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The rise of tip-based optical force microscopy has transformed nanoscale optical and chemical imaging, enabling sub-10 nm spatial resolution by integrating optical excitation with atomic force microscopy. Photoinduced force microscopy (PiFM) stands out as a key technique, with applications in single-molecule imaging, Raman spectroscopy, and near-field electromagnetic characterization. However, the contrast mechanisms underlying homodyne PiFM, particularly in the infrared regime, remain underexplored. This study provides novel insights into the interpretation of PiF signals, focusing on homodyne IR-PiFM. Contrary to previous assumptions, we demonstrate that the linear dependence of the PiF signal on sample thickness in homodyne mode originates from global photoacoustic forces rather than localized photothermal effects. By minimizing global interactions through power reduction and tip–sample proximity, we achieve a spatial resolution of 11 nm, comparable to heterodyne PiFM. Our findings reveal that both homodyne and heterodyne modes are fundamentally sensitive to tip-enhanced near-field optical intensity, with similar dependencies on sample thickness. This work advances the understanding of the contrast mechanism of PiFM and demonstrates that both homodyne and heterodyne modes can achieve high-resolution imaging, paving the way for broader applications in nanoscale optical and chemical analysis. [ABSTRACT FROM AUTHOR]

We report a model to predict equilibrium density profiles for different shaped colloids in two-dimensional liquid, nematic, and crystal states in nonuniform external fields. The model predictions are validated against Monte Carlo simulations and optical microscopy experiments for circular, square, elliptical, and rectangular colloidal particles in AC electric fields between parallel electrodes. The model to predict the densities of all states of different shaped particles is based on a balance of the local quasi-2D osmotic pressure against a compressive force due to induced dipole-field interactions. The osmotic force balance employs equations of state for hard ellipse liquid, nematic, and crystal state osmotic pressures, which are extended to additional particle shapes. The resulting simple analytical model is shown to accurately predict particle densities within liquid, liquid crystal, and crystal states for a broad range of particle shapes, system sizes, and field conditions. These findings provide a basis for quantitative design and control of fields to assemble and reconfigure colloidal particles in interfacial materials and devices. [ABSTRACT FROM AUTHOR]

The article discusses periodontal and implant surgery, which refers to a refinement of a surgical technique by which normal vision is enhanced through magnification. Topics include technical advantages of utilizing the operating microscope (OM) in periodontics, one of the main indications for periodontal and implant-related surgery, and concern on adopting a microsurgical approach.

In this study, a novel optical super-resolution imaging technique, grating and deep neural network assisted super-resolution microscopy, is proposed. The technique utilizes a sub-wavelength grating, placed between the sample and the microscope objective, to convert the evanescent waves of a sample surface into propagating waves, allowing more high spatial-frequency information of the sample to be detected in the far field. Then, the far-field image of the sample is captured and trained end-to-end with a customized deep neural network model to heuristically reconstruct a clear image of the sample with structural features smaller than λ/3. Compared with the existing super-resolution imaging techniques, the proposed technique has the advantages of label-free, large field of view, one-time direct imaging, and white light illumination and observation in an atmospheric environment. Moreover, it has the flexibility to replace raster and network rendering components according to specific inspection requirements to meet diverse application scenarios. [ABSTRACT FROM AUTHOR]

We present a comprehensive guide to light-sheet microscopy (LSM) to assist scientists in navigating the practical implementation of this microscopy technique. Emphasizing the applicability of LSM to image both static microscale and nanoscale features, as well as diffusion dynamics, we present the fundamental concepts of microscopy, progressing through beam profile considerations, to image reconstruction. We outline key practical decisions in constructing a home-built system and provide insight into the alignment and calibration processes. We briefly discuss the conditions necessary for constructing a continuous 3D image and introduce our home-built code for data analysis. By providing this guide, we aim to alleviate the challenges associated with designing and constructing LSM systems and offer scientists new to LSM a valuable resource in navigating this complex field. [ABSTRACT FROM AUTHOR]

Purpose: Marginal bone resorption (MBR) around dental implants may sometimes be a self-limiting condition due to balancing immunologic reactions against utilized materials rather than a progressive bacterial infection. Contrary to previous assumptions from ligature-induced experimental peri-implantitis studies, a recent 8-week experiment by the present authors showed that marginal ligatures trigger an inflammatory immune response, resulting in bone resorption around implants in the absence of plaque. The present study aimed to investigate whether this inflammatory/immunologic reaction attenuates or progresses toward implant failure after a longer healing time (12 weeks). Materials and Methods: Sterile silk ligatures were placed around the top of titanium (Ti) implants and compressed against the femoral cortical bone plate of six rabbits. A nonligated implant was used as a control. After 12 weeks of submerged healing, ground sections of implants and surrounding tissues were investigated with light microscopy. The marginal soft tissues were also analyzed using selected quantitative polymerase chain reaction (qPCR) markers. Results: Histologically, the ligatures were outlined by immune cells, including multinucleated giant cells (MNGCs), with adjacent fibrous encapsulation and resorbed peripheral bone that contrasted from the osseointegrated nonligated control implants. The difference in expression of qPCR markers was not significant, but > two-fold upregulation of markers CDIIb, IL1β, ARG1, NCF1, and CD4 and > twofold downregulation of CD8 indicated a mild, focal inflammatory/immune response against the ligatures compared to controls, with upregulation of M1 and M2 macrophages, neutrophils, and helper T-cells as well as downregulation of killer T cells. Further, the bone formation markers OC and ALPL were > two-fold downregulated (consistent with the lack of osseointegration of the ligatures) compared to control implants. Conclusions: Marginal silk ligatures trigger an inflammatory/immune response and aseptic bone resorption around implants. Compared to the previous 8-week study, the inflammatory reaction against the silk appears to attenuate with time, with only a mild persisting inflammation that may block osseointegration; instead, a fibrous tissue encapsulation-type reaction is maintained. This may explain why traditional ligature experiments have required regular exchange of ligatures for the bone resorption to progress. [ABSTRACT FROM AUTHOR]

Gram staining has been a frequently used staining protocol in microbiology. It is vulnerable to staining artifacts due to, e.g., operator errors and chemical variations. Here, we introduce virtual Gram staining of label-free bacteria using a trained neural network that digitally transforms dark-field images of unstained bacteria into their Gram-stained equivalents matching bright-field image contrast. After a one-time training, the virtual Gram staining model processes an axial stack of dark-field microscopy images of label-free bacteria (never seen before) to rapidly generate Gram staining, bypassing several chemical steps involved in the conventional staining process. We demonstrated the success of virtual Gram staining on label-free bacteria samples containing Escherichia coli and Listeria innocua by quantifying the staining accuracy of the model and comparing the chromatic and morphological features of the virtually stained bacteria against their chemically stained counterparts. This virtual bacterial staining framework bypasses the traditional Gram staining protocol and its challenges, including stain standardization, operator errors, and sensitivity to chemical variations.

Here, we present a protocol for using Myotally, a user-friendly software for fast, automated quantification of muscle fiber size, number, and central nucleation from immunofluorescent stains of mouse skeletal muscle cross-sections. We describe steps for installing the software, preparing compatible images, finding the file path, and selecting key parameters like image quality and size limits. We also detail optional features, such as measuring mean fluorescence. By automating these traditionally labor-intensive processes, Myotally improves research efficiency and data consistency.

As light sheet fluorescence microscopy (LSFM) becomes widely available, reconstruction of time-lapse imaging will further our understanding of complex biological processes at cellular resolution. Here, we present a comprehensive workflow for in toto capture, processing, and analysis of multi-view LSFM experiments using the ex vivo mouse embryo as a model system of development. Our protocol describes imaging on a commercial LSFM instrument followed by computational analysis in discrete segments, using open-source software. Quantification of migration and morphodynamics is included. For complete details on the use and execution of this protocol, please refer to Dominguez et al.1.

Cryogenic transmission electron microscopy (cryo-TEM) combined with single particle analysis (SPA) is an emerging imaging approach for soft materials. However, the accuracy of SPA-reconstructed nanostructures, particularly those formed by synthetic polymers, remains uncertain due to potential packing heterogeneity of the nanostructures. In this study, the combination of molecular dynamics (MD) simulations and image simulations is utilized to validate the accuracy of cryo-TEM 3D reconstructions of self-assembled polypeptoid fibril nanostructures. Using CryoSPARC software, image simulations, 2D classifications, ab initio reconstructions, and homogenous refinements are performed. By comparing the results with atomic models, the recovery of molecular details is assessed, heterogeneous structures are identified, and the influence of extraction location on the reconstructions is evaluated. These findings confirm the fidelity of single particle analysis in accurately resolving complex structural characteristics and heterogeneous structures, exhibiting its potential as a valuable tool for detailed structural analysis of synthetic polymers and soft materials.

Dynamin 1 (Dyn1) GTPase, a principal driver of membrane fission during synaptic endocytosis, self-assembles into short mechanoactive helices cleaving the necks of endocytic vesicles. While structural information about Dyn1 helix is abundant, little is known about the nanoscale dynamics of the helical scaffolding at the moment of fission, complicating mechanistic understanding of Dyn1 action. To address the role of the helix dynamics in fission, we used High-Speed Atomic Force Microscopy (HS-AFM) and fluorescence microscopy to track and compare the spatiotemporal characteristics of the helices formed by wild-type Dyn1 and its K44A mutant impaired in GTP hydrolysis on minimal lipid membrane templates. In the absence of nucleotide, membrane-bound WTDyn1 and K44ADyn1 self-assembled into tubular protein scaffolding of similar diameter encaging the lipid bilayer. In both cases, the GTP addition caused scaffold constriction coupled with formation of 20 to 30 nm nanogaps in the protein coverage. While both proteins reached scaffold diameters characteristic for membrane superconstriction causing fission, the fission was detected only with WTDyn1. We associated the fission activity with the dynamic evolution of the nanogaps: K44ADyn1 gaps were static, while WTDyn1 gaps actively evolved via repetitive nonaxisymmetric constriction-bending deformations caused by localized GTP hydrolysis. Modeling of the deformations implicated filament twist as an additional deformation mode which combines with superconstriction to facilitate membrane fission. Our results thus show that the dynamics of the Dyn1 helical scaffold goes beyond radial constriction and involves nonaxisymmetric deformations, where filament twist emerges as a critical driver of membrane fission.