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Microscopy Methods for Life Detection on Ocean Worlds

On Earth, light microscopy is commonly used in microbiology to identify organisms and observe their interactions with the environment; this makes it an attractive technique for in situ life detection methods on ocean worlds. As a standalone technique, brightfield microscopy, while able to provide important contextual information, has limited usefulness as a life detection technique because it is often challenging to differentiate between abiotic and biotic particles based solely on their size and shape, which may introduce risks of false positive or false negative interpretations. However, these risks can be reduced by combining brightfield microscopy with fluorescence microscopy to provide a method that correlate sample chemistry with sample morphology. In this work, we have used the Luminescence Imager for Exploration (LIfE), a brightfield and epifluorescence microscope with an integrated sample processing system (matured under the Concepts for Ocean worlds Life Detection Technology and Instrument Concepts of Europa Exploration programs) to develop methods that increase the fidelity of in situ microscopy life detection measurements through two main approaches. First, native fluorescence is excited in molecules that contain aromatic moieties such as proteins (using deep UV excitation), and energy carrying molecules and endogenous chromophores (using visible-light excitation), to correlate the location of these species with cell-like structural features (brightfield imaging). Second, fluorescent stains are used to selectively image cells and cell fragments by targeting proteins, lipids, and nucleic acids. We discuss the results of tests, obtained using ocean world analog samples, that have examined trades associated with implementing these methods autonomously in planetary missions, including the intrinsic properties of candidate fluorescence dyes and long-term storage and radiation stability.

Pavel E. Z. Klier↗

Microscopy Methods for Life Detection on Ocean Worlds

On Earth, light microscopy is commonly used in microbiology to identify organisms and observe their interactions with the environment; this makes it an attractive technique for in situ life detection methods on ocean worlds. As a standalone technique, brightfield microscopy, while able to provide important contextual information, has limited usefulness as a life detection technique because it is often challenging to differentiate between abiotic and biotic particles based solely on their size and shape, which may introduce risks of false positive or false negative interpretations. However, these risks can be reduced by combining brightfield microscopy with fluorescence microscopy to provide a method that correlate sample chemistry with sample morphology. In this work, we have used the Luminescence Imager for Exploration (LIfE), a brightfield and epifluorescence microscope with an integrated sample processing system (matured under the Concepts for Ocean worlds Life Detection Technology and Instrument Concepts of Europa Exploration programs) to develop methods that increase the fidelity of in situ microscopy life detection measurements through two main approaches. First, native fluorescence is excited in molecules that contain aromatic moieties such as proteins (using deep UV excitation), and energy carrying molecules and endogenous chromophores (using visible-light excitation), to correlate the location of these species with cell-like structural features (brightfield imaging). Second, fluorescent stains are used to selectively image cells and cell fragments by targeting proteins, lipids, and nucleic acids. We discuss the results of tests, obtained using ocean world analog samples, that have examined trades associated with implementing these methods autonomously in planetary missions, including the intrinsic properties of candidate fluorescence dyes and long-term storage and radiation stability.

Pavel E Z Klier↗

Machine learning models for segmentation and classification of cyanobacterial cells

Abstract Timelapse microscopy has recently been employed to study the metabolism and physiology of cyanobacteria at the single-cell level. However, the identification of individual cells in brightfield images remains a significant challenge. Traditional intensity-based segmentation algorithms perform poorly when identifying individual cells in dense colonies due to a lack of contrast between neighboring cells. Here, we describe a newly developed software package called Cypose which uses machine learning (ML) models to solve two specific tasks: segmentation of individual cyanobacterial cells, and classification of cellular phenotypes. The segmentation models are based on the Cellpose framework, while classification is performed using a convolutional neural network named Cyclass. To our knowledge, these are the first developed ML-based models for cyanobacteria segmentation and classification. When compared to other methods, our segmentation models showed improved performance and were able to segment cells with varied morphological phenotypes, as well as differentiate between live and lysed cells. We also found that our models were robust to imaging artifacts, such as dust and cell debris. Additionally, the classification model was able to identify different cellular phenotypes using only images as input. Together, these models improve cell segmentation accuracy and enable high-throughput analysis of dense cyanobacterial colonies and filamentous cyanobacteria.

Huffine, Clair A.↗

Cellulose Nanocrystals’ Role in Critical Mineral Beneficiation: Dual Aggregate-Dispersant Behavior Supports Environmentally Benign Nickel Processing

Environmentally unsustainable and toxic chemical flocculants and dispersants from nickel (Ni) processing contribute to industrial effluents that greatly impact biodiversity and aquatic life. Despite the industry’s efforts to reduce its ecological footprint—primarily due to the lack of commercially available biodegradable, environmentally benign, and non-toxic reagents—these reagents continue to harm natural ecosystems. Chemicals used during the processing operations often target a specific unit operation that negatively affects downstream operations. In this study, we discover the concentration-dependent behavior of cellulose nanocrystals (CNCs) as a dispersant and flocculant, avoiding the use of various harmful chemicals in the nickel processing stages. Electrophoretic, optical brightfield microscopy, and quartz crystal microbalance-dissipation studies detected charge neutralization behavior by renewable and biodegradable CNCs, which can greatly benefit Ni processing operations. Microflotation experiments demonstrated that CNCs enhanced Ni recovery from 62 to 77 wt.% and concentrate grade from 15 to 20 wt.%. Settling and turbidity studies demonstrated the dual flocculant-dispersant behavior of CNCs resulting from the alignment of CNC fibers along the octahedral brucite basal and amphoteric edge plane of serpentine. CNCs can be used as dispersants during froth flotation to improve Ni beneficiation and as post-processing flocculants for tailings management and dewatering, which is one of the major environmental and social concerns facing the mining and mineral processing industry. This study paves the way for CNCs to be used as flocculants and dispersants in a range of industries from healthcare to pharmaceuticals to semiconductor devices.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

A bi-channel aided stitching of atomic force microscopy images

Microscopy is an essential tool in scientific research, enabling the visualization of structures at micro- and nanoscale resolutions. However, the field of microscopy often encounters limitations in field-of-view (FOV), restricting the amount of sample that can be imaged in a single capture. To overcome this limitation, image stitching techniques have been developed to seamlessly merge multiple overlapping images into a single, high-resolution composite. The images collected from microscope need to be optimally stitched before accurate physical information can be extracted from post analysis. However, the existing stitching tools either struggle to stitch images together when the microscopy images are feature sparse or cannot address all the transformations of images when performing image stitching. To address these issues, we propose a bi-channel aided feature-based image stitching method and demonstrate its use on Atomic Force Microscopy (AFM) generated Pantoea sp. YR343 biofilm and PTO thin film sample images as experimental data. The topographical channel image of AFM data captures the morphological details of the sample, and a stitched topographical image is desired for researchers. We utilize the amplitude and phase channels of AFM data to maximize the matching features and to estimate the position of the original topographical images and show that the proposed bi-channel aided stitching method outperforms the traditional direct stitching approach in AFM topographical image stitching task. Here, we demonstrated the application on AFM, but similar approaches could be employed of optical microscopy with brightfield and fluorescence channels. We believe this proposed workflow can serve as a valuable augmentation strategy for microscopy image stitching tasks and will benefit the experimentalist to avoid erroneous analysis and discovery due to incorrect stitching.

Atomic force microscopy↗

Ray-tracing image simulations of transparent objects with complex shape and inhomogeneous refractive index

Optical images of transparent three-dimensional objects can be different from a replica of the object’s cross section in the image plane, due to refraction at the surface or in the body of the object. Simulations of the object’s image are thus needed for the visualization and validation of physical models. We report ray tracing image simulations that achieved high physical fidelity, reproducing optical behaviors and image features not rendered in previous studies. We replicated brightfield microscopy images of drops with complex shapes, and images of pressure and shock waves traveling inside them. For high physical fidelity, the simulations must replicate the spatial and angular distribution of illumination rays, and both the experiment and the simulation must be designed for accurate optical modeling. The simulations are highly sensitive to the properties of the drops and can be used to diagnose and refine fluid dynamics models. The simulated images can also be optimized to extract multiple 3D properties from experimental images. Compared to specialized single-shot 3D imaging methods, this approach has the advantage that it preserves the experimental simplicity, the high resolution, and the visual interpretability characteristic to basic optical imaging. The techniques introduced here are directly applicable to optical microscopy, so they can be used in other fields, such as microfluidics and biology, to expand the type and the accuracy of three-dimensional information that can be extracted from basic optical images.

Cavitation↗

Multi-contrast machine learning improves schistosomiasis diagnostic performance

Schistosomiasis currently affects over 250 million people and remains a public health burden despite ongoing global control efforts. Conventional microscopy is a practical tool for diagnosis and screening ofSchistosoma haematobium, but identification of eggs requires a skilled microscopist. Here we present a machine learning (ML)-based strategy for automated detection ofS. haematobiumthat combines two imaging contrasts, brightfield (BF) and darkfield (DF), to improve diagnostic performance. We collected BF and DF images of urine samples, many of them containingS. haematobiumeggs, during two different field studies in Côte d’Ivoire using a mobile phone-based microscope, the SchistoScope. We then trained separate egg-detection ML models and compared the patient-level performance of BF and DF models alone to combinations of BF and DF models, using annotations from trained microscopists as the gold standard. We found that models trained on DF images, and almost all BF and DF combinations, performed significantly better than models trained on BF images only. When models were trained on images from the first field study (n = 349 patients, 748 images of each contrast), patient-level classification performance on patient images from the second study (n = 375 patients, 752 images of each contrast) met the WHO Diagnostic Target Product Profile (TPP) sensitivity and specificity for the monitoring and evaluation use case (sensitivity for all models and combinations was >75% when evaluated at a confidence score threshold that resulted in specificity >96.5%). When we used images from both field studies for the training set, performance of the models was improved. Overall, this work shows that the use of DF and BF increases the performance of ML models on images from devices with low-cost optics, while retaining the portability, power, and time-to-results of the WHO’s diagnostic TPP. DF requires no additional sample preparation and does not increase the complexity of the imaging system. It thus offers a practical means to improve performance of automated diagnostics forS. haematobiumas well as other microscopy-based diagnostics.

Infectious Diseases↗

In Vivo Entombment of Bacteria and Fungi during Calcium Oxalate, Brushite, and Struvite Urolithiasis

Human kidney stones form via repeated events of mineral precipitation, partial dissolution, and reprecipitation, which are directly analogous to similar processes in other natural and manmade environments, where resident microbiomes strongly influence biomineralization. High-resolution microscopy and high-fidelity metagenomic (microscopy-to-omics) analyses, applicable to all forms of biomineralization, have been applied to assemble definitive evidence of in vivo microbiome entombment during urolithiasis. Stone fragments were collected from a randomly chosen cohort of 20 patients using standard percutaneous nephrolithotomy (PCNL). Fourier transform infrared (FTIR) spectroscopy indicated that 18 of these patients were calcium oxalate (CaOx) stone formers, whereas one patient formed each formed brushite and struvite stones. This apportionment is consistent with global stone mineralogy distributions. Stone fragments from seven of these 20 patients (five CaOx, one brushite, and one struvite) were thin sectioned and analyzed using brightfield (BF), polarization (POL), confocal, super-resolution autofluorescence (SRAF), and Raman techniques. DNA from remaining fragments, grouped according to each of the 20 patients, were analyzed with amplicon sequencing of 16S rRNA gene sequences (V1–V3, V3–V5) and internal transcribed spacer (ITS1, ITS2) regions. Bulk-entombed DNA was sequenced from stone fragments in 11 of the 18 patients who formed CaOx stones, and the patients who formed brushite and struvite stones. These analyses confirmed the presence of an entombed low-diversity community of bacteria and fungi, including Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, and Aspergillus niger. Bacterial cells approximately 1 μm in diameter were also optically observed to be entombed and well preserved in amorphous hydroxyapatite spherules and fans of needle-like crystals of brushite and struvite. In conclusion, these results indicate a microbiome is entombed during in vivo CaOx stone formation. Similar processes are implied for brushite and struvite stones. This evidence lays the groundwork for future in vitro and in vivo experimentation to determine how the microbiome may actively and/or passively influence kidney stone biomineralization.

59 BASIC BIOLOGICAL SCIENCES↗

Identification of dislocation etch pits in n-type GaAs by NIR transmission microscopy

An optical method is described for identifying dislocation etch pits in n-type GaAS, using near-IR brightfield transmission microscopy. Dislocations are revealed in a nondestructive manner through contrasts that are likely due to impurity decoration of the dislocation lines. By subjecting the same wafers to a photoetching technique, it was established that each etch pit on the surface is associated with a dislocation and that the termination of each (decorated) dislocation is an etch pit.

Cao, X. Z.↗

Light Microscopy Module Fan Disturbance Characterized Through Microgravity Emissions Laboratory Testing

A Light Microscopy Module (LMM) is being engineered, designed, and developed at the NASA Glenn Research Center. The LMM is planned as a remotely controllable on-orbit microscope subrack facility, allowing flexible scheduling and control of physical science and biological science experiments within Glenn s Fluids Integrated Rack on the International Space Station. The LMM concept is a modified commercial research imaging light microscope with powerful laser-diagnostic hardware and interfaces, creating a one-of-a-kind, state-of-the-art microscopic research facility. The microscope will house several different objectives, corresponding to magnifications of 10, 40, 50, 63, and 100. Features of the LMM include high-resolution color video microscopy, brightfield, darkfield, phase contrast, differential interference contrast, spectrophotometry, and confocal microscopy combined in a single configuration. Also, laser tweezers are integrated with the diagnostics as a sample manipulation technique. As part of the development phase of the LMM, it was necessary to quantify the microgravity disturbances generated by the control box fan. Isolating the fan was deemed necessary to reduce the fan speed harmonic amplitudes and to eliminate any broadband disturbances across the 60- to 70-Hz and 160- to 170-Hz frequency ranges. The accelerations generated by a control box fan component of the LMM were measured in the Microgravity Emissions Laboratory (MEL). The MEL is a low-frequency measurement system developed to simulate and verify the on-orbit International Space Station (ISS) microgravity environment. The accelerations generated by various operating components of the ISS, if too large, could hinder the science performed onboard by disturbing the microgravity environment. The MEL facility gives customers a test-verified way of measuring their compliance with ISS limitations on vibratory disturbance levels. The facility is unique in that inertial forces in 6 degrees of freedom can be characterized simultaneously for an operating test article. Vibratory disturbance levels are measured for engineering or flight-level hardware following development from component to subassembly through the rack-level configuration. The MEL can measure accelerations as small as 10-7g, the accuracy needed to confirm compliance with ISS requirements.

McNelis, Anne M.↗

Advancing Launch Readiness of the Europa Light Microscope

The search for life in our solar system relies on our ability to measure key biomarkers in situ and identify them as likely of biotic origin. One class of biomarkers that would be indicative of life is cell-like features such as the colocalization of key biochemicals, spatial organization of native UV fluorescent compounds, and polarization across membrane compartments. The Europa Lander Science Definition Team has emphasized the need for a light microscope to search for these features on Europa, and the Europa Light Microscope (ELM) has been developed to address this need. This work presents the development towards high launch readiness of ELM by demonstrating its optical resolution under brightfield illumination, deep UV fluorescence imaging of unstained living samples, and multicolor imaging of samples stained with exogenous fluorophores targeting proteins, lipids, and nucleic acids. This work also seeks to extend the capabilities of ELM by incorporating fluorescence-based assays for membrane potential as a key biomarker for living cellular material.

Pavel Erich Zivanovic Klier↗

The Dark Annulus of a Drop in a Hele-Shaw Cell Is Caused by the Refraction of Light through Its Meniscus

Knowing the meniscus shape of confined drops is important for understanding how they make first contact and then coalesce. When imaged from the top view by brightfield microscopy, a liquid drop (e.g., corn syrup) confined in a Hele-Shaw cell, surrounded by immiscible liquid (e.g., mineral oil), had a dark annulus, and the width of the annulus decreased with increasing concentration of corn syrup. Since the difference in the annulus width was presumed to be related to the meniscus shape of the drops, three-dimensional images of the drops with different concentrations were obtained using confocal fluorescence microscopy, and their cross-sectional meniscus profile was determined by image processing. The meniscus of the drops remained circular despite varying concentration. Since the refractive index of corn syrup increased with concentration, while the surface tension coefficient between corn syrup and mineral oil remained unchanged, the observed change in the annulus width was then attributed to the refraction of light passing through the drop’s meniscus. As such, a ray optics model was developed, which predicted that the annulus width of the drop would decrease as the refractive index of the drop approached that of the surrounding liquid. Therefore, the dark annulus of the drops in the Hele-Shaw cell was caused by the refraction of light passing through the circular meniscus of the drop.

Sangjin Ryu↗

Minnesota Solar Pathways: Illuminating Pathways to 10% Solar (Final Technical Report)

The Minnesota Solar Pathways project, funded in part by the Department of Energy’s Solar Energy Technologies Office, was a three-year project designed to explore least-risk, best-value strategies for meeting the State of Minnesota’s solar goals. As part of this aim, the Pathways Team modeled renewable generation costs, examined ways to streamline interconnection, and evaluated technologies that can increase solar hosting capacity on the distribution grid. Midway through the project period, the Pathways Team expanded the scope of study using the same modeling framework from the Minnesota Solar Potential Analysis to estimate and compare generation costs for high renewables penetration scenarios across the Midcontinent Independent System Operator (MISO) region. The analysis concludes that it can be cheaper to overbuild solar + wind and curtail surplus than to store all generation. In addition, the analysis of individual MISO subregions compared with the whole region highlights the opportunities of enabling market potential compared with impact of ongoing transmission constraints. Beyond the analysis of future scenarios, the partnerships initiated and supported through this project underscored the ongoing need for work with stakeholders to address key issues, particularly around siting, and leverage symbiotic strategies to meet state goals.

14 SOLAR ENERGY↗