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

Mobility of Nano-Particles in Rock Based Micro-Models

A confocal micro-particle image velocimetry (C-μPIV) technique along with associated post-processing algorithms is detailed for obtaining three dimensional distributions of nano-particle velocity and concentrations at select locations of the 2.5D (pseudo 3D) Poly(methyl methacrylate) (PMMA) and ceramic micro-model. The designed and fabricated 2.5D micro-model incorporates microchannel networks with 3D wall structures with one at observation wall which resembles fourteen morphological and flow parameters to those of fully 3D actual reservoir rock (Boise Sandstone) at resolutions of 5 and 10 μm in depth and 5 and 25 μm on plane. In addition, an in-situ, non-destructive method for measuring the geometry of low and high resolution PMMA and ceramic micro-models, including its depth, is described and demonstrated. The flow experiments use 860 nm and 300 nm fluorescence-labeled polystyrene particles, and the data is acquired using confocal laser scanning microscopy. Regular fluorescence microscopy is used for the in-situ geometry measurement along with the use of Rhodamine dye and a depth-to-fluorescence-intensity calibration, which is linear. Monochromatic excitation at a wavelength of 544 nm (green) produced by a HeNe continuous wave laser was used to excite the fluorescence-labeled nanoparticles emitting at 612 nm (red). Confocal images were captured by a highly sensitive fluorescence detector photomultiplier tube. Results of detailed three dimensional velocity, particle concentration distributions, and particle deposition rates from experiments conducted at flow rates of 0.5 nL/min, 1 nL/min, 10 nL/min and 100 nL/min are presented and discussed. The three dimensional micro-model geometry reconstructed from fluorescence data is used as the computational domain to conduct numerical simulations of the flow in the as-tested micro-model for comparisons to experimental results using dimensionless Navier-Stokes model. The flow simulation results are also used to qualitatively compare with velocity distributions of the flowing particles at selected locations. The comparison is qualitative because the particle sizes used in these experiments may not accurately follow the flow itself given the geometry of the micro-models. These larger particles were used for proof of concept purposes, and the techniques and algorithms used permit future use of particles as small as 50 nm

3D Architectural Analysis of Neurons, Astrocytes, Vasculature & Nuclei in the Motor and Somatosensory Murine Cortical Columns

Characterization of the complex cortical structure of the brain at a cellular level is a fundamental goal of neuroscience which can provide a better understanding of both normal function as well as disease state progression. Many challenges exist however when carrying out this form of analysis. Immunofluorescent staining is a key technique for revealing 3-dimensional structure, but subsequent fluorescence microscopy is limited by the quantity of simultaneous targets that can be labeled and intrinsic lateral and isotropic axial point-spread function (PSF) blurring during the imaging process in a spectral and depth-dependent manner. Even after successful staining, imaging and optical deconvolution, the sheer density of filamentous processes in the neuropil significantly complicates analysis due to the difficulty of separating individual cells in a highly interconnected network of tightly woven cellular arbors. In order to solve these problems, a variety of methodologies were developed and validated for improved analysis of cortical anatomy. An enhanced immunofluorescent staining and imaging protocol was utilized to precisely locate specific functional regions within brain slices at high magnification and collect four-channel, complete cortical columns. A powerful deconvolution routine was established which collected depth variant PSFs using an optical phantom for image restoration. Fractional volume analysis (FVA) was used to provide preliminary data of the proportions of each stained component in order to statistically characterize the variability within and between the functional regions in a depth-dependent and depth-independent manner. Finally, using machine learning techniques, a supervised learning model was developed that could automatically classify neuronal and astrocytic nuclei within the large cortical column datasets based on perinuclear fluorescence. These annotated nuclei were then used as seed points within their corresponding fluorescent channel for cell individualization in a highly interconnected network. For astrocytes, this technique provides the first method for characterization of complex morphology in an automated fashion over large areas without laborious dye filling or manual tracing

Cardiac multiscale bioimaging: from nano- through micro- to mesoscales.

Cardiac multiscale bioimaging is an emerging field that aims to provide a comprehensive understanding of the heart and its functions at various levels, from the molecular to the entire organ. It combines both physiologically and clinically relevant dimensions: from nano- and micrometer resolution imaging based on vibrational spectroscopy and high-resolution microscopy to assess molecular processes in cardiac cells and myocardial tissue, to mesoscale structural investigations to improve the understanding of cardiac (patho)physiology. Tailored super-resolution deep microscopy with advanced proteomic methods and hands-on experience are thus strategically combined to improve the quality of cardiovascular research and support future medical decision-making by gaining additional biomolecular information for translational and diagnostic applications

Biological applications of multimodal imaging involving Raman and 4Pi Raman microscopy

Raman microscopy is becoming an increasingly important label-free imaging technique. It proved to be a viable tool for life science applications allowing to analyze bacteria, cells, and tissues at the molecular level. Combining Raman microscopy with complementary imaging modalities and techniques is explored here to: (1) analyze mild traumatic brain injury (mTBI) in a combination with magnetic resonance imaging (MRI) for detecting mild, and invisible to medical imaging techniques, brain tissue damage; (2) reveal complementarity of Raman and fluorescence microscopy approaches for investigating and tracking bovine lactoferrin inside calf rectal epithelial cells in the presence of enterohemorrhagic Escherichia coli (EHEC); (3) apply Raman microscopy along-side the molecular analysis approaches (such as scanning transmission electron microscopy-energy dispersive X-ray (STEM-EDX), low energy X-ray fluorescence (LEXRF), nanoscale secondary ion mass spectrometry (Nano-SIMS)) to uncover the origin of the long-range conductance in cable bacteria; (4) develop multifunctional surface enhanced Raman scattering (SERS) platform based on calcium carbonate particles for enhancing a weak Raman scattering signal of biomolecules as well as to apply Raman microscopy for particle detection in vivo in Caenorhabditis elegans (C. elegans) worms; and (5) combine Raman microscopy and atomic force microscopy (AFM) to track Chlamydia psittaci in cells. Analysis of described above samples and phenomena is based on Raman molecular fingerprint images, where, similarly to fluorescence light microscopy, the resolution is limited by diffraction of light. Therefore, efforts are also put to enhance the resolution of Raman microscopy-based imaging by adding a 4Pi configuration to a confocal Raman microscope. As a result, a possibility to enhance the axial (also called longitudinal) resolution is investigated by constructing a 4Pi confocal Raman microscope, which is also applied to study bacteria inside cells. Results presented in this work emphasize the added value of multimodal microscopy approaches, particularly involving Raman microscopy, in a broad range of applications in bioengineering, biomedicine, and biology

Micro/Nano Manufacturing

Micro- and nano-scale manufacturing has been the subject of ever more research and industrial focus over the past 10 years. Traditional lithography-based technology forms the basis of micro-electro-mechanical systems (MEMS) manufacturing, but also precision manufacturing technologies have been developed to cover micro-scale dimensions and accuracies. Furthermore, these fundamentally different technology platforms are currently combined in order to exploit the strengths of both platforms. One example is the use of lithography-based technologies to establish nanostructures that are subsequently transferred to 3D geometries via injection molding. Manufacturing processes at the micro-scale are the key-enabling technologies to bridge the gap between the nano- and the macro-worlds to increase the accuracy of micro/nano-precision production technologies, and to integrate different dimensional scales in mass-manufacturing processes. Accordingly, this Special Issue seeks to showcase research papers, short communications, and review articles that focus on novel methodological developments in micro- and nano-scale manufacturing, i.e., on novel process chains including process optimization, quality assurance approaches and metrology

Measurement of micro burr and slot widths through image processing: Comparison of manual and automated measurements in micro‐milling

In this study, the burr and slot widths formed after the micro‐milling process of Inconel 718 alloy were investigated using a rapid and accurate image processing method. The measurements were obtained using a user‐defined subroutine for image processing. To determine the accuracy of the developed imaging process technique, the automated measurement results were compared against results measured using a manual measurement method. For the cutting experiments, Inconel 718 alloy was machined using several cutting tools with different geometry, such as the helix angle, axial rake angle, and number of cutting edges. The images of the burr and slots were captured using a scanning electron microscope (SEM). The captured images were processed with computer vision software, which was written in C++ programming language and open‐sourced computer library (Open CV). According to the results, it was determined that there is a good correlation between automated and manual measurements of slot and burr widths. The accuracy of the proposed method is above 91%, 98%, and 99% for up milling, down milling, and slot measurements, respectively. The conducted study offers a user‐friendly, fast, and accurate solution using computer vision (CV) technology by requiring only one SEM image as input to characterize slot and burr formation

The BrightEyes-TTM: an open-source time-tagging module for fluorescence lifetime imaging microscopy applications

The aim of this Ph.D. work is to reason and show how an open-source multi-channel and standalone time-tagging device was developed, validated and used in combination with a new generation of single-photon array detectors to pursue super-resolved time-resolved fluorescence lifetime imaging measurements. Within the compound of time-resolved fluorescence laser scanning microscopy (LSM) techniques, fluorescence lifetime imaging microscopy (FLIM) plays a relevant role in the life-sciences field, thanks to its ability of detecting functional changes within the cellular micro-environment. The recent advancements in photon detection technologies, such as the introduction of asynchronous read-out single-photon avalanche diode (SPAD) array detectors, allow to image a fluorescent sample with spatial resolution below the diffraction limit, at the same time, yield the possibility of accessing the single-photon information content allowing for time-resolved FLIM measurements. Thus, super-resolved FLIM experiments can be accomplished using SPAD array detectors in combination with pulsed laser sources and special data acquisition systems (DAQs), capable of handling a multiplicity of inputs and dealing with the single-photons readouts generated by SPAD array detectors. Nowadays, the commercial market lacks a true standalone, multi-channel, single-board, time-tagging and affordable DAQ device specifically designed for super-resolved FLIM experiments. Moreover, in the scientific community, no-efforts have been placed yet in building a device that can compensate such absence. That is why, within this Ph.D. project, an open-source and low-cost device, the so-called BrightEyes-TTM (time tagging module), was developed and validated both for fluorescence lifetime and time-resolved measurements in general. The BrightEyes-TTM belongs to a niche of DAQ devices called time-to-digital converters (TDCs). The field-gate programmable array (FPGA) technology was chosen for implementing the BrightEyes-TTM thanks to its reprogrammability and low cost features. The literature reports several different FPGA-based TDC architectures. Particularly, the differential delay-line TDC architecture turned out to be the most suitable for this Ph.D. project as it offers an optimal trade-off between temporal precision, temporal range, temporal resolution, dead-time, linearity, and FPGA resources, which are all crucial characteristics for a TDC device. The goal of the project of pursuing a cost-effective and further-upgradable open-source time-tagging device was achieved as the BrigthEyes-TTM was developed and assembled using low-cost commercially available electronic development kits, thus allowing for the architecture to be easily reproduced. BrightEyes-TTM was deployed on a FPGA development board which was equipped with a USB 3.0 chip for communicating with a host-processing unit and a multi-input/output custom-built interface card for interconnecting the TTM with the outside world. Licence-free softwares were used for acquiring, reconstructing and analyzing the BrightEyes-TTM time-resolved data. In order to characterize the BrightEyes-TTM performances and, at the same time, validate the developed multi-channel TDC architecture, the TTM was firstly tested on a bench and then integrated into a fluorescent LSM system. Yielding a 30 ps single-shot precision and linearity performances that allows to be employed for actual FLIM measurements, the BrightEyes-TTM, which also proved to acquire data from many channels in parallel, was ultimately used with a SPAD array detector to perform fluorescence imaging and spectroscopy on biological systems. As output of the Ph.D. work, the BrightEyes-TTM was released on GitHub as a fully open-source project with two aims. The principal aim is to give to any microscopy and life science laboratory the possibility to implement and further develop single-photon-based time-resolved microscopy techniques. The second aim is to trigger the interest of the microscopy community, and establish the BrigthEyes-TTM as a new standard for single-photon FLSM and FLIM experiments

Parameter optimisation of coaxial Melt Electrowriting

Melt electrowriting (MEW) is an additive manufacturing technique that combines the advantages of both, solution electrospinning and fused filament fabrication by allowing the deposition of ultra-fine fibres in a controlled way without the necessity for dispersion of the material being prepared in toxic solvents. This approach is promising for the research field of tissue engineering that requires not only different types of materials but also different manufacturing techniques for scaffolds that are supposed to provide robust but flexible support, which facilitates cell growth without causing damage to the surrounding tissue. Recent advances in MEW technology (Mar 2018) that allow the processing through coaxial extrusion geometries are examined in the context of composite materials and their possible application as for example drug delivery systems. In this study, poly(ε-caprolactone) (PCL) containing different percentages of different fluorophores was prepared using a precipitation process. The resulting composites were printed uniaxially as a means to investigate the effects of the added salts and the emerging additional charges on the jet. It was shown that the acceleration of the jet was increased with an increasing fluorophore loading, which could be seen in the resulting critical translation speeds. The uniaxial investigation was also important to validate the thermal stabilities for the dyes, which were used in the coaxial process. For the latter, DiOC18 and Rhodamine B were chosen because of their stability as well as their compatibility with PCL and each other. The feasibility of core-shell structures was examined by varying process and material parameters. Amongst other things, the green and red dyed polymers were processed through the inner and the outer syringe in order to get a clear distinction between the core and the shell. While these structures were observed occasionally using confocal laser scanning microscopy, they were found to be unstable due to diffusion and mixing at the Taylor cone during the MEW process. The in-process-mixing of the materials can be of interest for the application-specific compounding of heat- and strain-sensitive polymers while having control over their composition depending on the chosen flow rates. Janus fibres were also shown possible through adjustment of the axial location of the core syringe used, although mixing of the polymer prior to solidification affected this effect. Nevertheless, MEW is a technique with a vast amount of possible parameter combinations. Hence, the production of core-shell and Janus fibres is shown possible and offers a variety of applications such as in microelectromechanical and drug delivery systems as well as robotics. When applying air through the core syringe, the insertion of air into the melt leading to hollow channels in the fibres was shown feasible and reproducible. This technique offers the printing of fibres with outer diameters as big as 90 μm while containing a 25 μm cavity, as well as fibres with an outer and inner diameter of 10 and 2 μm, respectively. While the production of hollow fibres usually demands for a fabrication of core-shell structures first with a later dissolving of the core material, the coaxial MEW of hollow fibres is a one-step approach that works without toxic solvents. Furthermore, it enables the fabrication of structures that are known to be lighter while having improved mechanical properties compared to dense materials, hence offering a wide spectrum of applications such as in regenerative medicine and microfluidics