3D Microscopy Research Articles

Comparison of neck angle and musculoskeletal discomfort of surgeon in cataract surgery between three-dimensional heads-up display system and conventional microscope

The three-dimensional heads-up display system (3D HUDS) is increasingly utilized by ophthalmologists and suggested to offer ergonomic benefits compared to conventional operating microscopes. We aimed to quantitatively assess the surgeon’s neck angle and musculoskeletal discomfort during cataract surgery using commercially available 3D HUDS and conventional microscope. In this single-center comparative observational study, the surgeon conducted routine phacoemulsification surgeries using Artevo® 800 and Opmi Lumera® 700 (both from Carl Zeiss Meditec, Jena, Germany). The surgeon’s intraoperative neck angle was measured using the Cervical Range of Motion device. Postoperative musculoskeletal discomfort was assessed using the Visual Analog Scale (VAS) score after each surgery. A total of 80 cataract surgeries were analyzed, with 40 using Artevo® 800 and 40 using Opmi Lumera® 700. The neck angle was extended when using Artevo® 800 and flexed when using Opmi Lumera® 700 during continuous curvilinear capsulorhexis (CCC), phacoemulsification, and intraocular lens (IOL) placement (− 8.18 ± 2.85° vs. 8.27 ± 2.93° in CCC, − 7.83 ± 3.30° vs. 8.87 ± 2.83° in phacoemulsification, − 7.43 ± 3.80° vs. 7.67 ± 3.73° in IOL placement, respectively; all p < 0.001). The VAS score was significantly lower in surgeries performed with Artevo® 800 (1.27 ± 0.55 vs. 1.73 ± 0.64, p < 0.001). The findings suggest that 3D HUDS help reduce neck flexion and lower work-related musculoskeletal discomfort through ergonomic improvements.

Hyaluronic Acid and Calcium Hydroxyapatite in the Context of Hypertrophic Photoaging. Evaluation by 2D, 3D Photographs and Reflectance Confocal Microscopy (RCM).

Hyaluronic Acid and Calcium Hydroxyapatite in the Context of Hypertrophic Photoaging. Evaluation by 2D, 3D Photographs and Reflectance Confocal Microscopy (RCM).

Experimental study on tribological behavior of a binderless cemented carbide at high contact stress

Knowledge of frictional evolution and associated damage mechanism during sliding wear conditions using binderless WC-based cemented carbide is lacking. In this study, the frictional evolution and corresponding transformation of microstructure, and wear mechanisms of Al2O3/WC-based cemented carbide due to the effect of different contact pressure, especially high pressure have been explored using a reciprocating ball-on-flat sliding wear tester, which was experimentally simulated following the ASTM G133–02 standard. The dynamic curves in friction coefficient with the sliding time were described. The microscopic 3D topography of contact surface was obtained by the MFP-3D atomic force microscope. The material removal and the wear rate were discussed. Worn surface morphologies at different moments, cross-sectional images of wear tracks at different loads, and wear debris were taken by field emission scanning electron microscope. The results suggested that the pressure plays a decisive role in frictional evolution and wear characteristics. Two models of frictional evolution were declared. A novel “surge” phenomena in friction coefficient was found and explained at high contact pressure. Wear transition from mild wear to severe wear was confirmed. More than one wear mechanism was observed, including micro-cutting (polishing), generation and propagation of cracks, cracking-induced spalling, plastic deformation, and the formation of tribolayer.

Comparison of surface micro‐roughness and adaptation of titanium and cobalt chrome implant abutment fabricated by selective laser melting and conventional techniques

AbstractPurposeThe objective of this study was to assess the surface micro‐roughness and abutment adaptation of selective laser melting (SLM) implant abutments in comparison to cast and machined implant abutments.MethodsForty abutment specimens were divided equally into four groups according to the fabrication technique as follows (n = 10), Machined Ti alloy abutments (Control), Cast CoCr abutments, SLM‐CoCr abutments, and SLM‐Ti alloy abutments. Forty internal connection implants (Ø 4.0 ×10 mm, Superline™, Dentium Co., Seoul, Korea) were mounted in clear acrylic resin. Fabricated abutments were assessed for surface micro‐roughness using a 3D optical noncontact surface microscope. Vertical and horizontal adaptation of the abutment with implant interface was assessed by using Bruker micro‐CT. Data was assessed using analysis of variance and Tukey post hoc comparison tests for all the variables except vertical misfit was assessed using Kruskal–Wallis test. Pearson correlation was used to assess dependence between independent variable (surface roughness) and dependent variables (Horizontal misfit and vertical misfit).ResultsSLM‐Ti abutments showed significantly rougher surface (p &lt; 0.05) among the study groups. While SLM‐CoCr abutments were smoother than Cast abutments with mean Ra of 1.30 ± 0.11 and 1.58 ± 0.17 μm, respectively (p &lt; 0.05). For abutment adaptation, SLM‐Ti abutments showed the highest horizontal misfit among the groups (p &lt; 0.05). While, SLM‐CoCr abutments (29.24 ± 11.11 μm) showed comparable (p &gt; 0.05) horizontal misfit results with Cast (26.08 ± 3.93 μm) and machined (26.45 ± 7.33 μm) abutments. Comparable (p &gt; 0.05) vertical misfit values between Cast CoCr (487.01 ± 40.34 μm), SLM‐CoCr (358.38 ± 114.93 μm) and SLM‐Ti (299.85 ± 172.88 μm) abutments were observed. A positive correlation was found between the surface roughness of the mating surfaces and abutment adaptation.ConclusionSLM CoCr abutments exhibited low roughness and comparable abutment adaptation (vertically and horizontally) than cast (control) abutments. Therefore, SLM CoCr abutments should be clinically investigated as potential implant abutments for clinical use.

Damage and fracture studies of continuous flax fiber-reinforced composites 3D printed by in-nozzle impregnation additive manufacturing

Additive manufacturing (AM) of continuous yarn-reinforced biobased composites presents multi-functional properties and low environmental impact of this technology. Few studies focused on the mechanical damage mechanisms of continuous biobased composites obtained by AM processes, while it is a topic of high interest for the mastery of mechanical behaviors and optimization of the materials for high requirement applications. This study aims to assess the damage and fracture modes of continuous flax yarn-reinforced PLA manufactured by AM, with different yarn orientations. The additively manufactured biobased composites were characterized by tensile test, 3D microscopy and micro-tomography to link the process-structure-properties relationships regarding the damage and fracture modes. The results showed that the 0° manufactured composite had a significant enhancement of tensile properties compared to other configurations. The damage mechanism presented fiber rupture with polymer transverse cracks at 0°, while the 45° and 90°-oriented composites showed premature fiber/matrix interface debonding. This study aims to find the relationship between damage mechanisms, deposition strategy, and anisotropy of the additively manufactured long vegetal fibers-reinforced biobased composite materials. The results bring a new understanding of the anisotropy and defects in printed composite materials regarding their mechanical behaviors during damage.

Multimodal three-dimensional characterization of murine skeletal muscle micro-scale elasticity, structure, and composition: Impact of dysferlinopathy, Duchenne muscular dystrophy, and age on three hind-limb muscles

Skeletal muscle tissue function is governed by the mechanical properties and organization of its components, including myofibers, extracellular matrix, and adipose tissue, which can be modified by the onset and progression of many disorders. This study used a novel combination of quantitative micro-elastography and clearing-enhanced three-dimensional (3D) microscopy to assess 3D micro-scale elasticity and micro-architecture of muscles from two muscular dystrophies: dysferlinopathy and Duchenne muscular dystrophy, using male BLA/J and mdx mice, respectively, and their wild-type (WT) controls. We examined three muscles with varying proportions of slow- and fast-twitch myofibers: the soleus (predominantly slow), extensor digitorum longus (EDL; fast), and quadriceps (mixed), from BLA/J and WTBLA/J mice aged 3, 10, and 24 months, and mdx and WTmdx mice aged 10 months. Both dysferlin deficiency and age reduced the elasticity and variability of elasticity of the soleus and quadriceps, but not EDL. Overall, the BLA/J soleus was 20% softer than WT and less mechanically heterogeneous (−14% in standard deviation of elasticity). The BLA/J quadriceps at 24 months was 72% softer than WT and less mechanically heterogeneous (−59% in standard deviation), with substantial adipose tissue accumulation. While mdx muscles did not differ quantitatively from WT, regional heterogeneity was evident in micro-scale elasticity and micro-architecture of quadriceps (e.g., 11.2 kPa in a region with marked pathology vs 3.8 kPa in a less affected area). These results demonstrate differing biomechanical changes in hind-limb muscles of two distinct muscular dystrophies, emphasizing the potential for this novel multimodal technique to identify important differences between various myopathies.

DATA DRIVEN AND CELL SPECIFIC DETERMINATION OF NUCLEI-ASSOCIATED ACTIN STRUCTURE

In the specialized field of cellular mechanosignaling, the intricate relationship between the cytoskeleton and nucleus is crucial for determining the behavior and fate of mesenchymal stem cells (MSCs). Existing analytical methods for filamentous actin fibers (F-actin) suffer from issues of accuracy and reproducibility. To address this, our research introduces a significant advancement in image analysis by utilizing two deep-learning segmentation Convolutional Neural Networks (CNNs) based on U-Net architecture. This algorithm precisely quantifies F-actin and offers reproducible data from 3D confocal microscopy images. Our CNNs automatically extract the 3D shape of both the nucleus and individual actin fibers, thereby enabling the geometric reconstruction of these critical cellular components. We applied this methodology to MSCs exposed to low-intensity vibration, and hypothesized that mechanical stimuli will induce perinuclear actin remodeling. Confocal Z-stack images of MSCs were captured using a 40x oil lens and a Zeiss LSM 900 microscope. Nucleus and F-actin structures were stained with Hoechst 33342 and Phalloidin, respectively. Nucleus-based images were employed to mask and isolate actin fibers within the nuclear vicinity. A specialized deep-learning segmentation model was engineered within the PyTorch framework. For model training, a selective 2% of nuclei and F-actin images were annotated. Specifically, three layers from each of six representative nuclei, among approximately 500 cross-sectional layers, were meticulously selected; a similar approach was employed for actin. The model underwent 200 epochs of training. Post-training, the deep-learning model was employed to process all collected cross-sectional images of F-actin and nuclei. A subsequent analytical step involved the scrutiny of intersecting segmented fibers between consecutive layers for geometrically accurate actin fiber reconstruction. We analyzed 102 control and 110 vibration-treated mesenchymal stem cells (MSCs) by utilizing our deep-learning-based reconstruction algorithm on confocal Z-stack images. Our data revealed an 8% decrease in nuclear height from 5.73 µm to 5.28 µm (p &lt; 0.001) upon application of low-intensity vibration. In contrast, the two groups observed no significant difference in nuclear volume. Accompanying these changes at the nuclear level, the following changes in the F-actin cytoskeleton were observed upon LIV application. The total number of actin filaments per cell increased from 117 to 150. The average volume of individual actin fibers rose from 0.40 µm³ to 0.43 µm³. Notably, the volume of fibers at the apical nuclear surface increased from 0.35 µm³ to 0.46 µm³. While the average length of actin fibers in the apical nuclear surface remained largely unchanged due to the addition of smaller fibers in LIV groups, the LIV group had a larger number of fibers that were larger than 15µm.

DIRT/µ - Automated extraction of root hair traits using combinatorial optimization.

Similar to any microscopic appendages, such as cilia or antennae, phenotyping of root hairs has been a challenge due to their complex intersecting arrangements in two-dimensional (2D) images and the technical limitations of automated measurements. Digital Imaging of Root Traits at Microscale (DIRT/μ) addresses this issue by computationally resolving intersections and extracting individual root hairs from 2D microscopy images. This solution enables automatic and precise trait measurements of individual root hairs. DIRT/μ rigorously defines a set of rules to resolve intersecting root hairs and minimizes a newly designed cost function to combinatorically identify each root hair in the microscopy image. As a result, DIRT/μ accurately measures traits such as root hair length (RHL) distribution and root hair density (RHD), which are impractical for manual assessment. We tested DIRT/μ on three datasets to validate its performance and showcase potential applications. By measuring root hair traits in a fraction of the time manual methods require, DIRT/μ eliminates subjective biases from manual measurements. Automating individual root hair extraction accelerates phenotyping and quantifies trait variability within and among plants, creating new possibilities to characterize root hair function and their underlying genetics.

ECLiPSE: a versatile classification technique for structural and morphological analysis of 2D and 3D single-molecule localization microscopy data

Single-molecule localization microscopy (SMLM) has gained widespread use for visualizing the morphology of subcellular organelles and structures with nanoscale spatial resolution. However, analysis tools for automatically quantifying and classifying SMLM images have lagged behind. Here we introduce Enhanced Classification of Localized Point clouds by Shape Extraction (ECLiPSE), an automated machine learning analysis pipeline specifically designed to classify cellular structures captured through two-dimensional or three-dimensional SMLM. ECLiPSE leverages a comprehensive set of shape descriptors, the majority of which are directly extracted from the localizations to minimize bias during the characterization of individual structures. ECLiPSE has been validated using both unsupervised and supervised classification on datasets, including various cellular structures, achieving near-perfect accuracy. We apply two-dimensional ECLiPSE to classify morphologically distinct protein aggregates relevant for neurodegenerative diseases. Additionally, we employ three-dimensional ECLiPSE to identify relevant biological differences between healthy and depolarized mitochondria. ECLiPSE will enhance the way we study cellular structures across various biological contexts.

Lens-free on-chip 3D microscopy based on wavelength-scanning Fourier ptychographic diffraction tomography

Lens-free on-chip microscopy is a powerful and promising high-throughput computational microscopy technique due to its unique advantage of creating high-resolution images across the full field-of-view (FOV) of the imaging sensor. Nevertheless, most current lens-free microscopy methods have been designed for imaging only two-dimensional thin samples. Lens-free on-chip tomography (LFOCT) with a uniform resolution across the entire FOV and at a subpixel level remains a critical challenge. In this paper, we demonstrated a new LFOCT technique and associated imaging platform based on wavelength scanning Fourier ptychographic diffraction tomography (wsFPDT). Instead of using angularly-variable illuminations, in wsFPDT, the sample is illuminated by on-axis wavelength-variable illuminations, ranging from 430 to 1200 nm. The corresponding under-sampled diffraction patterns are recorded, and then an iterative ptychographic reconstruction procedure is applied to fill the spectrum of the three-dimensional (3D) scattering potential to recover the sample’s 3D refractive index (RI) distribution. The wavelength-scanning scheme not only eliminates the need for mechanical motion during image acquisition and precise registration of the raw images but secures a quasi-uniform, pixel-super-resolved imaging resolution across the entire imaging FOV. With wsFPDT, we demonstrate the high-throughput, billion-voxel 3D tomographic imaging results with a half-pitch lateral resolution of 775 nm and an axial resolution of 5.43 μm across a large FOV of 29.85 mm2 and an imaging depth of >200 μm. The effectiveness of the proposed method was demonstrated by imaging various types of samples, including micro-polystyrene beads, diatoms, and mouse mononuclear macrophage cells. The unique capability to reveal quantitative morphological properties, such as area, volume, and sphericity index of single cell over large cell populations makes wsFPDT a powerful quantitative and label-free tool for high-throughput biological applications.

Three-dimensional microscope skill acquisition: A randomised controlled study comparing two-dimensional laboratory microscope training, video gaming and virtual reality gaming

IntroductionFine microsurgical motor skill acquisition can be challenging. With increasing technological innovation the method of microsurgical skills acquisition may change. Studies show that laboratory-based microsurgical training programs on a 2D microscope significantly improves novice’s microsurgical skill acquisition. However, are these skills transferable to a 3D microscope or is gaming agility more important? We present a randomised control trial of three interventions, namely laboratory tabletop microscope training (LM), high-fidelity video gaming (Sony PlayStation 4 console) (VG) and high-fidelity virtual reality gaming (Sony PlayStation VR console) (VR) versus a control group. MethodsForty novice medical students were block randomised to four groups: control (no intervention) n=10, LM n=10, VG n=10 and VR n=10. Participants performed chicken femoral artery anastomosis using the Aesculap Aeos® 3D microscope platform at baseline and again after intervention. Performance was evaluated using a modified structured assessment of microsurgery skills (mSAMS) score, time taken to complete anastomosis and time for suture placement by two blinded independent assessors. ResultNo statistically significant difference was noted between the groups at baseline. There was a statistically significant improvement in the LM arm between baseline and post-training for mSAMS score and time for suture placement. In the VG, VR and control groups no statistically significant difference was observed. ConclusionOur study demonstrates that during early microsurgical training, an intense laboratory-based microsurgical training program significantly improves novice’s anastomotic performance on a 2D microscope, and these skills are transferable when a 3D anastomosis is carried out. However, focussed gaming had no significant effect, and the results were akin to the non-intervention group.

Unveiling Contact-Electrification Effect on Interfacial Water Oscillation.

Water is crucial for various physicochemical processes at the liquid-solid interfaces. In particular, the interfacial water, mediating the electric field and solvation effect along with the solid, corporately determine the electrochemical properties. Understanding the interaction between solid properties and the interface water holds significant importance in interfacial dynamics. However, the impact of alterations in the charged state of solid surfaces induced by contact electrification on interfacial water remains unknown. Here, the evolution of atomic-level resolution maps of hydration layers are reported on charged surfaces using 3D atomic force microscopy (3D-AFM). These findings demonstrate that electrostatic interactions can reinforce, distort, or collapse the characteristic structure of hydration layers. More importantly, these interactions exhibit interlayer differences and sample specificity in hydration layer structures of different substrates. In addition, similar oscillations of the hydration layer are observed at the electrochemical interface under different voltage biases. This suggests that contact-electrification has the potential to serve as a novel method for manipulating and regulating chemical reactions at the interface.

Laser-patterning bacterial nanocellulose for cell-controlled interaction

The interfacial topography of biomaterials has been identified as a major biophysical regulator of cell behavior and function, a role played through the interplay with biochemical cues. In this work, we demonstrate the potential of laser as a versatile technology for the direct fine-tuning of the topography of Bacterial nanocellulose (BNC) with bioinspired topographies and micropatterns on a cell size scale. Two lasers were used, with different wavelengths—IR (CO2, 10600 nm) and UV (tripled Nd: YVO4, 355 nm) —attempting to reproduce the Pitcher-plant topography and to create cell-contact guidance patterns, respectively. Different topographies with parallel grooves featuring a 20–300 μm period were generated on the BNC surface with high fidelity and reliability of the generated microstructures, as demonstrated by 3D optical profilometry and scanning electron microscopy. Moreover, it was demonstrated by X-ray photoelectron spectroscopy that laser processing does not result in detectable chemical modification of BNC. The developed anisotropic microstructures can control cell behavior, particularly regarding morphology, alignment, and spatial distribution. Thus, this proof-of-concept study on the high-resolution laser patterning of BNC opens new perspectives for the development of cell-modulating laser-engineered BNC interfaces, scaffolds, and other advanced medical devices, which can potentially broaden the application of BNC in the biomedical field.

Evidence for the Presence of Borrelia burgdorferi Biofilm in Infected Mouse Heart Tissues.

Borrelia burgdorferi, the bacterium responsible for Lyme disease, has been shown to form antimicrobial-tolerant biofilms, which protect it from unfavorable conditions. Bacterial biofilms are known to significantly contribute to severe inflammation, such as carditis, a common manifestation of Lyme disease. However, the role of B. burgdorferi biofilms in the development of Lyme carditis has not been thoroughly investigated due to the absence of an appropriate model system. In this study, we examined heart tissues from mice infected with B. burgdorferi for the presence of biofilms and inflammatory markers using immunohistochemistry (IHC), combined fluorescence in situ hybridization FISH/IHC, 3D microscopy, and atomic force microscopy techniques. Our results reveal that B. burgdorferi spirochetes form aggregates with a known biofilm marker (alginate) in mouse heart tissues. Furthermore, these biofilms induce inflammation, as indicated by elevated levels of murine C-reactive protein near the biofilms. This research provides evidence that B. burgdorferi can form biofilms in mouse heart tissue and trigger inflammatory processes, suggesting that the mouse model is a valuable tool for future studies on B. burgdorferi biofilms.

Comparison of misfit and roughness of CAD-CAM ZrO, selective laser sintered CoCr and preformed Ti implant abutment crowns

BackgroundMarginal misfit and surface roughness of customized implant abutments is critical for restorative success. However, little is known about the comparison of misfit and surface roughness of CAD-CAM Zirconium oxide (ZrO), selective laser melting (SLM) Cobalt Chrome (CoCr) and preformed abutments. The aim of the study is to investigate the relation of misfit and micro-roughness of selective laser melting (SLM), preformed and CAD-CAM implant abutments.MethodsThirty internal connection, endosseous dental implants (Ø 4.0 mm x 10 mm, Dentium) were mounted in Polymethyl methacrylate vertically. Ten preformed Titanium alloy (Ti) abutments with 1 mm soft tissue height and Ø 4.5 mm were included as controls. Ten each of Y-TZP and SLM-CoCr, abutment/crowns were fabricated using CAD-CAM milling (CAD-CAM-ZrO) and SLM techniques. Surface micro-roughness (Ra) of the fabricated implant abutment/crown was evaluated with a 3D optical non-contact microscope. All implant restorations were torqued to implants (30 Ncm) using a Tohnichi BTGE digital torque gauge and were analyzed with Bruker micro-CT (Skyscan 1173) to detect micro-gaps at pre-selected points at implant abutment interface. The Ra and misfit data were compared using ANOVA, Tukey-Kramer, Kruskal-Wallis test and Pearson correlation (p < 0.05).ResultsMean Ra among SLM CoCr abutments [0.88 (0.09) µm] were lower than CAD-CAM-ZrO and higher than preformed Ti abutments. Horizontal misfit among SLM-CoCr [45.43 (9.41) µm] and preformed Ti [36.87 (13.23) µm] abutments was not statistically different (p > 0.05). Misfit was significantly higher in Y-TZP samples compared to SLM-CoCr (p = 0.031) and preformed Ti abutments (p = 0.01). Preformed Ti abutments showed significantly lower misfit compared to SLM-CoCr abutments (p = 0.01). A positive linear correlation was observed between the surface roughness (Ra) and vertical misfit (r = 0.61, p < 0.05).ConclusionSLM CoCr abutments showed rough surface compared to preformed Ti abutments, while horizontal misfit was comparable among SLM-CoCr and preformed abutments. Misfit was significantly greater in Y-TZP abutments, compared to SLM and preformed abutments. SLM abutment fabrication technique needs further improvement to provide better fit and surface topography.

Marginal integrity and physicomechanical properties of a thermoviscous and regular bulk-fill resin composites.

To evaluate the marginal integrity (MI%) and to characterize specific properties of a thermoviscous bulk-fill resin composite, two regular bulk-fill resin composites, and a non-bulk-fill resin composite. VisCalor bulk (VBF), Filtek One Bulk Fill (OBF), and Aura Bulk Fill (ABF) were evaluated. Filtek Z250 XT (ZXT) was used as non-bulk-fill control. MI% was evaluated in standardized cylindrical cavities restored with the composites by using a 3D laser confocal microscope. The following properties were characterized: volumetric polymerization shrinkage (VS%), polymerization shrinkage stress (Pss), degree of conversion (DC%), microhardness (KHN), flexural strength (FS), and elastic modulus (EM). Data were analyzed by one-way and two-way ANOVA, and Tukey HSD post-hoc test (α = 0.05). VBF presented the highest MI% and the lowest VS% and Pss (p < 0.05). DC% ranged from 59.4% (OBF) to 71.0% (ZXT). ZXT and VBF presented similar and highest KHN than OBF and ABF (p < 0.05). ABF presented the lowest FS (p < 0.05). EM ranged from 5.5 GPa to 7.7 GPa, with the values of ZXT and VBF being similar and statistically higher than those of OBF and ABF (p < 0.05). Thermoviscous technology employed by VisCalor bulk was able to improve its mechanical behavior comparatively to regular bulk-fill resin composites and to contribute to a better marginal integrity in restorations built up in cylindrical cavities with similar geometry to a class I cavity as well. Although presenting overall better physicomechanical properties, Z250 XT presented the worst MI%. The marginal integrity, which is pivotal for the success of resin composite restorations, could be improved using VisCalor bulk-fill. The worst MI% presented by Z250 XT reinforces that non-bulk-fill resin composites shall not be bulk-inserted in the cavity to be restored.

Actomyosin-mediated inhibition of synaptic vesicle release under CB1R activation

Long-term synaptic plasticity is critical for adaptive function of the brain, but presynaptic mechanisms of functional plasticity remain poorly understood. Here, we show that changes in synaptic efficacy induced by activation of the cannabinoid type-1 receptor (CB1R), one of the most widespread G-protein coupled receptors in the brain, requires contractility of the neuronal actomyosin cytoskeleton. Specifically, using a synaptophysin-pHluorin probe (sypH2), we show that inhibitors of non-muscle myosin II (NMII) ATPase as well as one of its upstream effectors Rho-associated kinase (ROCK) prevent the reduction of synaptic vesicle release induced by CB1R activation. Using 3D STORM super-resolution microscopy, we find that activation of CB1R induces a redistribution of synaptic vesicles within presynaptic boutons in an actomyosin dependent manner, leading to vesicle clustering within the bouton and depletion of synaptic vesicles from the active zone. We further show, using sypH2, that inhibitors of NMII and ROCK specifically restore the release of the readily releasable pool of synaptic vesicles from the inhibition induced by CB1R activation. Finally, using slice electrophysiology, we find that activation of both NMII and ROCK is necessary for the long-term, but not the short-term, form of CB1R induced synaptic plasticity at excitatory cortico-striatal synapses. We thus propose a novel mechanism underlying CB1R-induced plasticity, whereby CB1R activation leads to a contraction of the actomyosin cytoskeleton inducing a reorganization of the functional presynaptic vesicle pool, preventing vesicle release and inducing long-term depression.

Fluorescence Lifetime Multiplexing (FLEX) for simultaneous high dimensional spatial biology in 3D

Immunohistochemistry is a crucial method for detecting specific proteins within tissue samples, yet constrained to one biomarker per tissue section. Multiplexed immunofluorescence, while allowing simultaneous visualization of multiple proteins, faces limitations in the number of simultaneous fluorescent labels due to spectral overlap. Although cyclic immunofluorescence techniques have successfully broadened antibody staining capacities in a single tissue sample, they are plagued by time-consuming and labor-intensive procedures, sample degradation risks, and inability to scale beyond thin sections. In this study, we introduce the use of 3D confocal Fluorescence Lifetime Imaging Microscopy as a high-throughput, multiplexed immunofluorescence platform that can differentiate 11 or more biomarkers in 3D tissue volumes. Leveraging both spectral and lifetime information, this approach allows for practical spatial biology in thin sections that can readily scale to larger volumes of tissue. We believe that this highly multiplexed and versatile biomarker imaging platform will significantly expedite cancer research and enable new translational approaches in the future.

Membrane-Active Antibiotics Affect Domains in Bacterial Membranes as the First Step of Their Activity.

The need to combat antimicrobial resistance is becoming more and more pressing. Here we investigate the working mechanism of a small cationic agent, N-alkylamide 3d, by conventional and high-speed atomic force microscopy. We show that N-alkylamide 3d interacts with the membrane of Staphylococcus aureus, where it changes the organization and dynamics of lipid domains. After this initial step, supramolecular structures of the antimicrobial agent attach on top of the affected membrane gradually, covering it entirely. These results demonstrate that lateral domains in the bacterial membranes might be affected by small antimicrobial agents more often than anticipated. At the same time, we show a new dual-step activity of N-alkylamide 3d that not only destroys the lateral membrane organization but also effectively covers the whole membrane with aggregates. This final step could render the membrane inaccessible from the outside and possibly prevent signaling and waste disposal of living bacteria.

Automated cell lineage reconstruction using label-free 4D microscopy.

Patterns of lineal descent play a critical role in the development of metazoan embryos. In eutelic organisms that generate a fixed number of somatic cells, invariance in the topology of their cell lineage provides a powerful opportunity to interrogate developmental events with empirical repeatability across individuals. Studies of embryonic development using the nematode Caenorhabditis elegans have been drivers of discovery. These studies have depended heavily on high-throughput lineage tracing enabled by 4D fluorescence microscopy and robust computer vision pipelines. For a range of applications, computer-aided yet manual lineage tracing using 4D label-free microscopy remains an essential tool. Deep learning approaches to cell detection and tracking in fluorescence microscopy have advanced significantly in recent years, yet solutions for automating cell detection and tracking in 3D label-free imaging of dense tissues and embryos remain inaccessible. Here, we describe embGAN, a deep learning pipeline that addresses the challenge of automated cell detection and tracking in label-free 3D time-lapse imaging. embGAN requires no manual data annotation for training, learns robust detections that exhibits a high degree of scale invariance, and generalizes well to images acquired in multiple labs on multiple instruments. We characterize embGAN's performance using lineage tracing in the C. elegans embryo as a benchmark. embGAN achieves near-state-of-the-art performance in cell detection and tracking, enabling high-throughput studies of cell lineage without the need for fluorescent reporters or transgenics.