing process using low-cost hardware and in a high-speed mode, this dissertation discussed the newly developed strobe-stereoscopy (SS) technique for in- motion targets examination. Stereoscopy is utilized for 3D reconstruction from recorded image pairs based on the triangulation of the display pixels, test target, and cameras. Stroboscopy is introduced to lock the moving target at different locations by frequency matching between the light source and the controlled motor. Fluorescent fluid was introduced and implemented to the SS system for high-gloss reflective surface inspection. Stereoscopy technique is limited on the diffused surface because of the sensitivity to illumination dispersion, fluorescent strobe-stereoscopy (FSS) technique overcomes the limitation to polished surface inspection and is applied to step- by-step fabrication process monitoring thus complete the metrology-in-loop for the automated production. The surface filtering-based image selection and extraction approach (ISE) is created for quick pattern extraction from the freeform base structure, which was integrated into the built hardware configuration. In this dissertation, the performance of inspection systems has been analyzed and validated with comprehensive experiment results. Potential and future work of the proposed technique was included as well

Optical MEMS

Optical microelectromechanical systems (MEMS), microoptoelectromechanical systems (MOEMS), or optical microsystems are devices or systems that interact with light through actuation or sensing at a micro- or millimeter scale. Optical MEMS have had enormous commercial success in projectors, displays, and fiberoptic communications. The best-known example is Texas Instruments’ digital micromirror devices (DMDs). The development of optical MEMS was impeded seriously by the Telecom Bubble in 2000. Fortunately, DMDs grew their market size even in that economy downturn. Meanwhile, in the last one and half decade, the optical MEMS market has been slowly but steadily recovering. During this time, the major technological change was the shift of thin-film polysilicon microstructures to single-crystal–silicon microsructures. Especially in the last few years, cloud data centers are demanding large-port optical cross connects (OXCs) and autonomous driving looks for miniature LiDAR, and virtual reality/augmented reality (VR/AR) demands tiny optical scanners. This is a new wave of opportunities for optical MEMS. Furthermore, several research institutes around the world have been developing MOEMS devices for extreme applications (very fine tailoring of light beam in terms of phase, intensity, or wavelength) and/or extreme environments (vacuum, cryogenic temperatures) for many years. Accordingly, this Special Issue seeks to showcase research papers, short communications, and review articles that focus on (1) novel design, fabrication, control, and modeling of optical MEMS devices based on all kinds of actuation/sensing mechanisms; and (2) new developments of applying optical MEMS devices of any kind in consumer electronics, optical communications, industry, biology, medicine, agriculture, physics, astronomy, space, or defense

Smart optical imaging systems with automated electronics

In this dissertation, proposed and demonstrated are several novel smart electronically automated optical designs to efficiently solve existing real-world problems in the field of shape sensing and imaging. First half of the thesis proposes shape sensing techniques that use an Electronically Controlled Variable Focus Lens (ECVFL) within a smart optical design suitable for a wide range of applications including shape sensing and projection displays. The second part of this dissertation involves the use of the Digital Micromirror Device (DMD) deployed within several smart optical designs including an embedded laser beam profiler and a new camera idea which is inspired by the Telecommunication science field. Specifically, proposed and demonstrated is the design and implementation of the novel imaging device called Coded Access Optical Sensor (CAOS) where CAOS is able of operating with different application dependent working modes. Experimentally and successfully demonstrated for the first time are its use for coherent light laser imaging as well as for incoherent imaging of a high dynamic range white light scenario. It is also shown how its design can be further extended for multispectral and hyperspectral imaging applications

Adaptive Optics Progress

For over four decades there has been continuous progress in adaptive optics technology, theory, and systems development. Recently there also has been an explosion of applications of adaptive optics throughout the fields of communications and medicine in addition to its original uses in astronomy and beam propagation. This volume is a compilation of research and tutorials from a variety of international authors with expertise in theory, engineering, and technology. Eight chapters include discussion of retinal imaging, solar astronomy, wavefront-sensorless adaptive optics systems, liquid crystal wavefront correctors, membrane deformable mirrors, digital adaptive optics, optical vortices, and coupled anisoplanatism

MEMS Technology for Biomedical Imaging Applications

Biomedical imaging is the key technique and process to create informative images of the human body or other organic structures for clinical purposes or medical science. Micro-electro-mechanical systems (MEMS) technology has demonstrated enormous potential in biomedical imaging applications due to its outstanding advantages of, for instance, miniaturization, high speed, higher resolution, and convenience of batch fabrication. There are many advancements and breakthroughs developing in the academic community, and there are a few challenges raised accordingly upon the designs, structures, fabrication, integration, and applications of MEMS for all kinds of biomedical imaging. This Special Issue aims to collate and showcase research papers, short commutations, perspectives, and insightful review articles from esteemed colleagues that demonstrate: (1) original works on the topic of MEMS components or devices based on various kinds of mechanisms for biomedical imaging; and (2) new developments and potentials of applying MEMS technology of any kind in biomedical imaging. The objective of this special session is to provide insightful information regarding the technological advancements for the researchers in the community

MEMS-Based Endomicroscopes for High Resolution in vivo Imaging

Intravital microscopy is an emerging methodology for performing real time imaging in live animals. This technology is playing a greater role in the study of cellular and molecular biology because in vitro systems cannot adequately recapitulate the microenvironment of living tissues and systems. Conventional intravital microscopes use large, bulky objectives that require wide surgical exposure to image internal organs and result in terminal experiments. If these instruments can be reduced sufficiently in size, biological phenomena can be observed in a longitudinal fashion without animal sacrifice. The epithelium is a thin layer of tissue in hollow organs, and is the origin of many types of human diseases. In vivo assessment of biomarkers expressed in the epithelium in animal models can provide valuable information of disease development and drug efficacy. The overall goal of this work is to develop miniature imaging instruments capable of visualizing the epithelium in live animals with subcellular resolution. The dissertation is divided into four projects, where each contains an imaging system developed for small animal imaging. These systems are all designed using laser beam scanning technology with tiny mirrors developed with microelectromechanical systems (MEMS) technology. By using these miniature scanners, we are able to develop endomicroscopes small enough for hollow organs in small animals. The performance of these systems has been demonstrated by imaging either excised tissue or colon of live mice. The final version of the instrument can collect horizontal/oblique plane images in the mouse colon in real time (>10 frames/sec) with sub-micron resolution (<1 um), deep tissue penetration (~200 um) and large field of view (700 x 500 um). A novel side-viewing architecture with distal MEMS scanning was developed to create clear and stable image in the mouse colon. With the use of the instrument, it is convenient to pinpoint location of interest and create a map of the colon using image mosaicking. Multispectral fluorescence images can by collected at excitation wavelength ranging from 445 nm to 780 nm. The instruments have been used to 1) validate specific binding of a cancer targeting agent in the mouse colon and 2) study the tumor development in a mouse model with endogenous fluorescence protein expression. We use these studies to show that we have developed an enabling technology which will allow biologist to perform longitudinal imaging in animal models with subcellular resolution.PHDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/136954/2/dxy_1.pd

Conference on Binary Optics: An Opportunity for Technical Exchange

The papers herein were presented at the Conference on Binary Optics held in Huntsville, AL, February 23-25, 1993. The papers were presented according to subject as follows: modeling and design, fabrication, and applications. Invited papers and tutorial viewgraphs presented on these subjects are included