Shedding light on melanins within in situ human eye melanocytes using 2-photon microscopy profiling techniques.

Choroidal melanocytes (HCMs) are melanin-producing cells in the vascular uvea of the human eye (iris, ciliary body and choroid). These cranial neural crest-derived cells migrate to populate a mesodermal microenvironment, and display cellular functions and extracellular interactions that are biologically distinct to skin melanocytes. HCMs (and melanins) are important in normal human eye physiology with roles including photoprotection, regulation of oxidative damage and immune responses. To extend knowledge of cytoplasmic melanins and melanosomes in label-free HCMs, a non-invasive 'fit-free' approach, combining 2-photon excitation fluorescence lifetimes and emission spectral imaging with phasor plot segmentation was applied. Intracellular melanin-mapped FLIM phasors showed a linear distribution indicating that HCM melanins are a ratio of two fluorophores, eumelanin and pheomelanin. A quantitative histogram of HCM melanins was generated by identifying the image pixel fraction contributed by phasor clusters mapped to varying eumelanin/pheomelanin ratio. Eumelanin-enriched dark HCM regions mapped to phasors with shorter lifetimes and longer spectral emission (580-625 nm) and pheomelanin-enriched lighter pigmented HCM regions mapped to phasors with longer lifetimes and shorter spectral emission (550-585 nm). Overall, we demonstrated that these methods can identify and quantitatively profile the heterogeneous eumelanins/pheomelanins within in situ HCMs, and visualize melanosome spatial distributions, not previously reported for these cells

A holographic system for subsea recording and analysis of plankton and other marine particles

We report here details of the design, development, initial testing and field-deployment of the HOLOMAR system for in-situ subsea holography and analysis of marine plankton and nonliving particles. HOLOMAR comprises a submersible holographic camera ("HoloCam") able to record in-line and off-axis holograms at depths down to 100 m, together with specialised reconstruction hardware ("HoloScan") linked to custom image processing and classification software. The HoloCam consists of a laser and power supply, holographic recording optics and holographic plate holders, a water-tight housing and a support frame. It utilises two basic holographic geometries, in-line and off-axis such that a wide range of species, sizes and concentrations can be recorded. After holograms have been recorded and processed they are reconstructed in full three-dimensional detail in air in a dedicated replay facility. A computer-controlled microscope, using video cameras to record the image at a given depth, is used to digitise the scene. Specially written software extracts a binarised image of an object in its true focal plane and is classified using a neural network. The HoloCam was deployed on two separate cruises in a Scottish sea loch (Loch Etive) to a depth of 100 m and over 300 holograms were recorded

Detection of leukocytes stained with acridine orange using unique spectral features acquired from an image-based spectrometer

A leukocyte differential count can be used to diagnosis a myriad blood disorders, such as infections, allergies, and efficacy of disease treatments. In recent years, attention has been focused on developing point-of-care (POC) systems to provide this test in global health settings. Acridine orange (AO) is an amphipathic, vital dye that intercalates leukocyte nucleic acids and acidic vesicles. It has been utilized by POC systems to identify the three main leukocyte subtypes: granulocytes, monocytes, and lymphocytes. Subtypes of leukocytes can be characterized using a fluorescence microscope, where the AO has a 450 nm excitation wavelength and has two peak emission wavelengths between 525 nm (green) and 650 nm (red), depending on the cellular content and concentration of AO in the cells. The full spectra of AO stained leukocytes has not been fully explored for POC applications. Optical instruments, such as a spectrometer that utilizes a diffraction grating, can give specific spectral data by separating polychromatic light into distinct wavelengths. The spectral data from this setup can be used to create object-specific emission profiles. Yellow-green and crimson microspheres were used to model the emission peaks and profiles of AO stained leukocytes. Whole blood was collected via finger stick and stained with AO to gather preliminary leukocyte emission profiles. A MATLAB algorithm was designed to analyze the spectral data within the images acquired using the image-based spectrometer. The algorithm utilized watershed segmentation and centroid location functions to isolate independent spectra from an image. The output spectra represent the average line intensity profiles for each pixel across a slice of an object. First steps were also taken in processing video frames of manually translated microspheres. The high-speed frame rate allowed objects to appear in multiple consecutive images. A function was applied to each image cycle to identify repeating centroid locations. The yellow-green (515 nm) and crimson (645 nm) microspheres exhibited a distinct separation in colorimetric emission with a peak-to-peak difference of 36 pixels, which is related to the 130 nm peak emission difference. Two AO stained leukocytes exhibited distinct spectral profiles and peaks across different wavelengths. This could be due to variations in the staining method (incubation period and concentration) effecting the emissions or variations in cellular content indicating different leukocyte subtypes. The algorithm was also effective when isolating unique centroids between video frames. We have demonstrated the ability to extract spectral information from data acquired from the image-based spectrometer of microspheres, as a control, and AO stained leukocytes. We determined that the spectral information from yellow-green and crimson microspheres could be used to represent the wavelength range of AO stained leukocytes, thus providing a calibration tool. Also, preliminary spectral information was successfully extracted from yellow-green microspheres translated under the linear slit using stationary images and video frames, thus demonstrating the feasibility of collecting data from a large number of objects

Challenges and opportunities for quantifying roots and rhizosphere interactions through imaging and image analysis

The morphology of roots and root systems influences the efficiency by which plants acquire nutrients and water, anchor themselves and provide stability to the surrounding soil. Plant genotype and the biotic and abiotic environment significantly influence root morphology, growth and ultimately crop yield. The challenge for researchers interested in phenotyping root systems is, therefore, not just to measure roots and link their phenotype to the plant genotype, but also to understand how the growth of roots is influenced by their environment. This review discusses progress in quantifying root system parameters (e.g. in terms of size, shape and dynamics) using imaging and image analysis technologies and also discusses their potential for providing a better understanding of root:soil interactions. Significant progress has been made in image acquisition techniques, however trade-offs exist between sample throughput, sample size, image resolution and information gained. All of these factors impact on downstream image analysis processes. While there have been significant advances in computation power, limitations still exist in statistical processes involved in image analysis. Utilizing and combining different imaging systems, integrating measurements and image analysis where possible, and amalgamating data will allow researchers to gain a better understanding of root:soil interactions

Structure-Property Relationships in Aluminum-Copper alloys using Transmission X-Ray Microscopy (TXM) and Micromechanical Testing

abstract: Aluminum alloys are ubiquitously used in almost all structural applications due to their high strength-to-weight ratio. Their superior mechanical performance can be attributed to complex dispersions of nanoscale intermetallic particles that precipitate out from the alloy’s solid solution and offer resistance to deformation. Although they have been extensively investigated in the last century, the traditional approaches employed in the past haven’t rendered an authoritative microstructural understanding in such materials. The effect of the precipitates’ inherent complex morphology and their three-dimensional (3D) spatial distribution on evolution and deformation behavior have often been precluded. In this study, for the first time, synchrotron-based hard X-ray nano-tomography has been implemented in Al-Cu alloys to measure growth kinetics of different nanoscale phases in 3D and reveal mechanistic insights behind some of the observed novel phase transformation reactions occurring at high temperatures. The experimental results were reconciled with coarsening models from the LSW theory to an unprecedented extent, thereby establishing a new paradigm for thermodynamic analysis of precipitate assemblies. By using a unique correlative approach, a non-destructive means of estimating precipitation-strengthening in such alloys has been introduced. Limitations of using existing mechanical strengthening models in such alloys have been discussed and a means to quantify individual contributions from different strengthening mechanisms has been established. The current rapid pace of technological progress necessitates the demand for more resilient and high-performance alloys. To achieve this, a thorough understanding of the relationships between material properties and its structure is indispensable. To establish this correlation and achieve desired properties from structural alloys, microstructural response to mechanical stimuli needs to be understood in three-dimensions (3D). To that effect, in situ tests were conducted at the synchrotron (Advanced Photon Source) using Transmission X-Ray Microscopy as well as in a scanning electron microscope (SEM) to study real-time damage evolution in such alloys. Findings of precipitate size-dependent transition in deformation behavior from these tests have inspired a novel resilient aluminum alloy design.Dissertation/ThesisDoctoral Dissertation Materials Science and Engineering 201