3D Microscopy Research Articles

Synthesis and Characterization of a Novel Intrinsically Fluorescent Analog of Cholesterol with Improved Photophysical Properties.

Live-cell imaging of cholesterol trafficking depends on suitable cholesterol analogs. However, existing fluorescent analogs of cholesterol either show very different physicochemical properties compared to cholesterol or demand excitation in the ultraviolet spectral region. We present a strategy to synthesize two novel intrinsically fluorescent sterol probes with a close resemblance of cholesterol. The analogs contain four conjugated double bonds in the ring system and either a keto group (probe 5) or a hydroxy group (probe 6) in the C3 position. The emission of 5 is in the visible range of the spectrum, i.e., red-shifted by 150 nm compared to the widely used dehydroergosterol. Together with its high multiphoton absorption, this allows for imaging of 5 on conventional microscopes, including multicolor 3D and time-lapse microscopy. Molecular dynamics simulations and nuclear magnetic resonance spectroscopy reveal that 5 can condense the fatty acyl chains of phospholipids in model membranes. In giant unilamellar vesicles, 5 partitions equally into the liquid-ordered and disordered phases. In contrast, 6 emits in the ultraviolet range and is unstable in solution, preventing its use in live-cell imaging applications. The good photophysical properties of 5 make it a suitable analogue for improved live-cell imaging of sterol transport.

Three-dimensional random-access confocal microscopy with 3D remote focusing system.

Understanding biological activities in cells or deep tissues requires high-speed three-dimensional (3D) imaging. Substantial progress has been made with the emergence of 3D random-access microscopy. However, current solutions for fast 3D random-access imaging remain complex and costly. Herein we propose a simple, cost-effective, and fast 3D random-access confocal microscopy with remote focusing system. Our system shows isotropic response times across the x, y, and z axes, with a 34-fold improvement in axial response time over traditional piezo stages. We demonstrate its volumetric imaging performance with fluorescent particles and live cells. Furthermore, we validate the 3D random-access imaging capability of this system by continuously monitoring the signals in three different planes, showing a refresh rate of 500 Hz on two different positions in 3D. The simplicity, versatility, and affordability of our system promise widespread applications in research and industry.

EXTH-48. ADENO-ASSOCIATED VIRUS TARGETING OF THE TUMOR MICROENVIRONMENT TO IMPROVE LYMPHOCYTE RECRUITMENT IN GLIOBLASTOMA

Abstract BACKGROUND Glioblastoma (GBM) is notoriously devoid of lymphocytes driven in part by a paucity of lymphocyte trafficking factors necessary to prompt their recruitment, infiltration, and activation. We have developed a novel recombinant adeno-associated virus (AAV) gene therapy strategy that enables focal and stable reconstitution of the GBM tumor microenvironment (TME) with C-X-C motif ligand 9 (CXCL9), a powerful call-and-receive chemokine for cytotoxic T lymphocytes (CTLs). By increasing lymphocyte recruitment in GBM, we HYPOTHESIZE that these tumors will be more responsive to immunotherapy. METHODS 3D fluorescence microscopy was used to characterize the distribution, tropism, and durability of AAV6 transgene expression in preclinical GBM. Flow cytometry and 3D fluorescence microscopy was used to quantify T cell infiltration and visualize their biodistribution in preclinical GBM treated with AAV-CXCL9 in combination with anti-PD-1 immune checkpoint blockade (ICB). Single-cell RNA sequencing was used to characterize intratumor lymphocyte subsets following treatment. Survival studies with and without lymphocyte depletion were done to evaluate immunogenic responsiveness of therapy. RESULTS Focal delivery of CXCL9 with AAV6 results in stable transduction of peri-tumoral astrocytes. When combined with anti-PD-1 ICB, lymphocyte infiltration was improved, with cells found disseminated across the tumor stroma and within the tumor parenchyma. CXCR3, the chemokine receptor for CXCL9, was expressed across lymphocyte subsets and NKT cells, validating treatment selectivity of AAV6-CXCL9 for lymphocytes. Combination treatment considerably improved overall survival in two distinct GBM models, with durable responses dependent on CD8 T-lymphocyte infiltration. CONCLUSIONS By manipulating local chemokine directional guidance, AAV-CXCL9 increases tumor infiltration by CD8-postive cytotoxic lymphocytes, sensitizing GBM to anti-PD-1 ICB. These effects are accompanied by immunologic signatures evocative of an inflamed and responsive TME. These findings support targeted AAV gene therapy as a promising adjuvant strategy for reconditioning GBM immunogenicity given its excellent safety profile, TME-tropism, modularity, and off-the-shelf capability.

Ontogenetic and evolutionary trends on cephalopod digestive systems

AbstractMeropelagic octopuses hatch as planktonic paralarvae, being the subject of progressive morphological and behavioural changes ending with settlement on the seafloor as juveniles. The comparative morphological study of digestive systems can help to understand the adaptation to particular niches during this challenging plankton-benthos transition. Here, the morphometric development of the digestive system of the common octopus (Octopus vulgaris) is described through 3D microscopy techniques over the first two months of life. This morphological development is compared with those of adults and the holobenthic cuttlefish Sepia officinalis, as well as with the holopelagic squids Loligo vulgaris and Illex coindetii. Based on present and published results, we suggest four stages for the early development of O. vulgaris: (1) post embryonic, (2) early paralarval, (3) advanced paralarval and (4) early juvenile. The digestive system development was variable over time, with faster growth during the first days of planktonic life and after settlement. All the paralarvae-bearing species showed very proportionally enlarged posterior salivary glands at hatching compared both to more developed conspecifics and to the cuttlefish hatchling. This could reflect a potential common feeding mechanism through external digestion and probably a compensatory mechanism for an early digestive gland underperformance. The proportionally smallest digestive system has been found in L. vulgaris, mainly because of their remarkably smaller digestive glands. All species showed major changes in digestive organs between hatchlings and developed phases, indicating how different ecological contexts over ontogeny are reflected in their digestive structures. Graphical abstract

3D surgical microscopes for oral surgical procedures

We look at how 3D microscopes can aid oral surgeons in real time and allow for more accurate treatment.

Trophoblast Proliferation is higher in Female than in Male Preeclamptic Placentas

IntroductionPreeclampsia (PE) is a pregnancy-specific hypertensive disorder with inflammatory complications. There are no known placental histopathological features, which are unique to PE. It is often pooled with the fetal growth restriction (FGR) under a single umbrella pathophysiology, the maternal vascular malperfusion (MVM). The aim of this study is to assess the villous trophoblast and the villous tree quantitatively in PE placentas and to identify morpholgical correlates unique to PE. Methods20 PE placentas (10 female and 10 male) and 20 Control placentas (10 female and 10 male) were included in the study. The villous trophoblast and the villous tree were assessed quantitatively by Stereology and 3D Microscopy. For Stereology measurements, the villous tree was classified in contractile and non-contractile parts based on immunohistochemical detection of perivascular myofibroblasts. ResultsThe density of proliferative trophoblast nuclei is increased, whereas the density of non-proliferative trophoblast nuclei is decreased in female PE placentas. The male PE placentas do not show this effect. Though no significant difference in the diffusion distance was observed, the non-contractile villi and the fetal vessels inside show a significantly reduced volume in PE placentas. The branching index of the villous tree is lower in PE placentas in general. However, in female PE placentas the deviation is accentuated. ConclusionIn PE, the villous trophoblast shows a sexually dimorphic alteration in the density of proliferative and non-proliferative nuclei, which is inherently different from FGR.

Human milk fat globule size distributions: comparison between laser diffraction and 3D confocal laser scanning microscopy

Milk fat globules (MFGs) in human milk provide energy to breastfed infants and support infant development. Accurate measurements of MFG size distributions are important to better understand MFG function and origin, as well as the influence of MFG size on milk composition analysis methods. Nevertheless, commonly used laser diffraction systems have never been thoroughly validated for size distribution measurements in human milk. Here, we introduce a new method for determining the size distribution of milk fat globules in human milk, using 3D confocal laser scanning microscopy (CLSM) in combination with fluorescent labeling of MFGs. We validate and compare 3D CLSM to laser diffraction (Mastersizer 2000, Malvern Panalytical), using polystyrene microsphere size standards. Next, we apply both methods to evaluate MFG size distributions in human milk. We show that 3D CLSM can be used to obtain more accurate size distributions between 500 nm and 10 μm compared to laser diffraction. Importantly, MFG size distributions obtained with 3D CLSM contain no secondary population around 1 μm, in contrast to laser diffraction measurements. This suggests that the bimodal MFG distribution obtained by laser diffraction can be an artifact of the built-in fitting algorithm, instead of an actual feature of human milk. This work demonstrates that care should be taken when interpreting size distributions of MFGs measured with laser diffraction and that 3D CLSM is an accurate alternative for measuring size distributions in lactation and dairy research.

Identification of CD44 as a key engager to hyaluronic acid-rich extracellular matrices for cell traction force generation and tumor invasion in 3D

Mechanical properties of the extracellular matrix (ECM) critically regulate a number of important cell functions including growth, differentiation and migration. Type I collagen and glycosaminoglycans (GAGs) are two primary components of ECMs that contribute to mammalian tissue mechanics, with the collagen fiber network sustaining tension, and GAGs withstanding compression. The architecture and stiffness of the collagen network are known to be important for cell-ECM mechanical interactions via integrin cell surface adhesion receptors. In contrast, studies of GAGs in modulating cell-ECM interactions are limited. Here, we present experimental studies on the roles of hyaluronic acid (HA) in single tumor cell traction force generation using a recently developed 3D cell traction force microscopy method. Our work reveals that CD44, a cell surface receptor to HA, is engaged in cell traction force generation in conjunction with β1-integrin. We find that HA significantly modifies the architecture and mechanics of the collagen fiber network, decreasing tumor cells’ propensity to remodel the collagen network, attenuating traction force generation, transmission distance, and tumor invasion. Our findings point to a novel role for CD44 in traction force generation, which can be a potential therapeutic target for diseases involving HA rich ECMs such as breast cancer and glioblastoma.

Transfer learning-based techniques for efficient 3D-reconstruction of functionally graded materials

The various properties and phenomena in materials significantly depend on the high-quality three-dimensional (3D) microstructure. This paper proposes a novel method for 3D reconstruction of functionally graded materials (FGMs) using transfer learning techniques to overcome the limitations of experimental 3D microscopy. The approach begins by interpolating 2D cross-sectional images of FGM microstructures through a hybrid deep learning method combining StyleGAN and Gatys techniques, generating intermediate images based on style and content. In the next step, transfer learning is applied using convolutional occupancy networks to reconstruct the 3D microstructure from the 2D image depth layers, with the model trained on point clouds obtained from 2D images. To validate the method, the reconstructed microstructures are compared to actual material images at the same depth using various metrics, including lineal path, two-point correlation, and quality of connection functions. The results show a high degree of accuracy, indicating that the reconstructed images closely match the target microstructures. Finally, to show the application of model the approach is implemented to the functionally graded lanthanum strontium manganite (LSM) and 3D microstructure of this FGM has been extracted using the approach

In-depth morphological and functional analysis of ATTR-CA myocardial cells and niche by electron microscopic 3D reconstruction

Abstract Background Cell-cell interactions and interplay with extracellular matrix are known to be important in the steady state and pathological conditions, including wild-type transthyretin cardiac amyloidosis (wtATTR-CA). However, alterations of the microstructure of cardiomyocytes (CMs) and cardiac niches have not been well analyzed. Normal histological staining is usually limited in resolution, and 2D electron microscopy cannot capture stereoscopic images or the distribution of target particles and organelles in intra- and extracellular 3D space, so novel methods are being attempted. Methods and Results Using Serial Block Face-Scanning Electron Microscopy (SBF-SEM) and a 3D structural reconstruction system, we for the first time investigated the microstructure of human myocardial tissue from control hypertrophied heart (hypertensive heart disease) and wtATTR-CA patients. The microstructure of myocardial niche, cell morphology/numbers, and the distribution of intracellular organelles such as intercalated discs (ICDs) were compared to control endomyocardial biopsy (EMB) samples (2 controls vs 5 wtATTR-CA patients). In all wtATTR-CA samples, amyloid fibers were deposited originating around the capillary structures with various electron densities, compressing myocardial cells not only from the periphery but also dividing them from the center of the cell body, and CMs showed degeneration and dwarfing according to the severity of amyloid fiber deposition (Figure 1). The capillary structures showed degeneration and narrowing with stratified basement membrane, and rarefaction (3378±216.5 vs 1448±116.8/mm2, p<0.0001). Notably, the stromal cells (pericytes, fibroblasts, macrophages) were more likely buried by amyloid fibers than CMs, and their numbers were also reduced, indicating a loss of adjacent and intercellular interaction with capillaries and CMs (Fig. 2, 1892±194.4 vs 1189±107.4 mm2, p=0.0043). This method can also be used to analyze intracellular organelles and microstructures in CMs. In ATTR-CA, morphological abnormalities were observed in ICDs (Figure 3) and mitochondria. Conclusion In the ATTR-CM patients, there was a significant decrease in the number of capillaries in accordance with the deposition of myocardial amyloid fibers, as well as a decrease in stromal cells. These results support the hypothesis that amyloid fibril deposition not only causes chronic myocardial ischemia, but also reduces the number of niche cells, which play a supportive role for CM function and entirely exacerbate the damage. The SBF-SEM is an innovative tool for 3D imaging and quantitative analysis of myocardial ultrastructure and functions.Methods & Figure 1Figure 2 and Figure 3

A flexible porous polyimide/copper composite film toward high-mass-loading anodes in lithium-ion batteries

Conventional metal foil current collectors are essential for electronic conduction in lithium-ion batteries, but the planar structure limits the fast transporting of electron and the power density. Herein, a light, conductive, self-extinguishing, and flexible porous polyimide/copper composite film (PIF/Cu) derived from a polyimide foam was fabricated via hot pressing and electroless plating methods. The honeycomb structure was illustrated in PIF/Cu under a 3D X-ray tomography microscope, revealing the successful construction of a 3D conductive network. PIF/Cu-30 possessed an electronic conductivity of as high as 3.6 × 105 S m−1 at a bulk density of 0.26 g cm−3 and exhibited exceptional structural stability even after successive and harsh mechanical loads. Graphite with a mass load of 12 mg cm−2 was loaded into the PIF/Cu-30 to prepare the anode for cell assembly. The cells using PIF/Cu-30 achieved a better performance in capacity retention (98.2 %@60 cycles) than those using traditional Cu foils (55.1 %@60 cycles), which resulted from the fast transport of electrons and Li-ion through the 3D conductive networks in the porous PIF/Cu-30.

3D Micropatterned Traction Force Microscopy: A Technique to Control 3D Cell Shape While Measuring Cell-Substrate Force Transmission.

Cell shape and function are intimately linked, in a way that is mediated by the forces exerted between cells and their environment. The relationship between cell shape and forces has been extensively studied for cells seeded on flat 2D substrates, but not for cells in more physiological 3D settings. Here, a technique called 3D micropatterned traction force microscopy (3D-µTFM) to confine cells in 3D wells of defined shape, while simultaneously measuring the forces transmitted between cells and their microenvironment is demonstrated. This technique is based on the 3D micropatterning of polyacrylamide wells and on the calculation of 3D traction force from their deformation. With 3D-µTFM, it is shown that MCF10A breast epithelial cells exert defined, reproducible patterns of forces on their microenvironment, which can be both contractile and extensile. Cells switch from a global contractile to extensile behavior as their volume is reduced are further shown. The technique enables the quantitative study of cell mechanobiology with full access to 3D cellular forces while having accurate control over cell morphology and the mechanical conditions of the microenvironment.

3D Cryo-Electron Microscopy Reveals the Structure of a 3-Fluorenylmethyloxycarbonyl Zipper Motif Ensuring the Self-Assembly of Tripeptide Nanofibers.

Short peptide-based supramolecular hydrogels appeared as highly interesting materials for applications in many fields. The optimization of their properties relies mainly on the design of a suitable hydrogelator through an empirical trial-and-error strategy based on the synthesis of various types of peptides. This approach is in part due to the lack of prior structural knowledge of the molecular architecture of the various families of nanofibers. The 3D structure of the nanofibers determines their ability to interact with entities present in their surrounding environment. Thus, it is important to resolve the internal structural organization of the material. Herein, using Fmoc-FFY tripeptide as a model amphiphilic hydrogelator and cryo-EM reconstruction approach, we succeeded to obtain a 3.8 Å resolution 3D structure of a self-assembled nanofiber with a diameter of approximately 4.1 nm and with apparently "infinite" length. The elucidation of the spatial organization of such nano-objects addresses fundamental questions about the way short amphiphilic N-Fmoc peptides lacking secondary structure can self-assemble and ensure the cohesion of such a lengthy nanostructure. This nanofiber is organized into a triple-stranded helix with an asymmetric unit composed of two Fmoc-FFY peptides per strand. The three identical amphiphilic strands are maintained together by strong lateral interactions coming from a 3-Fmoc zipper motif. This hydrophobic core of the nanofiber is surrounded by 12 phenyl groups from phenylalanine residues, nonplanar with the six Fmoc groups. Polar tyrosine residues at the C-term position constitute the hydrophilic shell and are exposed all around the external part of the assembly. This fiber has a highly hydrophobic central core with an internal diameter of only 2.4 Å. Molecular dynamics simulations highlight van der Waals and hydrogen bonds between peptides placed on top of each other. We demonstrate that the self-assembly of Fmoc-FFY, whether induced by annealing or by the action of a phosphatase on the phosphorylated precursor Fmoc-FFpY, results in two nanostructures with minor differences that we are unable to distinguish.

A low-temperature process for lithium extraction from lithium-bearing mineral: Conversion behavior and mechanisms

Lithium extraction from lithium-bearing clay mineral (LiBC) has received significant attention due to the increasing demand of lithium for the advanced renewable energy industry. However, the low contents of valuable elements and stable physiochemical properties of LiBC result in high energy consumption and low efficiency in extracting lithium from LiBC. This study demonstrates a low-temperature strategy using (NH4)2SO4 as an additive to effectively extract lithium from LiBC. In details, the thermodynamic analysis verifies that Li, Al, and Fe can be converted to sulfates at 250–450 °C during thermal treatment, indicating the possibility for extraction of Li under low-temperature conditions. Subsequently, the leaching behavior of lithium under different roasting temperatures and water leaching conditions was investigated. The results show that 83.43 % of Li can be extracted under optimized conditions. The mechanism study was carried out through a number of characterization techniques including 3D microscope, TG-DSC, XRD, FTIR, and SEM. It reveals that formation of liquidus phase of molten salt is the key to achieve low-temperature roasting and transfer and reactions at the interface of molten salt and LiBC can be significantly enhanced that can easily breakdown the interlayer structures. Consequently, it offers distinct advantages, since the roasting temperature can be significantly decreased with minor impurity ions being leached into the solutions. This research therefore provides an efficient and environmentally friendly method for lithium extraction from minerals.

Single-Shot 3D Imaging Meta-Microscope.

Three-dimensional (3D) imaging enables high-precision and high-resolution axial positioning, which is crucial for biological imaging, semiconductor defect monitoring, and other applications. Conventional implementations rely on bulky optical elements or scanning mechanisms, resulting in low speed and complicated setups. Here, we generate the double-helix (DH) point spread function with an all-dielectric metasurface and thus innovate the 3D imaging microscope (hence dubbed meta-microscope), both in 4f and 2f imaging systems. The 4f-meta-microscope with a numerical aperture of 0.7 achieves an axial localization accuracy below 0.12 μm within a 15.47 μm detection range, while the 2f-DH meta-microscope with a numerical aperture of 0.3 shows a 1.12 μm accuracy within a 227.33 μm range. We also demonstrate single-shot and accurate 3D biological imaging of the mouse kidney tissue and peach anther, providing a comprehensive and efficient approach for 3D bioimaging and other applications through a single-shot 3D meta-microscope.

A Simple and Fast Optical Clearing Method for Whole-Mount Fluorescence In Situ Hybridization (FISH)Imaging.

We report a single-step optical clearing method that is compatible with RNA fluorescence in situ hybridization (FISH) imaging. We previously demonstrated microscopy imaging with immunohistochemistry and genetic reporters using a technique called lipid-preserving refractive index matching for prolonged imaging depth (LIMPID). Our protocol reliably produces high-resolution three-dimensional (3D) images with minimal aberrations using high magnification objectives, captures large field-of-view images of whole-mount tissues, and supports co-labeling with antibody and FISH probes. We also custom-designed FISH probes for quail embryos, demonstrating the ease of fabricating probes for use with less common animal models. Furthermore, we show high-quality 3D images using a conventional fluorescence microscope, without using more advanced depth sectioning instruments such as confocal or light-sheet microscopy. For broader adoption, we simplified and optimized 3D-LIMPID-FISH to minimize the barrier to entry, and we provide a detailed protocol to aid users with navigating the thick and thin of 3D microscopy.

Comparative Analysis of Layer Thickness Measurement Methods for Photovoltaic Modules: A Comprehensive Study

The increasing volume of end‐of‐life (EoL) photovoltaic (PV) modules poses a significant challenge, necessitating efficient and sustainable recycling processes. In the PVReValue project, it is aimed to develop a comprehensive methodology for the systematic separation and recycling of EoL PV modules. Central to this effort is the precise determination of layer thicknesses, critical for effective characterization, and separation of input material. In this study, a comparative analysis of various industrial‐applicable methods is conducted for measuring layer thicknesses in PV modules. Both destructive and nondestructive techniques are evaluated based on criteria such as time, cost, accuracy, and applicability. Methods such as optical coherence tomography (OCT), coaxial multicolor confocal measurement, and ultrasonic measurements are assessed alongside traditional approaches like calotte grinding and optical 3D microscopy. In these findings, it is indicated that while OCT and confocal measurement offer high accuracy and nondestructiveness, they are complex and costly. Conversely, calotte grinding, although less costly, provides only localized information and can be challenging for polymers. In this study, it is concluded with recommendations for the possible integration of these methods into an industrial recycling workflow, highlighting the potential for improving PV module recycling efficiency and sustainability.

3D X-ray microscope acts as an accurate and effective equipment of pathological diagnosis in craniofacial imaging

Craniofacial structure and dental hard tissue used to be researched on by traditional imaging tools such as light microscope, electron microscope and micro-CT. Due to the limitations of imaging principle, resolution and 3D rendering reconstruction technique, traditional imaging tools are constrained for presenting fine structure and precise measurements. Here a brand-new imaging equipment–3D X-ray microscope is introduced to realize a more efficient scanning by demonstrating the comparison of the craniofacial structures and dental hard tissue of diabetes and normal DBA mouse. To explore a higher resolution, more efficient imaging measurement and 3D reconstruction method on craniofacial structure and dental hard tissue. The study included 12 DBA mice which were divided into two groups (control group and diabetes group). The heads were separated and scanned by 3D X-ray microscope, after which regions of interest were selected, followed by measurement and 3D reconstruction based on microscope attached software Dragonfly pro©. Hemi-mandibles were collected for enamel mineral density assessment supported by QRM-MicroCT-HA phantom. Data was submitted to paired t-tests at a 95% confidence level. The automatic assessed enamel thickness of diabetes mice decreased on average, whereas the rest of manual measurements and automatic assessed density showed no statistical difference. We constructed HA phantom assisted enamel density procedure in Dragonfly software. Craniofacial structure and dental hard tissue were well-presented both in 2D slide and 3D reconstruction viewport by 3D X-ray microscope which can be routinely used as craniofacial structure and dental hard tissue imaging tool.

Diagnosis of dental erosion using intraoral scanner in comparison to 3D confocal laser microscope

Diagnosis of dental erosion using intraoral scanner in comparison to 3D confocal laser microscope

A proposal for the combined analysis of bone quantity and quality of human cortical bone by quasi-brittle fracture mechanics

Quasi-brittle fracture mechanics is used to evaluate fracture of human cortical bone in aging. The approach is demonstrated using cortical bone bars extracted from one 92-year-old human male cadaver. In-situ fracture mechanics experiments in a 3D X-ray microscope are conducted. The evolution of the fracture process zone is documented. Fully developed fracture process zone lengths at peak load are found to span about three osteon diameters. Crack deflection and arrest at cement lines is a key process to build extrinsic toughness. Strength and toughness are found as size-dependent, not only for laboratory-scale experimental specimens but also for the whole femur. A scaling law for the length fracture process zone is used. Then, size-independent, tissue fracture properties are calculated. Linear elastic fracture mechanics applied to laboratory beam specimens underestimates the tissue toughness by 60%. Tissue fracture properties are used to predict the load capacity of the femur in bending within the range of documented data. The quasi-brittle fracture mechanics approach allows for the assessment of the combined effect of bone quantity and bone quality on fracture risk. However, further work is needed considering a larger range of subjects and in the model validation at the organ length scale.