Search Results (142)

The mineralogical characteristics and formation mechanism of the green phase within moss agate remain debated. With large quantities of green mineral inclusions, the moss agate from Fuxin (China) provides an excellent natural laboratory for resolving these issues. This study presents petrographic, spectroscopic, scanning electron microscopy (SEM), and electron probe microanalysis (EPMA) data to determine the composition and texture of these green inclusions. The agate predominantly occurs in pores and fractures of andesitic breccias of the Lower Cretaceous Yixian Formation. Microscopic observations reveal various morphologies (filamentous, clustered, platy, globular, and irregular) and tones (light, dark, true, and yellowish green) of the inclusions. X-ray diffraction (XRD), infrared, Raman spectroscopic, and EPMA analyses demonstrate that the inclusions are rich in celadonite. Compositional or color zones are observed for both celadonite-rich regions and the agate. SEM imaging reveals euhedral celadonite crystals increase progressively in size across older-to-younger growth domains of each zone. The euhedral habits of celadonite, the systematic grain-size increase in celadonite-rich zones, and the zoned structure of the agate collectively suggest that the agate and celadonite within it precipitated from hydrothermal fluids. Full article

Hydroxyapatite coatings containing graphene oxide and palladium were hydrothermally deposited on a β-type Ti30Zr5Mo alloy in order to establish how the organization of graphene oxide and the incorporation of palladium influence the structural, electrochemical, and biological performance of the coating system. Six compositions—hydroxyapatite, hydroxyapatite with 1 wt% palladium, hydroxyapatite with 2 wt% palladium, hydroxyapatite/graphene oxide, and the corresponding graphene oxide composites containing 1 and 2 wt% palladium—were compared using X-ray diffraction, Fourier-transform infrared spectroscopy, Raman spectroscopy, X-ray photoelectron spectroscopy, scanning electron microscopy combined with energy-dispersive X-ray spectroscopy, electrochemical impedance spectroscopy, potentiodynamic polarization, cell-density analysis with A7R5 cells, and bioluminescence imaging of Staphylococcus aureus Xen36. The apatite framework was retained in every composition, and Raman spectroscopy confirmed that the carbon phase derived from graphene oxide survived hydrothermal processing. X-ray photoelectron spectroscopy of the graphene oxide coating containing 2 wt% palladium revealed oxygenated carbon environments and predominantly oxidized, oxygen-coordinated palladium species rather than metallic palladium. Electrochemical behavior depended strongly on composition. The hydroxyapatite coating containing 2 wt% palladium showed the lowest corrosion current density, 1.46 ± 0.02 µA cm⁻² (0.0127 ± 0.0002 mm year⁻¹), and a mean total resistance of 15.73 ± 0.13 kΩ cm², close to that of the unmodified hydroxyapatite coating (1.95 ± 0.04 µA cm⁻²; 16.52 ± 0.40 kΩ cm²). The hydroxyapatite/graphene oxide coating showed the weakest barrier behavior, with a corrosion current density of 4.21 ± 0.07 µA cm⁻² (0.0366 ± 0.0006 mm year⁻¹) and a mean total resistance of only 8.50 ± 0.15 kΩ cm². Microscopic and electrochemical evidence indicated that the expected barrier effect of graphene oxide was lost because the graphene oxide-containing layer was structurally heterogeneous. This layer contained folded, sheet-like domains and pathways that remained accessible to the electrolyte, and restacking or aggregation of the sheets induced by calcium ions is proposed as a plausible origin of this morphology. Adding 1 wt% palladium partially restored coating coverage and raised the mean total resistance to 14.97 ± 0.20 kΩ cm² (2.15 ± 0.04 µA cm⁻²), whereas 2 wt% palladium lowered the interfacial charge-transfer resistance and reduced the mean total resistance to 11.73 ± 0.30 kΩ cm² (2.91 ± 0.06 µA cm⁻²), even though the outer part of the coating remained intact. All coatings supported higher short-term A7R5 cell densities than the uncoated substrate, and the highest value, 330.2% of the control, was obtained for the graphene oxide coating containing 2 wt% palladium. The same graphene-oxide/palladium coatings, however, produced considerably stronger bioluminescent responses from Staphylococcus aureus than the remaining compositions. Overall, the hydroxyapatite coating containing 2 wt% palladium offered the most balanced combination of electrochemical and biological performance. The results show that the behavior of these coatings is governed by the organization of graphene oxide, by the interfacial chemistry associated with palladium, by the pathways available to the electrolyte, and by buried charge-transfer processes, rather than by nominal additive content alone. Their architecture is characterized by folded sheet-like domains and accessible electrolyte pathways, with Ca²⁺-mediated restacking/aggregation proposed as a plausible origin. Incorporation of 1 wt% Pd partially restored coating coverage and increased the mean total resistance to 14.97 ± 0.20 kΩ cm², whereas increasing Pd to 2 wt% reduced the interfacial charge-transfer resistance and decreased the mean total resistance to 11.73 ± 0.30 kΩ cm² despite retention of the outer-coating barrier. All coatings supported higher short-term A7R5 cell densities than the uncoated substrate, with HA/GO-2Pd reaching 330.2% of the control. However, the GO/Pd coatings produced substantially higher Xen36 bioluminescent responses than the remaining compositions. Overall, HA-2Pd provided the most balanced electrochemical and biological performance, while the results demonstrate that the behavior of HA/GO/Pd coatings is governed by GO organization, Pd-associated interfacial chemistry, electrolyte-access pathways, and buried interfacial charge-transfer processes rather than nominal additive content alone. Full article

Background/Objectives: Estimating the post-mortem interval (PMI) of human skeletal remains remains challenging because bone undergoes structural, molecular and optical changes that evolve differently over time and are strongly influenced by taphonomic conditions. Rather than assuming that a single multimodal classifier performs equally well across all PMI intervals, this study aimed to determine which imaging or spectroscopic modality is most informative for specific forensic decision tasks and to derive an exploratory task-specific diagnostic decision-support framework based on internally evaluated sample-level analyses. Methods: Human femoral bone samples were assigned to five PMI classes ranging from 0–2 weeks to >100 years and examined using micro-computed tomography, hyperspectral imaging, handheld and microscopic Raman spectroscopy, and NIR-ONE spectroscopy. Repeated acquisitions were aggregated at the physical-sample level. Four targeted diagnostic contrasts were defined for the present reanalysis: archaeological class 5 versus classes 1–4, early classes 1 + 2 versus later classes 4 + 5, classes 1 + 2 versus class 4 within the Raman-compatible forensic range, and class 1 versus class 2. In addition, an exploratory direct pairwise comparison of class 4 versus class 5 was performed to specifically assess the boundary between the latest forensic interval and archaeological material. Parameter-wise ROC analysis, bootstrap confidence intervals, exploratory operating points at approximately 95% specificity, and repeated stratified cross-validation were used. All preprocessing for multivariate modelling was performed within the respective cross-validation folds. Results: Diagnostic performance was strongly task-dependent. Archaeological class 5 was distinguished from classes 1–4 by micro-CT Mean2 (AUC 0.984), NIR-ONE reflectance at 1944 nm (AUC 0.980), and HSI-derived tissue water index (TWI; AUC 0.961). In the additional exploratory direct C4-versus-C5 analysis, micro-CT Mean2 showed complete separation of the available samples (AUC 1.000), while NIR-ONE reflectance at 1944 nm retained excellent discriminatory performance (AUC 0.963). In contrast, HSI-derived TWI showed only moderate direct C4-versus-C5 discrimination (AUC 0.742). For early classes 1 + 2 versus later classes 4 + 5, the device-derived HSI StO₂ index showed the highest univariate performance (AUC 0.940), followed by TWI (AUC 0.894) and the NIR spectral slope between 1550 and 1950 nm (AUC 0.860). Within the Raman-compatible forensic range, HSI remained highly informative, while Raman carbonate/phosphate and crystallinity parameters provided complementary molecular information. Class 1 versus class 2 discrimination remained moderate. Corrected five-class modelling achieved only moderate balanced accuracy and did not consistently improve upon NIR-ONE alone. Conclusions: Diagnostic performance was strongly task-dependent. In this internally evaluated cohort, micro-CT Mean2 and high-wavelength NIR-ONE features showed the strongest discrimination of archaeological-compatible C5 material, whereas HSI-derived optical indices were most informative for the separated early-versus-later contrast and Raman spectroscopy provided complementary molecular information within C1–C4. Because C5 comprised only six unique physical samples and archaeological context was confounded with chronological age, the very high C5-related AUC estimates should be regarded as exploratory, hypothesis-generating estimates rather than validated measures of chronological PMI. The proposed decision-support framework is likewise exploratory and requires independent external validation before forensic implementation. Full article

Raman spectroscopy is a commonly used label-free technique for the chemical analysis of microplastics (MPs), which are defined as plastic particles larger than 1 μm but smaller than 5 mm size; given its small signal strength and diffraction-limited spatial resolution, Raman spectroscopy has limited applicability in the study of nanoplastics (NPs), which are defined as particles smaller than 1 μm. Tip-enhanced Raman scattering (TERS) is a promising technique that can overcome these limitations by using a single plasmonic tip of an atomic force microscope (AFM) to enhance the Raman signal from a small sample volume. Here we used TERS for the analysis of NPs in human bronchoalveolar lavage (BAL) fluid. We developed a sub-sampling procedure for nanoscale TERS imaging on an SiO₂/Si substrate and performed control experiments. We investigated the experimental coffee-ring effect on the spatial distribution of NPs on the substrate. We compared the results of TERS imaging with the conventional confocal Raman microscopy and observed the presence of similar types of plastic, pigments, and mineral particles, revealing the origin of NPs from the corresponding MPs. The total nanoparticle density observed in BAL using TERS was ~50 billion particles per liter, most of which were NPs. Our TERS approach may be extended to other types of human fluids and tissue samples to provide insights into the health effects of NPs. Full article

Lignite-derived activated carbon (L-AC) was fabricated via a microwave-assisted KOH activation process using a low-rank Mae Moh lignite and explored its potential as an adsorbent solid for post-combustion CO₂ capture. Optimization of the KOH ratio, microwave irradiation power, and activation time gave rise to a product with a BET surface area of 1349 m² g⁻¹ and total pore volume of 0.78 cm³ g⁻¹, which represented 165 times and 78 times enhancement compared with that of the initial lignite, respectively. Scanning electron microscope (SEM) images proved the formation of a hierarchical macropore–mesopore–micropore structure, whereas Raman (I_D/I_G = 1.83) and Fourier-transform infrared spectroscopy analyses revealed a graphitic-like structure rich in defects with the existence of C=O and C–O–C functional groups involved in the Lewis acid–base interaction between L-AC and CO₂ molecules. Dynamic fixed-bed breakthrough tests performed at temperatures of 298, 328, and 353 K under post-combustion relevant conditions (CO₂ concentration: 15%, pressure: 1 atm) yielded CO₂ equilibrium uptake capacities of 47.34, 34.37, and 21.34 mg g⁻¹, respectively, with outstanding cyclic stability achieved after six consecutive adsorption–desorption cycles of temperature swing adsorption–desorption at 393 K. Among the seven nonlinear kinetic models, the Avrami, FL-PFO, and general-order models exhibited the highest fitting accuracy (R² = 0.9994–0.9998), suggesting that CO₂ adsorption onto L-AC proceeds through heterogeneous, multi-stage adsorption kinetics. A Weber–Morris intra-particle diffusion analysis identified a three-stage sequential transport mechanism in which mesopore diffusion constitutes the primary rate-limiting step. Thermodynamic parameters confirmed spontaneous (ΔG° = −24.20 to −26.87 kJ mol⁻¹), exothermic (ΔH° = −9.42 kJ mol⁻¹), and entropy-assisted adsorption (ΔS° = +49.93 J mol⁻¹ K⁻¹) consistent with a physisorption mechanism, corroborated by a low activation energy of 9.11 kJ mol⁻¹. These findings demonstrate the viability of low-rank lignite as a low-cost precursor for the scalable synthesis of high-performance carbonaceous CO₂ adsorbents for post-combustion capture applications. Full article

Background/Objectives: The precise characterization of the key microstructural and physicochemical attributes in sustained-release microspheres remains a technical bottleneck, hindering the understanding of drug release mechanisms, and limiting insights into the “process–structure–performance” relationship. To address this, we developed novel methods to conduct in-depth research on the microscopic properties of microspheres. Methods: Scanning electron microscopy (SEM) combined with a deep learning-based image segmentation (DLIS) algorithm was established for quantitative analysis of the pore structure. Laser confocal Raman spectroscopy (LCRS) was employed for in situ, non-destructive, three-dimensional (3D) visualization and quantitative mapping of the active pharmaceutical ingredient (API) distribution within microspheres. Results: This study successfully developed and applied SEM-DLIS and LCRS as reliable tools for microstructural and physicochemical characterization. SEM-DLIS analysis revealed significant differences in surface and internal pore structure among microspheres from different manufacturers and between particles of different sizes from the same batch. LCRS imaging further identified distinct API distribution patterns: uniform dispersion, outer-layer enrichment, and heterogeneous distribution. The combined data elucidate that the initial burst release is governed by the synergistic effect of surface porosity and API surface enrichment, whereas the sustained release kinetics are jointly regulated by the internal pore structure, particle size, and API spatial distribution. Conclusions: The findings establish that microstructure dictates release behavior and that all observed structural variations are linked to critical process parameters (CPPs), validating the “process determines structure” hypothesis. The established methodology provides a critical technical framework for the reverse engineering and quality equivalence assessment of generic microspheres, as well as for the quality-by-design-based optimization of innovative drug products, thereby advancing both pharmaceutical development and regulatory science. Full article

Basic calcium phosphate (BCP) and calcium pyrophosphate dihydrate (CPPD) crystals drive the degenerative and inflammatory pathways that lead to crystal arthropathy. Although recent classification criteria and imaging recommendations have improved standardisation for CPPD disease, it remains underdiagnosed in practice. In contrast, BCP disease lacks validated classification criteria and standardised diagnostic descriptors, limiting translational research and clinical recognition. Accurate diagnoses are limited by varied reference standards, operator-dependent techniques, and the absence of validated bedside tools. As the population ages and the burden of crystal disease rises, there will be increased demand for reliable diagnostic tools in clinical practice. This review summarises current and emerging diagnostic tools for identifying BCP and CPPD, with emphasis on bridging the diagnostic gap from research to routine care. Polarised light microscopy remains a highly specific test for the diagnosis of CPPD but is limited by inter-observer variability and access to polarising light microscopes. Imaging tools such as ultrasound, conventional radiography, computed tomography (CT) and dual-energy CT can identify CPPD, although performance varies by anatomical site, disease stage and technical parameters. Ultrasound is a useful bedside tool for identifying BCP disease, whereas CT and DECT also have diagnostic utility but lack validation for routine use. Emerging technologies such as computational polarised light microscopy and Raman spectroscopy are promising techniques, but require further research to evidence their use in clinical practice. Development of clear diagnostic reference standards, classification criteria for BCP disease, and evidence-based multimodal diagnostic pathways are essential to bridge the gap between research and routine clinical diagnosis. Full article

Glioblastoma (GBM) remains the most aggressive primary malignant brain tumor in adults, with poor survival largely driven by diffuse cellular infiltration, profound heterogeneity, and near-universal recurrence following standard therapy. Although maximizing the extent of resection is a key determinant of patient outcome, current clinical imaging modalities lack the spatial resolution necessary to detect microscopic tumor invasion and therapy-resistant cell populations. Emerging in vivo imaging technologies capable of cellular and near-single-cell resolution have therefore become a major focus in preclinical neuro-oncology research, with growing relevance for surgical guidance, treatment adaptation, and translational discovery. This review evaluates multiple optical imaging modalities, including multi-photon microscopy, near-infrared II fluorescence imaging, bioluminescence imaging, photoacoustic imaging, optical coherence tomography, confocal laser endomicroscopy, Raman spectroscopy, autofluorescence microscopy, and fluorescence macroscopy with a focus on their ability to detect residual GBM cells. Despite significant advances, these approaches remain constrained by limitations in molecular target availability, probe delivery across the blood–brain barrier, and signal variability within heterogeneous tumor regions. The biological complexity of GBM further challenges detection, as residual tumor cells are spatially dispersed and phenotypically diverse, limiting the effectiveness of single-marker or single-modality strategies. Together, these findings highlight the need for integrated, biologically informed imaging approaches to improve detection of residual disease and guide surgical decision making. Full article

Background: Glioblastoma (GBM) remains the most aggressive primary brain tumor, and intraoperative frozen section analysis is the current standard for rapid histopathological assessment. However, this approach is time-consuming and resource-intensive. Stimulated Raman scattering (SRS) imaging has emerged as a label-free technique enabling near real-time microscopic evaluation of fresh tissue. This study compares the visualization of selected histopathological features in a newly developed intraoperative SRS system with conventional hematoxylin–eosin (HE) staining in confirmed GBM. Methods: Tumor samples from 30 patients with neuropathologically confirmed GBM were analyzed. For each case, both HE-stained frozen sections and SRS-generated virtual HE-like images were prepared from separate portions of the specimen. Twelve neuropathologists with varying levels of experience assessed 60 images according to seven predefined GBM criteria, resulting in 720 image evaluations. Feature detection was analyzed using cluster-adjusted generalized estimating equation models, and interobserver agreement was assessed using Fleiss’ κ. Results: Descriptively, hypercellularity and hypervascularization were identified at similar frequencies in both modalities, whereas pleomorphism, endothelial proliferation, mitotic activity, and necrosis were more often recognized in HE images. In cluster-adjusted analyses, SRS showed significantly lower detection rates for hypercellularity, pleomorphism, endothelial proliferation, and mitotic activity, while no significant difference was observed for hypervascularization, necrosis, or pseudopalisading after false discovery rate correction. Interobserver agreement was feature-dependent and generally higher for HE than SRS, particularly for hypercellularity. Conclusions: In this feature-level analysis of neuropathologically confirmed GBM, SRS imaging provided rapid, label-free morphological information and showed comparable visualization of selected histopathological features, particularly hypervascularization. While conventional HE-stained frozen sections remained superior for certain WHO-defining features, SRS represents a promising intraoperative adjunct that may complement established neuropathological workflows. Further studies including non-tumor tissue and a broader range of glioma grades are needed to determine the full diagnostic accuracy and clinical applicability of this technique. Full article

Gold-assisted exfoliation is an effective approach to obtain clean and large-area monolayers of transition metal dichalcogenides, yet the microscopic evolution of interfacial adhesion remains poorly understood. Here, we investigate temperature-controlled exfoliation of MoS₂ between 30 and 170 °C. Based on optical microscopy image analysis, mild heating slightly improves the exfoliation yield, which is associated with the release of interfacial contaminants and trapped gases—these substances enhance the adhesion between gold and molybdenum disulfide (Au-MoS₂). Unexpectedly, as revealed by AFM, SEM-EDS, and Raman analyses, parts of the Au film start to peel off from the underlying Ti adhesion layer at approximately 100 °C. This Au film detachment, resulting from the surprisingly weak Au-Ti adhesion, serves as a unique probe for interfacial strength: it preferentially occurs at the boundaries of MoS₂ flakes, indicating that the reinforcement of the Au-MoS₂ interaction originates at the edges rather than being uniformly distributed. At higher temperatures (>130 °C), Au detachment expands to larger areas, indicating that boundary-localized adhesion progressively extends across the entire interface. Additional STM/STS measurements further confirm that thermal annealing improves local Au-MoS₂ contact by removing interfacial species and enabling surface reconstruction. These findings establish a microscopic picture of temperature-assisted exfoliation, highlighting the dual roles of interfacial contaminant release and boundary effects, and offering guidance for more reproducible fabrication of high-quality 2D monolayers. Full article

The optical microscope is an indispensable observation instrument that has fundamentally contributed to progress in science and technology. Dark-field microscopy and scattered light imaging techniques enable high-contrast observation of nanoparticles in water. However, the scattered light is focused by the optical lenses, resulting in a blurred image of the nanoparticle structure. Here, we developed a projection optical microscope (PROM), which directly observes the scattered light from the nanoparticles without optical lenses. In this method, the sample is placed below the focus position of the microscope’s objective lens and the projected light is detected by an image sensor. This enables direct observation of the sample with a spatial resolution of approximately 20 nm. Using this method, changes in the aggregation state of nanoparticles in solution can be observed at a speed faster than the video frame rate. Moreover, the mechanism of such high-resolution observation may be related to the quantum properties of light, making it an interesting phenomenon from the perspective of optical engineering. We expect this method to be applicable to the observation and analysis of samples in materials science, biology and applied physics, and thus to contribute to a wide range of scientific, technological and industrial fields. Full article

Codex 462 of the Fondo Hortus Pisanus of the Biblioteca Universitaria of Pisa is a precious example of a 16th century illustrated herbal, Icones variarum plantarum, containing 35 tempera paintings by the German soldier Georg Dyckman, an amateur but highly talented artist. The manuscript was recently restored on the occasion of an international exhibition; the necessary preliminary studies for the restoration included a series of in situ diagnostic studies using contactless techniques (digital microscope, multispectral imaging, XRF, Raman and ER-FTIR). These analyses proved useful in deepening the knowledge of the materials and the execution technique of this type of illustrated herbals and in choosing the most appropriate solutions during the restoration phase. In view of the growing interest in this type of historical evidence, which involves both art history and the history of science, this study offers an interesting new perspective on the subject, useful both from a technical point of view for future conservation and for analytical and historical artistic studies. Full article

Background: The use of optical microscopic techniques has gained increasing attention in recent years for studying the bioavailability (BA) and bioequivalence (BE) of topical drugs. Stimulated Raman scattering (SRS), one type of optical imaging technique, probes chemical-specific information and has excellent spatiotemporal resolution. It uses intrinsic molecular vibrational signatures, and therefore, labeling samples or other treatments is unnecessary to track a molecule. Because of its unique advantages, studies have used SRS for BA evaluations and, more recently, for BE evaluations. In BE evaluation, low data variance within a treatment group is important to ensure sensitivity and specificity in comparing treatment groups. Methods: When measuring forward-direction SRS signals transmitted through skin, the signal intensity is susceptible to variance due to several factors, such as the microscope system’s performance, the different optical features of topical drug products, and the heterogeneity of skin in transmitting light. This work closely investigated the effects of these factors on an SRS signal and developed solutions to reduce their effects on the data variance. Specifically, we constructed a method using a dual-modality detector built in-house, which simultaneously measured both the SRS signal and total light transmission synchronized in time and co-registered in space. Results: We developed equations to normalize SRS signals using the transmission intensity, and the results demonstrated a clear improvement in the SRS signal via a reduction in the signal variance (up to a 9.46% CV value decrease) that is otherwise caused by various factors associated with the use of topical drugs and the composition of the skin. We carried out an exploratory BE study using tretinoin-containing topical products and observed improvements in BE assessment with the developed method (could achieve a reduction of 0.11 in the CI value). Conclusions: This work has led to a better understanding of the factors that affect SRS imaging and has provided an effective method to compensate for these factors in BE assessments. This is a critical initial effort for better practical implementation of SRS in cutaneous pharmacokinetics (cPKs) studies of topical drugs. Full article

Recent advancements in carbon nanotubes and graphene have driven significant research into other low-dimensional materials, with phosphorus-based materials emerging as a notable area of interest. Phosphorus nanowires and thin sheets show promise for applications in devices such as batteries, photodetectors, and field-effect transistors. However, the presence of multiple allotropes of phosphorus complicates their characterization. Accurate identification of these allotropes is essential for understanding their physical, optical, and electronic properties, which influence their potential applications. Researchers frequently encounter difficulties in consolidating literature for the confirmation of the structure of their materials, a process that can be time-consuming. This minireview addresses this issue by providing a comprehensive, side-by-side comparison of Raman and X-ray diffraction characteristic peaks, as well as electron microscopic images and lattice spacings, for the various phosphorus allotropes. To our knowledge, this is the first compilation to integrate all major structural fingerprints into unified summary tables, enabling rapid cross-referencing. This resource aims to support researchers in accurately identifying phosphorus phases during synthesis and device fabrication workflows. For example, distinguishing between red phosphorus polymorphs is crucial for optimizing anode materials in sodium-ion batteries, where electrochemical performance is phase-dependent. Full article

Magnetotactic bacteria (MTB), a unique group of Gram-negative prokaryotes, have the remarkable ability to biomineralize magnetic nanoparticles (MNPs) intracellularly, making them promising candidates for various biomedical applications such as biosensors, drug delivery, imaging contrast agents, and cancer-targeted therapies. To fully exploit the potential of MTB, a precise understanding of the structural, surface, and functional properties of these biologically produced nanoparticles is required. Given these concerns, this review provides a focused synthesis of the most widely used microscopic and spectroscopic methods applied in the characterization of MTB and their associated MNPs, covering the latest research from January 2022 to May 2025. Specifically, various optical microscopy techniques (e.g., transmission electron microscopy (TEM), scanning electron microscopy (SEM), and atomic force microscopy (AFM)) and spectroscopic approaches (e.g., localized surface plasmon resonance (LSPR), surface-enhanced Raman scattering (SERS), and X-ray photoelectron spectroscopy (XPS)) relevant to ultrasensitive MTB biosensor development are herein discussed and compared in term of their advantages and disadvantages. Overall, the novelty of this work lies in its clarity and structure, aiming to consolidate and simplify access to the most current and effective characterization techniques. Furthermore, several gaps in the characterization methods of MTB were identified, and new directions of methods that can be integrated into the study, analysis, and characterization of these bacteria are suggested in exhaustive manner. Finally, to the authors’ knowledge, this is the first comprehensive overview of characterization techniques that could serve as a practical resource for both younger and more experienced researchers seeking to optimize the use of MTB in the development of advanced biosensing systems and other biomedical tools. Full article