Conventional Microscopy Research Articles

Super‐Resolution Imaging in Collagen‐Abundant Thick Tissues

Expansion microscopy (ExM) has gained increasing popularity for 3D ultrastructural imaging of cultured cells and tissue slices at nanoscale resolution using conventional microscopes via physical expansion of biological tissues. However, its application to collagen‐abundant thick tissues is still challenging. Herein, a new method, collagen ExM (ColExM), optimized for expanding tissues containing more than 70% collagen, is demonstrated. ColExM succeeds in 4.5‐fold linear expansion with minimal structural distortion of corneal and skin tissues. It is compatible with immunostaining, allowing super‐resolution visualization of 3D neural structures innervating hair follicles, corneas, and pancreatic tumors with high stromal collagen content. The method succeeds in identifying individual mitochondria and previously unrecognized dendritic spinelike structures of corneal nerves. It also enables fine mapping of structural rearrangement of tight junctions and actin cytoskeletons. Therefore, ColExM can facilitate the exploration of 3D nanoscale structures in collagen‐rich tissues.

Comprehensive study of optical contrast, reflectance, and Raman spectroscopy of multilayer graphene

Graphene research has developed quite rapidly partially because even a monatomic layer can be visualized with a conventional optical microscope. Although optical properties of multilayer graphene such as optical contrast, reflectance (R), and Raman scattering have been well studied, they are studied independently and the thickness dependence is limited to a rather thin region. In this paper, the evolution of optical properties by thickness from monolayer to multilayer graphene up to 107 nm thick is studied comprehensively. The empirically known change of color of multilayer graphene is confirmed from the R, G and B intensities extracted from the optical images. It is also found that, as far as R for visible light is concerned, multilayer graphene is not necessarily considered as a layered material, and the refractive index for monolayer graphene is applicable even for the thickest multilayer graphene flake in this study. On the other hand, the layered structure and Raman scattering at each layer are essential to reproduce the G-band intensity of Raman scattering (I(G)). Not only the multiple reflection but also the interference of scattered Raman light should be considered for I(G) of multilayer graphene thicker than ∼30 nm.

A comparison of super-resolution microscopy techniques for imaging tightly packed microcolonies of an obligate intracellular bacterium.

Conventional optical microscopy imaging of obligate intracellular bacteria is hampered by the small size of bacterial cells, tight clustering exhibited by some bacterial species and challenges relating to labelling such as background from host cells, a lack of validated reagents, and a lack of tools for genetic manipulation. In this study we imaged intracellular bacteria from the species Orientia tsutsugamushi (Ot) using five different fluorescence microscopy techniques: standard confocal, Airyscan confocal, instant Structured Illumination Microscopy (iSIM), three-dimensional Structured Illumination Microscopy (3D-SIM) and Stimulated Emission Depletion Microscopy (STED). We compared the ability of each to resolve bacterial cells in intracellular clumps in the lateral (xy) axis, using full width half maximum (FWHM) measurements of a labelled outer membrane protein (ScaA) and the ability to detect small, outer membrane vesicles external to the cells. We next compared the ability of each technique to sufficiently resolve bacteria in the axial (z) direction and found 3D-STED to be the most successful method for this. We then combined this approach with a custom 3D cell segmentation and analysis pipeline using the open-source, deep learning software, Cellpose to segment the cells and subsequently the commercial software Imaris to analyze their 3D shape and size. Using this combination, we demonstrated differences in bacterial shape, but not their size, when grown in different mammalian cell lines. Overall, we compare the advantages and disadvantages of different super-resolution microscopy techniques for imaging this cytoplasmic obligate intracellular bacterium based on the specific research question being addressed.

Enhanced SERS performance of gold nanoparticle assemblies on a cysteine-mutant Tobacco mosaic virus scaffold

The employment of biomolecular templates for the synthesizing nanohybrid constructs is expanding, driven by their prospective uses in biosensing and biomedical fields. Gold nanoparticles (AuNPs) and, in particular, their assemblies are especially preferred for Surface Enhanced Raman Spectroscopy (SERS) because of their ability to amplify Raman signals through localized surface plasmon resonances, thus enabling the detection of molecules at exceedingly low concentrations. Our investigative approach is dedicated to studing the role of cysteine mutants in the nucleation and assembly of AuNPs on Tobacco mosaic virus (TMV-C, carrying T158C mutation) scaffolds. Employing biomineralization and direct grafting methods, we synthesized these nanohybrids and examined them using conventional transmission electron microscopy (TEM), in situ liquid TEM, and fluorescence spectroscopy. We demonstrated that the syntheses obtained with TMV-C give denser plasmonic nanostructures, with is ideal for SERS applications. The SERS performances of these novel nanohybrids with various AuNPs sizes and densities were evaluated, revealing excellent enhancement factors for the nanosystems obtained by direct grafting that highlight their potential for the detection of biomolecules in solution.

Role of the aging procedure on the ω + β microstructure in the metastable β Ti-30Nb-3Fe alloy

This report investigated the effect of low-temperature aging on the ω + β microstructure in the Ti-30Nb-3Fe alloy (wt%). Samples were subjected to several aging procedures. The ω + β microstructure remained quite stable under conventional transmission electron microscopy across most temperature/time regimes. Only the double aging treatment led to the growth of ω particles and an increased in Vickers hardness, indicating that the pre-aging treatment was necessary to promote rapid ω growth in the subsequent treatment. Furthermore, the early stages of solute partitioning that accompanies ω growth did not drastically increase hardness, suggesting that no embrittlement occurred.

Pathology students' perceptions of virtual learning: A case study of students in Saudi Arabia.

Pathology laboratory classes are traditionally conducted using a conventional light microscope. The Coronavirus Disease 2019 (COVID-19) pandemic and recent technological advances necessitated remote learning through online classes using virtual slides (VS) instead of glass slides (GS). The purpose of this study was to gauge the perception of learning pathology using virtual slides (VS) as opposed to glass slides (GS) for medical students in Saudi Arabia. This study would help modify teaching methods with the advancement of the application of newer methods in online teaching. This two-phased study evaluated learning outcomes and perceptions in pathology online education for medical students. Using a questionnaire, Phase one analyzed second and third-year students' perceptions of the teaching methods after an online pathology course. Phase Two assessed the learning outcomes of third-year students during online practical sessions using a pretest and post-test design. Statistical data were collected using a simple additive approach. Statistical tools were used to determine the factors affecting students' perceptions. The accessibility of VS at any possible time, location, or device was the most advantageous trait of virtual learning (mean = 2.94±0.9). Students agreed the least with virtual slides as the only optimal method of learning pathology (mean = 2.25±0.9). Most enjoyed the virtual lab experience (51.7%) but still prefer both laboratory-GS and virtual-VS classes (83.5%). VS had the benefit of accessibility and efficiency. The acceptance of VS was significantly affected by the orientation prior to the online class. Findings showed that VS cannot completely replace GS and more aspects such as technical difficulties and prior VS experience should be explored.

A Framework of Multi-View Machine Learning for Biological Spectral Unmixing of Fluorophores with Overlapping Excitation and Emission Spectra.

The accuracy in assigning fluorophore identity and abundance, termed spectral unmixing, in biological fluorescence microscopy images remains challenging due to the unavoidable and significant overlap in emission spectra among fluorophores. In conventional laser scanning confocal spectral microscopy, fluorophore information is acquired by recording emission spectra with a single combination of discrete excitation wavelengths. As a matter of fact, organic fluorophores have not only unique emission spectral signatures but also have unique and characteristic excitation spectra. In this paper, we propose a generalized multi-view machine learning approach, which makes use of both excitation and emission spectra to greatly improve the accuracy in differentiating multiple highly overlapping fluorophores in a single image. By recording emission spectra of the same field with multiple combinations of excitation wavelengths, we obtain data representing these different views of the underlying fluorophore distribution in the sample. We then propose a framework of multi-view machine learning methods, which allows us to flexibly incorporate noise information and abundance constraints, to extract the spectral signatures of fluorophores from their reference images and to efficiently recover their corresponding abundances in unknown mixed images. Numerical experiments on simulated image data demonstrate the method's efficacy in improving accuracy, allowing for the discrimination of 100 fluorophores with highly overlapping spectra. Furthermore, validation on images of mixtures of fluorescently labeled E. coli demonstrates the power of the proposed multi-view strategy in discriminating fluorophores with spectral overlap in real biological images.

(Invited) Nano-Characterization of the Internal Morphology of Polymer Electrolyte Membranes with Cryogenic Scanning Transmission Electron Microscopy

Polymer membranes play important roles in electrochemical devices such as polymer electrolyte fuel cells. In recent years, there has been a growing interest in alkaline anion exchange membrane (AAEM) fuel cells as the alkaline environment offers a potential low-cost alternative to commercial proton exchange membrane (PEM) fuel cells [1]. Nonetheless, optimization of polymer properties such as ion conductivity and mechanical durability is still necessary to achieve commercially viable AAEM based fuel cells. Often, semicrystalline polymer backbones are selected for AAEM design, aiming to replicate the exceptional performance of Nafion in PEM fuel cells where the crystalline domains are believed to act as cross-linkers, mitigating excessive water uptake and mechanical degradation [2,3].Characterizing the local polymer morphology at the nanoscale is a crucial step in understanding how to optimize the design of AAEMs with enhanced durability and ion conductivity. Imaging polymers using conventional transmission electron microscopy (TEM) techniques is challenging however, due to their inherently-high beam sensitivity and low image contrast. Here, we demonstrate nanometer-resolution mapping of the ordering in polymer electrolyte membranes using cryogenic four-dimensional scanning transmission electron microscopy (cryo-4D-STEM). Conventionally used for mapping inorganic crystalline materials, the 4D-STEM technique has more recently also found applications in mapping semicrystalline, soft materials as well [4,5]. Here, using a model system of semicrystalline hydrogenated polynorbornene (hPN) AAEMs [6], we demonstrate the effects of polymer synthesis including molecular weight and thermal history on crystalline morphology and in turn on the polymer performance. Further, we compare the crystalline architecture of the hPN copolymers to that of Nafion 212 (Figure 1). Our results suggest that polymers with homogeneously distributed, nanometer-size crystalline domains lead to improved water management, a property directly related to the mechanical integrity of the polymer, with possible impacts on ion conductivity. More broadly, the results give new insights into how we can tune AAEM design in order to improve their performance.[1] Dekel, Journal of Power Sources, 375, 158-169. (2018).[2] Yang, et al. Chemical Reviews, 122(6), 6117-6321. (2022).[3] Mauritz & Moore, Chemical reviews, 104(10), 4535-4586. (2004).[4] Panova, et al. Nature materials, 18(8), 860-865. (2019).[5] Bustillo, et al. Accounts of chemical research, 54(11), 2543-2551. (2021).[6] Treichel, et al. Macromolecules, 53(19), 8509-8518. (2020).Figure 1: Cryo-4D-STEM crystallinity maps of hPN-co-iPrMe copolymers with varying syntheses and Nafion 212. We observe improvement in the water management of the copolymer membranes as the crystalline domains are reduced in volume and distributed homogeneously. The chemical structures of the polymers are listed below the crystallinity maps. Figure 1

Rapid and Comprehensive Screening for Urogenital and Gastrointestinal Schistosomiasis with Handheld Digital Microscopy Combined with Circulating Cathodic Antigen Testing.

Novel methods are required to aid the monitoring of schistosomiasis control and elimination initiatives through mass drug administration. Portable digital and mobile phone microscopy is a promising tool for this purpose. This cross-sectional study evaluated the diagnostic operating characteristics of a converted mobile phone microscope (the SchistoScope) for the detection of Schistosoma haematobium eggs, as determined by community-based field workers and expert microscopists, compared with a field gold standard of light microscopy. Three hundred sixty-five urine samples were evaluated by conventional light microscopy, with 49 (13.4%) positive for S. haematobium. Compared with light microscopy, the sensitivity and specificity of S. haematobium detection by field microscopists trained to use the SchistoScope were 26.5% (95% CI: 14.9-41.1%) and 98.4% (95% CI: 96.3-99.5%), respectively. The sensitivity and specificity of S. haematobium detection by expert microscopists using the SchistoScope was 74% (95% CI: 59.7-85.4%) and 98.1% (95% CI: 95.9-99.3%), respectively, compared with light microscopy. The sensitivity rose to 96.1% and 100% when evaluating for egg counts greater than five and 10 eggs per 10 mL, respectively. A point-of-care circulating cathodic anion (POC CCA) test was used to evaluate Schistosoma mansoni; however, there were too few positive samples to reliably comment on diagnostic characteristics. This study demonstrated that a "urine-only" approach to rapidly screen for schistosomiasis at the point of sample collection can be conducted with mobile phone microscopy (S. haematobium) coupled with POC CCA (S. mansoni). Such an approach may aid in streamlined schistosomiasis control and elimination initiatives.

Parameter-free super-resolution structured illumination microscopy via a physics-enhanced neural network.

With full-field imaging and high photon efficiency advantages, structured illumination microscopy (SIM) is one of the most potent super-resolution (SR) modalities in bioscience. Regarding SR reconstruction for SIM, spatial domain reconstruction (SDR) has been proven to be faster than traditional frequency domain reconstruction (FDR), facilitating real-time imaging of live cells. Nevertheless, SDR relies on high-precision parameter estimation for reconstruction, which tends to suffer from low signal-to-noise ratio (SNR) conditions and inevitably leads to artifacts that seriously affect the accuracy of SR reconstruction. In this Letter, a physics-enhanced neural network-based parameter-free SDR (PNNP-SDR) is proposed, which can achieve SR reconstruction directly in the spatial domain. As a result, the peak-SNR (PSNR) of PNNP-SDR is improved by about 4 dB compared to the cross-correlation (COR) SR reconstruction; meanwhile, the reconstruction speed of PNNP-SDR is even about five times faster than the fast approach based on principal component analysis (PCA). Given its capability of achieving parameter-free imaging, noise robustness, and high-fidelity and high-speed SR reconstruction over conventional SIM microscope hardware, the proposed PNNP-SDR is expected to be widely adopted in biomedical SR imaging scenarios.

Development of the Interosseous Muscles of the Human Hand: Morphological and Functional Aspects of the Terminal Insertion.

To date, there have been no studies conducted on the development of interosseous muscles (IO) in the human hand. This study aimed to investigate the development of these muscles in order to clarify their terminal insertions and their relationship with the metacarpophalangeal joints. Serial sections of 25 human specimens (9 embryos and 16 fetuses) between the 7th and 14th weeks of development, sourced from the Collection of the Department of Anatomy and Embryology at UCM Faculty of Medicine, were analyzed bilaterally using a conventional optical microscope. Our findings revealed that, during the 7th week of development, the metacarpophalangeal interzone mesenchyme extended into the extensor apparatus of the fingers. Furthermore, we observed that the joint capsule and the tendon of the IO derive from the articular interzone mesenchyme. By the end of the 7th week, corresponding to Carnegie stage 21, the myotendinous junction appeared, initiating cavitation of the metacarpophalangeal joint. During the fetal period, the terminal insertions of the IO were identified: both the dorsal interosseous (DI) and palmar interosseous (PI) muscles insert into the metacarpophalangeal joint capsule and establish a connection with the volar plate located at the base of the proximal phalanx and the extensor apparatus. Some muscle fibers also attach to the joint capsule at the level of the proximal synovial cul-de-sac. The functional implications of these findings are discussed within this work. This study provides the first detailed description of the development of the interosseous muscles in the human hand.

Scanning Near-Field Optical Microscopy: Recent Advances in Disordered and Correlated Disordered Photonics

Disordered and correlated disordered photonic materials have emerged in the past few decades and have been rapidly proposed as a complementary alternative to ordered photonics. These materials have thrived in the field of photonics, revealing the considerable impact of disorder with and without structural correlations on the scattering, transport, and localization of light in matter. Scanning near-field optical microscopy (SNOM) has proven to be a fundamental tool for the study of the interaction between light and matter at the nanoscale in such systems, allowing for the investigation of optical properties and local electromagnetic fields with extremely high spatial resolution, surpassing the diffraction limit of conventional optical microscopy. In this review, the most important and recent advances obtained for disordered and correlated disordered luminescent structures by means of the aperture SNOM technique are addressed, showing how it allows the tailoring of local density of states (LDOS), as well as providing access to statistical analysis for multi-resonance disordered and hyperuniform disordered structures at telecom wavelengths.

Waveguide-based microscope slide for label-free high-resolution imaging

Waveguide-based total internal reflection fluorescence microscopy has been widely adopted due to its excellent signal-to-noise ratio over a large field of view. However, with the increasing demand for label-free imaging, waveguide-based evanescent light scattering microscopy (ESM) has also garnered significant attention. Here, we present a low-cost waveguide-based microscope slide that offers easier integration with conventional optical microscopy. This microscope slide uses an incoherent light source coupled to a lithium tantalate (LT) planar waveguide to generate an evanescent light that illuminates samples located within a few hundred nanometers of the waveguide surface. We perform its application for imaging chromium nanoholes and polystyrene nanospheres, demonstrating its label-free, high-resolution, high-contrast imaging performance. LT waveguide microscope slides provide a simple and effective solution for ESM.

Deep learning and defocus imaging for determination of three-dimensional position and orientation of microscopic objects

We present a method to determine the three-dimensional position and orientation of microscopic, non-spherical objects in microfluidic and laboratory-on-a-chip systems observed through conventional optical microscopes. The method is based on the combination of the General Defocusing Particle Tracking technique [Barnkob et al., “General defocusing particle tracking,” Lab Chip 15, 3556–3560 (2015)] and deep learning. It requires minimal input from the user, is suitable for real-time applications, and can be applied to any microscopic object with an approximately ellipsoidal shape, such as unicellular swimming organisms, red blood cells, or spheroidal colloids. The main challenge is linked to the construction of suitable training datasets for the neural network. We provide a procedure generally valid for active microswimmers and discuss possible strategies for other types of objects. An implementation using the Visual Geometry Group convolutional neural network (VGG-16) is presented and tested on synthetic images with different backgrounds and noise levels. The same implementation is used to track the position and orientation of different specimens of the heterotrophic ciliate Euplotes Vannus in free-swimming motion. The measurements were performed with a 10 × objective over a depth of 800 μm with an average estimated uncertainty in the orientation angles of 9.0%.

Comparison of Foldscope to optical microscope to identify basic histology

Introduction: The teaching of basic histology is crucial in the training of healthcare professionals. Traditionally, the optical microscope is used, but it has limitations such as cost and the need for specific infrastructure. The Foldscope (FS), a low-cost and easy-to-handle tool, emerges as a promising alternative. Objective: To validate the efficacy of the FS in teaching basic histology for healthcare training, comparing it with the traditional method of binocular optical microscopy. Method: We used a kit of prepared histology slides, first examined with an optical microscope and then with the FS. We used a smartphone for image capture, ensuring comparative accuracy between the methods. Results: The FS could observe structures seen with a 10x objective in conventional microscopy but with less sharpness and some peripheral distortions. The applicability of the FS proved effective in observing structures of various tissues, such as the thyroid, arteries, veins, and neurons, although it has limitations for teaching muscular and testicular tissues. Conclusion: The FS is an innovative educational tool that bridges the gap between theory and practice in histology teaching. It is a complementary resource to the optical microscope, effectively identifying structures with about 100x magnification. However, it does not completely replace the optical microscope due to its limitations in magnification and image quality.

Multiplexed spatial transcriptomics methods and the application of expansion microscopy.

While spatial transcriptomics has undeniably revolutionized our ability to study cellular organization, it has driven the development of a great number of innovative transcriptomics methods, which can be classified into in situ sequencing (ISS) methods, in situ hybridization (ISH) techniques, and next-generation sequencing (NGS)-based sequencing with region capture. These technologies not only refine our understanding of cellular processes, but also open up new possibilities for breakthroughs in various research domains. One challenge of spatial transcriptomics experiments is the limitation of RNA detection due to optical crowding of RNA in the cells. Expansion microscopy (ExM), characterized by the controlled enlargement of biological specimens, offers a means to achieve super-resolution imaging, overcoming the diffraction limit inherent in conventional microscopy and enabling precise visualization of RNA in spatial transcriptomics methods. In this review, we elaborate on ISS, ISH and NGS-based spatial transcriptomic protocols and on how performance of these techniques can be extended by the combination of these protocols with ExM. Moving beyond the techniques and procedures, we highlight the broader implications of transcriptomics in biology and medicine. These include valuable insight into the spatial organization of gene expression in cells within tissues, aid in the identification and the distinction of cell types and subpopulations and understanding of molecular mechanisms and intercellular changes driving disease development.

Predicting DNA damage response in non-small cell lung cancer organoids via simultaneous label-free autofluorescence multiharmonic microscopy

The DNA damage response (DDR) is a fundamental readout for evaluating efficacy of cancer therapeutics, many of which target DNA associated processes. Current techniques to evaluate DDR rely on immunostaining for phosphorylated histone H2AX (γH2AX), which is an indicator of DNA double-strand breaks. While γH2AX immunostaining can provide a snapshot of DDR in fixed cell and tissue samples, this method is technically cumbersome due to temporal monitoring of DDR requiring timepoint replicates, extensive assay development efforts for 3D cell culture samples such as organoids, and time-consuming protocols for γH2AX immunostaining and its evaluation. The goal of this current study is to reduce overall burden on assay duration and development in non-small cell lung cancer (NSCLC) organoids by leveraging label-free multiphoton imaging. In this study, simultaneous label-free autofluorescence multiharmonic (SLAM) microscopy was used to provide rich intracellular information based on endogenous contrasts. SLAM microscopy enables imaging of live samples eliminating the need to generate sacrificial sample replicates and has improved image acquisition in 3D space over conventional confocal microscopy. Predictive modeling between label-free SLAM microscopy and γH2AX immunostained images confirmed strong correlation between SLAM image features and γH2AX signal. Across multiple DNA targeting chemotherapeutics and multiple patient-derived NSCLC organoid lines, the optical redox ratio and third harmonic generation channels were used to robustly predict DDR. Imaging via SLAM microscopy can be used to more rapidly predict DDR in live 3D NSCLC organoids with minimal sample handling and without labeling.

The Alphabet Soup of Microscopy: An Introduction to Advanced Imaging Techniques. Part I: Super-Resolution’s STED, SIM, SMI, and SMLM

Abstract As technology advances, the field of microscopy offers unique opportunities for scientific exploration. However, the complex terminology used to describe these cutting-edge techniques often appears as a jumble of letters, akin to alphabet soup. Among the plethora of acronyms, some of the most prevalent are associated with super-resolution microscopy, including Stimulated Emission Depletion Microscopy (STED), Structured Illumination Microscopy (SIM), Spatially Modulated Illumination (SMI), and Single-Molecule Localization Microscopy (SMLM). These techniques push past the limitations of conventional light microscopy, achieving resolutions down to the tens of nanometers. This article aims to decode these common super-resolution techniques, fostering a deeper understanding of microscopy and igniting interest in their application.

Fast in-line failure analysis of sub-micron-sized cracks in 3D interconnect technologies utilizing acoustic interferometry

More than Moore technology is driving semiconductor devices towards higher complexity and further miniaturization. Device miniaturization strongly impacts failure analysis (FA), since it triggers the need for non-destructive approaches with high resolution in combination with cost and time efficient execution. Conventional scanning acoustic microscopy (SAM) is an indispensable tool for failure analysis in the semiconductor industry, however resolution and penetration capabilities are strongly limited by the transducer frequency. In this work, we conduct an acoustic interferometry approach, based on a SAM-setup utilizing 100 MHz lenses and enabling not only sufficient penetration depth but also high resolution for efficient in-line FA of Through Silicon Vias (TSVs). Accompanied elastodynamic finite integration technique-based simulations, provide an in-depth understanding concerning the acoustic wave excitation and propagation. We show that the controlled excitation of surface acoustic waves extends the contingency towards the detection of nm-sized cracks, an essential accomplishment for modern FA of 3D-integration technologies.

Orthogonal DNA Self-Assembly-Based Expansion Microscopy Platform for Amplified, Multiplexed Biomarker Imaging.

Expansion microscopy (ExM) facilitates nanoscale imaging under conventional microscopes, but it frequently encounters challenges such as fluorescence losses, low signal-to-noise ratio (SNR), and limited detection throughput. To address these issues, a method of orthogonal DNA self-assembly-based ExM (o-DAExM) platform is developed, which employs hybridization chain reaction instead of conventional fluorescence labeling units, showcasing signal amplification efficacy, enhancement of SNR, and expandable multiplexing capability at any stage of the ExM process. In this work, o-DAExM has been applied to compare with immunofluorescence-based ExM for cellular cytoskeleton imaging, and the resolved nanoscale spatial distributions of cytoskeleton show outstanding performance and reliability of o-DAExM. Furthermore, the study demonstrates the utility of o-DAExM in accurately revealing exosome heterogeneous information and multiplexed analysis of protein targets in single cells, which provides infinite possibilities in super-resolution imaging of cells and other samples. Therefore, o-DAExM offers a straightforward expansion and signal labeling method, highlighting future prospects to study nanoscale structures and functional networks in biological systems.