3D Microscopy Research Articles

Influence of cement type, excess removal, and polishing on the cement joint.

To compare marginal gap width and depth with different cementation systems, excess removal, and after polishing. In total, 80 composite crowns were milled, divided into ten groups, and cemented on identical artificial teeth. Eight crowns per group were fixed with (i) zinc phosphate cement (ZnOPh), (ii) glass-ionomer cement (GIC), (iii) resin-reinforced glass-ionomer cement (GIC mod), (iv) dual-curing adhesive composite (Comp dual), or (v) dual-curing self-adhesive composite (Comp SE dual). Excess removal was performed with a scaler after brief light-cure (tack-cure), final light-cure, during rubber or gel phase or by wiping with foam pellet. Curing was completed in chemical, dark cure, or light-curing modus. The specimens were polished and stored in water (37°C). The margins were digitized using a 3D laser-scanning microscope (VK-X100 series, Keyence). The width and the depth of the marginal gap were measured at 10 points between the crown margin and the preparation margin. The width after excess removal varied between 65.1 ± 15.7 µm (Comp dual, wipe, with polishing) and 208.6 ± 266.7 µm (Comp SE dual, dark cure, without polishing). The depth varied between 29.8 ± 22.2 µm (Comp dual, wipe, with polishing) and 89.5 ± 45.2 µm (Comp SE dual, dark cure, without polishing). The impact on gap width and depth was detected for fixation material, excess removal, and polishing. The gap depth and width depend on the luting material and the mode of access removal. Polishing can improve the gap quality, especially for GIC and resin-based systems.

Data-Driven and Cell-Specific Determination of Nuclei-Associated Actin Structure.

Quantitative and volumetric assessment of filamentous actin fibers (F-actin) remains challenging due to their interconnected nature, leading researchers to utilize threshold based or qualitative measurement methods with poor reproducibility. Here we introduce a novel machine learning based methodology for accurate quantification and reconstruction of nuclei-associated F-actin. Utilizing a Convolutional Neural Network (CNN), we segment actin filaments and nuclei from 3D confocal microscopy images and then reconstruct each fiber by connecting intersecting contours on cross-sectional slices. This allowed measurement of the total number of actin filaments and individual actin filament length and volume in a reproducible fashion. Focusing on the role of F-actin in supporting nucleocytoskeletal connectivity, we quantified apical F-actin, basal F-actin, and nuclear architecture in mesenchymal stem cells (MSCs) following the disruption of the Linker of Nucleoskeleton and Cytoskeleton (LINC) Complexes. Disabling LINC in mesenchymal stem cells (MSCs) generated F-actin disorganization at the nuclear envelope characterized by shorter length and volume of actin fibers contributing a less elongated nuclear shape. Our findings not only present a new tool for mechanobiology but introduce a novel pipeline for developing realistic computational models based on quantitative measures of F-actin.

Quantifying nanoscopic alterations associated with mitochondrial dysfunction using three-dimensional single-molecule localization microscopy.

Mitochondrial morphology provides unique insights into their integrity and function. Among fluorescence microscopy techniques, 3D super-resolution microscopy uniquely enables the analysis of mitochondrial morphological features individually. However, there is a lack of tools to extract morphological parameters from super-resolution images of mitochondria. We report a quantitative method to extract mitochondrial morphological metrics, including volume, aspect ratio, and local protein density, from 3D single-molecule localization microscopy images, with single-mitochondrion sensitivity. We validated our approach using simulated ground-truth SMLM images of mitochondria. We further tested our morphological analysis on mitochondria that have been altered functionally and morphologically in controlled manners. This work sets the stage to quantitatively analyze mitochondrial morphological alterations associated with disease progression on an individual basis.

Quantitative pulsatility measurements using 3D dynamic ultrasound localization microscopy

A rise in blood flow velocity variations (i.e. pulsatility) in the brain, caused by the stiffening of upstream arteries, is associated with cognitive impairment and neurodegenerative diseases. The study of this phenomenon requires brain-wide pulsatility measurements, with large penetration depth and high spatiotemporal resolution. The development of dynamic ultrasound localization microscopy (DULM), based on ULM, has enabled pulsatility measurements in the rodent brain in 2D. However, 2D imaging accesses only one slice of the brain and measures only 2D-projected and hence biased velocities . Herein, we present 3D DULM: using a single ultrasound scanner at high frame rate (1000–2000 Hz), this method can produce dynamic maps of microbubbles flowing in the bloodstream and extract quantitative pulsatility measurements in the cat brain with craniotomy and in the mouse brain through the skull, showing a wide range of flow hemodynamics in both large and small vessels. We highlighted a decrease in pulsatility along the vascular tree in the cat brain, which could be mapped with ultrasound down to a few tens of micrometers for the first time. We also performed an intra-animal validation of the method by showing consistent measurements between the two sides of the Willis circle in the mouse brain. Our study provides the first step towards a new biomarker that would allow the detection of dynamic abnormalities in microvessels in the brain, which could be linked to early signs of neurodegenerative diseases.

Durability Improvement of Pumice Lightweight Aggregate Concrete by Incorporating Modified Rubber Powder with Sodium Silicate.

To improve the durability of pumice lightweight aggregate concrete applied in cold and drought areas, sodium silicate-modified waste tire rubber powder is used to treat the pumice lightweight aggregate concrete. The pumice lightweight aggregate concrete studied is mainly used in river lining structures. It will be eroded by water flow and the impact of ice and other injuries, resulting in reduced durability, and the addition of modified rubber will reduce the damage. The durability, including mass loss rate and relative dynamic elastic modulus of pumice lightweight aggregate concrete with different sodium silicate dosages and rubber power particle sizes, is analyzed under freeze-thaw cycles, and the microstructure is further characterized by using microscopic test methods such as nuclear magnetic resonance tests, ultra-depth 3D microscope tests, and scanning electron microscopy tests. The results showed that the durability of pumice lightweight aggregate concrete is significantly improved by the addition of modified waste tire rubber powder, and the optimum durability is achieved when using 2 wt% sodium silicate modified rubber power with a particle size of 20, and then the mass loss rate decreased from 4.54% to 0.77% and the relative dynamic elastic modulus increased from 50.34% to 64.87% after 300 freeze-thaw cycles compared with other samples. The scanning electron microscopy test result showed that the surface of rubber power is cleaner after the modification of sodium silicate, so the bonding ability between rubber power and cement hydration products is improved, which further improved the durability of concrete under the freeze-thaw cycle. The results of the nuclear magnetic resonance test showed that the pore area increased with the number of freeze-thaw cycles, and the small pores gradually evolved into large pores. The effect of sodium silicate on the modification of rubber power with different particle sizes is different. After the treatment of 2 wt% sodium silicate, the relationship between the increased rate of pore area and the number of freezing-thawing cycles is 23.8/times for the pumice lightweight aggregate concrete containing rubber power with a particle size of 20 and 35.3/times for the pumice lightweight aggregate concrete containing a particle size of 80 rubber power, respectively.

3D Traction Force Microscopy in Biological Gels: From Single Cells to Multicellular Spheroids.

Cell traction force plays a critical role in directing cellular functions, such as proliferation, migration, and differentiation. Current understanding of cell traction force is largely derived from 2D measurements where cells are plated on 2D substrates. However, 2D measurements do not recapitulate a vital aspect of living systems; that is, cells actively remodel their surrounding extracellular matrix (ECM), and the remodeled ECM, in return, can have a profound impact on cell phenotype and traction force generation. This reciprocal adaptivity of living systems is encoded in the material properties of biological gels. In this review, we summarize recent progress in measuring cell traction force for cells embedded within 3D biological gels, with an emphasis on cell-ECM cross talk. We also provide perspectives on tools and techniques that could be adapted to measure cell traction force in complex biochemical and biophysical environments.

Abstract TMP120: Contralesional Corticospinal Tract Reinnervates Cervical Spinal Anatomical Regions With Highest Pre-Lesion Synapse Density After Unilateral M1 Stroke in Mice

Introduction: Stroke leaves many patients with significant motor impairment. Following ischemic lesions of the rodent primary motor cortex, sprouting of the contralesional corticospinal tract (cCST) into the injured hemicord is associated with improved motor recovery. In adult mice, most corticospinal neurons project to spinal interneurons, and few synapse directly on alpha motor neurons. We examined the distribution of newly formed corticospinal synapses in the mouse spinal cord, using genetically encoded tracers, 3D microscopy, and a spinal atlas with machine learning-based automated classification and registration. Hypothesis: We tested the hypothesis that cCST neurons innervate homotopic regions in the injured hemicord after unilateral motor cortex stroke in adult mice. Method: We induced a photothrombotic motor cortex stroke or performed sham surgery (n=4 per group) in 8-11 week old male C57/B6 mice and administered a contralesional motor cortex injection of an anterograde adeno-associated virus expressing both membrane-targeted tdTomato and synaptically-targeted eGFP. Cervical spinal cords were subjected to volumetric imaging via serial two-photon tomography (TissueCyte 1000) to characterize synaptic and axonal density of cCST collaterals in whole cervical spinal cords in both stroke and sham mice. Unbiased, global quantification of axonal and synaptic density across the entire cervical cord was accomplished by the development of a custom automated image analysis pipeline, incorporating a novel 3D spinal cord reference volume, published spinal cord annotations comprising 47 distinct anatomical regions (SpinalJ), and machine learning based pixel classification. Results: We observed cCST synapses in the 6-week post-stroke injured hemicord innervating lamina IV & IX most densely at C7 & C8 (See Figure). Conclusions: Contralesional CST reinnervation of the denervated hemicord is targeted to both lamina and cervical level specific.

Analysis of RYR2 distribution in HEK293 cells and mouse cardiac myocytes using 3D minflux microscopy achieves all optical subunit resolution

Analysis of RYR2 distribution in HEK293 cells and mouse cardiac myocytes using 3D minflux microscopy achieves all optical subunit resolution

Eclipse: A versatile approach for structural and morphological analysis of 2D and 3D super-resolution fluorescence microscopy data

Eclipse: A versatile approach for structural and morphological analysis of 2D and 3D super-resolution fluorescence microscopy data

Removal Characteristics of Ni-Ferrite Oxide Film Formed on the Stainless Steel Surface in Fiber Laser Decontamination

Every nuclear facility has a limited operating life, at the end of which they must be shut down and decommissioned. For decommissioning, the removal of radioactive contaminants and demolition should be considered. Before nuclear power plants are decommissioned, the primary system, which is the most heavily contaminated, is decontaminated first, followed by decontamination of the surfaces of containment containers and buildings. This study examined the removal characteristics of Ni-ferrite coated on the stainless steel (STS304) specimen surface using an eco-friendly laser that generates little secondary waste, and derived the optimal efficiency conditions for laser decontamination. In this study, simulated oxide film specimens were fabricated by coating the STS304 substrate using non-radioactive Ni-ferrite. The experimental equipment for laser decontamination was a Q-switched fiber laser with an average power of 100 W. The laser beam was irradiated on the surface of the specimen according to the beam overlap rate. In addition, the characteristics of the decontaminated area and removal of the coating layer were checked using digital 3D microscopy, scanning electron microscope (SEM), and X-ray diffraction (XRD). The results of laser decontamination experiment indicated the possibility of coating layer removal with fewer scans as the x- or y-axis directions beam overlap rate increased. However, the surface was discolored when excessive overlaps occurred. This study provided basic data for laser decontamination applicable to the removal of Ni-ferrite oxide films on STS304.

DeepFocus: fast focus and astigmatism correction for electron microscopy

High-throughput 2D and 3D scanning electron microscopy, which relies on automation and dependable control algorithms, requires high image quality with minimal human intervention. Classical focus and astigmatism correction algorithms attempt to explicitly model image formation and subsequently aberration correction. Such models often require parameter adjustments by experts when deployed to new microscopes, challenging samples, or imaging conditions to prevent unstable convergence, making them hard to use in practice or unreliable. Here, we introduce DeepFocus, a purely data-driven method for aberration correction in scanning electron microscopy. DeepFocus works under very low signal-to-noise ratio conditions, reduces processing times by more than an order of magnitude compared to the state-of-the-art method, rapidly converges within a large aberration range, and is easily recalibrated to different microscopes or challenging samples.

Fast light-field 3D microscopy with out-of-distribution detection and adaptation through conditional normalizing flows.

Real-time 3D fluorescence microscopy is crucial for the spatiotemporal analysis of live organisms, such as neural activity monitoring. The eXtended field-of-view light field microscope (XLFM), also known as Fourier light field microscope, is a straightforward, single snapshot solution to achieve this. The XLFM acquires spatial-angular information in a single camera exposure. In a subsequent step, a 3D volume can be algorithmically reconstructed, making it exceptionally well-suited for real-time 3D acquisition and potential analysis. Unfortunately, traditional reconstruction methods (like deconvolution) require lengthy processing times (0.0220 Hz), hampering the speed advantages of the XLFM. Neural network architectures can overcome the speed constraints but do not automatically provide a way to certify the realism of their reconstructions, which is essential in the biomedical realm. To address these shortcomings, this work proposes a novel architecture to perform fast 3D reconstructions of live immobilized zebrafish neural activity based on a conditional normalizing flow. It reconstructs volumes at 8 Hz spanning 512x512x96 voxels, and it can be trained in under two hours due to the small dataset requirements (50 image-volume pairs). Furthermore, normalizing flows provides a way to compute the exact likelihood of a sample. This allows us to certify whether the predicted output is in- or ood, and retrain the system when a novel sample is detected. We evaluate the proposed method on a cross-validation approach involving multiple in-distribution samples (genetically identical zebrafish) and various out-of-distribution ones.

Evaluation of tumor microvasculature with 3D ultrasound localization microscopy based on 2D matrix array.

Evaluation of tumor microvascular morphology is of great significance in tumor diagnosis, therapeutic effect prediction, and surgical planning. Recently, two-dimensional ultrasound localization microscopy (2DULM) has demonstrated its superiority in the field of microvascular imaging. However, it suffers from planar dependence and is unintuitive. We propose a novel three-dimensional ultrasound localization microscopy (3DULM) to avoid these limitations. We investigated 3DULM based on a 2D array for tumor microvascular imaging. After intravenous injection of contrast agents, all elements of the 2D array transmit and receive signals to ensure a high and stable frame rate. Microbubble signal extraction, filtering, positioning, tracking, and other processing were used to obtain a 3D vascular map, flow velocity, and flow direction. To verify the effectiveness of 3DULM, it was validated on double helix tubes and rabbit VX2 tumors. Cisplatin was used to verify the ability of 3DULM to detect microvascular changes during tumor treatment. In vitro, the sizes measured by 3DULM at 3mm and 13mm were 178 and 182 , respectively. In the rabbit tumors, we acquired 9000 volumes to reveal vessels about 30 in diameter, which surpasses the diffraction limit of ultrasound in traditional ultrasound imaging, and the results matched with micro-angiography. In addition, there were significant changes in vascular density and curvature between the treatment and control groups. The effectiveness of 3DULM was verified in vitro and in vivo. Hence, 3DULM may have potential applications in tumor diagnosis, tumor treatment evaluation, surgical protocol guidance, and cardiovascular disease. 3D ultrasound localization microscopy is highly sensitive to microvascular changes; thus, it has clinical potential for tumor diagnosis and treatment evaluation. • 3D ultrasound localization microscopy is demonstrated on double helix tubes and rabbit VX2 tumors. • 3D ultrasound localization microscopy can reveal vessels about 30 in diameter-far smaller than traditional ultrasound. • This form of imaging has potential applications in tumor diagnosis, tumor treatment evaluation, surgical protocol guidance, and cardiovascular disease.

3D evaluation of the extracellular matrix of hypoxic pancreatic islets using light sheet fluorescence microscopy

ABSTRACT Pancreatic islet transplantation is a promising treatment for type 1 diabetes, but the survival and function of transplanted islets are hindered by the loss of extracellular matrix (ECM) during islet isolation and by low oxygenation upon implantation. This study aimed to evaluate the impact of hypoxia on ECM using a cutting-edge imaging approach based on tissue clearing and 3D microscopy. Human and rat islets were cultured under normoxic (O2 21%) or hypoxic (O2 1%) conditions. Immunofluorescence staining targeting insulin, glucagon, CA9 (a hypoxia marker), ECM proteins (collagen 4, fibronectin, laminin), and E-cadherin (intercellular adhesion protein) was performed on fixed whole islets. The cleared islets were imaged using Light Sheet Fluorescence Microscopy (LSFM) and digitally analyzed. The volumetric analysis of target proteins did not show significant differences in abundance between the experimental groups. However, 3D projections revealed distinct morphological features that differentiated normoxic and hypoxic islets. Under normoxic conditions, ECM could be found throughout the islets. Hypoxic islets exhibited areas of scattered nuclei and central clusters of ECM proteins, indicating central necrosis. E-cadherin was absent in these areas. Our results, demonstrating a diminution of islets’ functional mass in hypoxia, align with the functional decline observed in transplanted islets experiencing low oxygenation after grafting. This study provides a methodology combining tissue clearing, multiplex immunofluorescence, Light Sheet Fluorescence Microscopy, and digital image analysis to investigate pancreatic islet morphology. This 3D approach allowed us to highlight ECM organizational changes during hypoxia from a morphological perspective.

Dynamic Heat Transfer Simulation in Textile for Practical Application: A Comparative Analysis of Microscopic and Macroscopic Approaches

Heat transfer simulation in textile materials has many practical applications, such as in the design of protective clothing for firefighters, the development of thermal insulation materials, and the optimization of temperature control in textiles for comfort and performance. This paper presents a study on heat transfer simulation in woven fabrics using the Comsol Multiphysics® application. The simulations were carried out for both microscopic (3D) and macroscopic (1D) scales and the heat flux variation was compared with experimental results. The steady-state average heat flow through the textile was determined, and this value was used to calculate the thermal resistance of the woven fabrics. The thermal resistance values obtained were within a deviation range of 4.2% to 6.3% from the values determined according to ISO 11092/1016, thus validating the proposed model. For the transient regime, the microscopic approach proved to be more accurate, the estimated time for the heat flow to reach a certain value depending essentially on the scale approached in the modeling. This finding has particular significance in the field of protective clothing, especially for firefighters exposed to radiant heat sources, where estimating the time for burns to occur is crucial.

Data Science for Health Image Alignment: A User-Friendly Open-Source ImageJ/Fiji Plugin for Aligning Multimodality/Immunohistochemistry/Immunofluorescence 2D Microscopy Images.

Most of the time, the deep analysis of a biological sample requires the acquisition of images at different time points, using different modalities and/or different stainings. This information gives morphological, functional, and physiological insights, but the acquired images must be aligned to be able to proceed with the co-localisation analysis. Practically speaking, according to Aristotle's principle, "The whole is greater than the sum of its parts", multi-modal image registration is a challenging task that involves fusing complementary signals. In the past few years, several methods for image registration have been described in the literature, but unfortunately, there is not one method that works for all applications. In addition, there is currently no user-friendly solution for aligning images that does not require any computer skills. In this work, DS4H Image Alignment (DS4H-IA), an open-source ImageJ/Fiji plugin for aligning multimodality, immunohistochemistry (IHC), and/or immunofluorescence (IF) 2D microscopy images, designed with the goal of being extremely easy to use, is described. All of the available solutions for aligning 2D microscopy images have also been revised. The DS4H-IA source code; standalone applications for MAC, Linux, and Windows; video tutorials; manual documentation; and sample datasets are publicly available.

3D Holo-tomographic Mapping of COVID-19 Microclots in Blood to Assess Disease Severity.

The coronavirus disease 2019 (COVID-19) has impacted health globally. Cumulative evidence points to long-term effects of COVID-19 such as cardiovascular and cognitive disorders, diagnosed in patients even after the recovery period. In particular, micrometer-sized blood clots and hyperactivated platelets have been identified as potential indicators of long COVID. Here, we resolve microclot structures in the plasma of patients with different subphenotypes of COVID-19 in a label-free manner, using 3D digital holo-tomographic microscopy (DHTM). Based on 3D refractive index (RI) tomograms, the size, dry mass, and prevalence of microclot composites were quantified and then parametrically differentiated from fibrin-rich microclots and platelet aggregates in the plasma of COVID-19 patients. Importantly, fewer microclots and platelet aggregates were detected in the plasma of healthy controls compared to COVID-19 patients. Our imaging and analysis workflow is built around a commercially available DHT microscope capable of operation in clinical settings with a 2 h time period from sample preparation and data acquisition to results.

Comparison of holotomographic microscopy and coherence-controlled holographic microscopy.

Quantitative phase imaging (QPI) is a powerful tool for label-free visualisation of living cells. Here, we compare two QPI microscopes - the Telight Q-Phase microscope and the Nanolive 3D Cell Explorer-fluo microscope. Both systems provide unbiased information about cell morphology, such as individual cell dry mass, perimeter and area. The Q-Phase microscope uses artefact-free, coherence-controlled holographic imaging technology to visualise cells in real time with minimal phototoxicity. The 3D Cell Explorer-fluo employs laser-based holotomography to reconstruct 3D images of living cells, visualising their internal structures and dynamics. Here, we analysed the strengths and limitations of both microscopes when examining two morphologically distinct cell lines - the cuboidal epithelial MDCK cells which form multicellular clusters and solitary growing Rat2 fibroblasts. We focus mainly on the ability of the devices to generate images suitable for single-cell segmentation by the built-in software, and we discuss the segmentation results and quantitative data generated from the segmented images. We show that both microscopes offer slightly different advantages, and the choice between them depends on the specific requirements and goals of the user.

Evidence of internalized microplastics in mussel tissues detected by volumetric Raman imaging

Microplastics are a global ecological concern due to their potential risk to wildlife and human health. Animals ingest microplastics, which can enter the trophic chain and ultimately impact human well-being. The ingestion of microplastics can cause physical and chemical damage to the animals' digestive systems, affecting their health. To estimate the risk to ecosystems and human health, it is crucial to understand the accumulation and localization of ingested microplastics within the cells and tissues of living organisms. However, analyzing this issue is challenging due to the risk of sample contamination, given the ubiquity of microplastics. Here, an analytical approach is employed to confirm the internalization of microplastics in cryogenic cross-sections of mussel tissue. Using 3D Raman confocal microscopy in combination with chemometrics, microplastics measuring 1 μm in size were detected. The results were further validated using optical and fluorescence microscopy. The findings revealed evidence of microplastics being internalized in the digestive epithelial tissues of exposed mussels (Mytilus galloprovincialis), specifically within the digestive cells forming digestive alveoli. This study highlights the need to investigate the internalization of microplastics in organisms like mussels, as it helps us understand the potential risks they pose to aquatic biota and ultimately to human health. By employing advanced imaging techniques, challenges associated with sample contamination can be overcome and valuable insights into the impact of microplastics on marine ecosystems and human consumers are provided.

Network response of brain microvasculature to neuronal stimulation

Neurovascular coupling (NVC), or the adjustment of blood flow in response to local increases in neuronal activity is a hallmark of healthy brain function, and the physiological foundation for functional magnetic resonance imaging (fMRI). However, it remains only partly understood due to the high complexity of the structure and function of the cerebrovascular network. Here we set out to understand NVC at the network level, i.e. map cerebrovascular network reactivity to activation of neighbouring neurons within a 500×500×500 μm3 cortical volume (∼30 high-resolution 3-nL fMRI voxels). Using 3D two-photon fluorescence microscopy data, we quantified blood volume and flow changes in the brain vessels in response to spatially targeted optogenetic activation of cortical pyramidal neurons. We registered the vessels in a series of image stacks acquired before and after stimulations and applied a deep learning pipeline to segment the microvascular network from each time frame acquired. We then performed image analysis to extract the microvascular graphs, and graph analysis to identify the branch order of each vessel in the network, enabling the stratification of vessels by their branch order, designating branches 1–3 as precapillary arterioles and branches 4+ as capillaries. Forty-five percent of all vessels showed significant calibre changes; with 85 % of responses being dilations. The largest absolute CBV change was in the capillaries; the smallest, in the venules. Capillary CBV change was also the largest fraction of the total CBV change, but normalized to the baseline volume, arterioles and precapillary arterioles showed the biggest relative CBV change. From linescans along arteriole-venule microvascular paths, we measured red blood cell velocities and hematocrit, allowing for estimation of pressure and local resistance along these paths. While diameter changes following neuronal activation gradually declined along the paths; the pressure drops from arterioles to venules increased despite decreasing resistance: blood flow thus increased more than local resistance decreases would predict. By leveraging functional volumetric imaging and high throughput deep learning-based analysis, our study revealed distinct hemodynamic responses across the vessel types comprising the microvascular network. Our findings underscore the need for large, dense sampling of brain vessels for characterization of neurovascular coupling at the network level in health and disease.