Chemical & Biomedical Imaging最新文献

Coupling electrochemistry with optical techniques gives in-depth insights into the interfacial processes in action. In that context, fluorescence confocal laser scanning microscopy (F-CLSM) enables an electrode surface characterization with spatial resolution in the lateral plane (xy) as well as in the axial direction (z), perpendicular to the electrode surface. However, like most optical techniques, fluorescence microscopy has intrinsic limitations, notably in terms of resolution and sensitivity, which are investigated in this contribution by conducting F-CLSM experiments with two disk electrodes of different sizes: a large microelectrode (LME, Ø = 250 μm) and a much smaller so-called ultramicroelectrode (UME, Ø = 18 μm). We demonstrated that the diffusion layers of both microelectrodes can be imaged with sufficient resolution and sensitivity to be quantitatively compared with the simulated concentration profiles. This work highlights the intrinsic technical challenges associated with this kind of coupled experiments, and it discusses the conditions that should be fulfilled to obtain reliable results at the microscale. These results pave the way toward reaction layer imaging down to micrometric resolution and could help decipher complex electrochemical reactions possibly involving transient species.

Diabetes is a complex metabolic disorder characterized by persistent hyperglycemia, which causes damage to multiple target organs and triggers a range of complications. Oxidative stress, driven by reactive oxygen species (ROS) such as hypochlorite (HClO) and hydrogen peroxide (H₂O₂), plays a crucial role in the onset and progression of diabetes and its associated complications. Therefore, the simultaneous and differential detection of HClO, H₂O₂, and their mixture is essential for accurately assessing oxidative stress status and understanding their synergistic roles in disease progression. In this study, we present a triple-signal fluorescent probe, probe 1, designed to simultaneously and selectively detect HClO, H₂O₂, and their combination with high specificity and sensitivity. The probe emits three distinct fluorescence signals, enabling precise real-time visualization of oxidative stress dynamics in complex biological systems. Probe 1 has been successfully applied to track both exogenous and endogenous levels of HClO and H₂O₂ in living cells and zebrafish models. Furthermore, its efficacy has been demonstrated in diabetic mouse models, where it facilitates the spatial and temporal monitoring of oxidative stress across different organs. These findings underscore the potential of probe 1 as a powerful tool for advancing the understanding of oxidative stress mechanisms and developing targeted therapeutic strategies for diabetes and related diseases.

We implemented a multimodal set of functional imaging techniques optimized for deep-tissue imaging to investigate how cancer cells invade surrounding tissues and how their physiological properties change in the process. As a model for cancer invasion of the extracellular matrix, we created 3D spheroids from triple-negative breast cancer cells (MDA-MB-231) and nontumorigenic breast epithelial cells (MCF-10A). We analyzed multiple hallmarks of cancer within the same spheroid by combining a number of imaging techniques, such as metabolic imaging of nicotinamide adenine dinucleotide by fluorescence lifetime imaging microscopy (NADH-FLIM), hyperspectral imaging of a solvatochromic lipophilic dye (Nile Red), and extracellular matrix imaging by second harmonic generation (SHG). We included phasor-based bioimage analysis of spheroids at three different time points, tracking both morphological and biological properties, including cellular metabolism, fatty acid storage, and collagen organization. Employing this multimodal deep-imaging framework, we observed and quantified cancer cell plasticity in response to changes in the environment composition.

Hyperspectral stimulated Raman scattering (SRS) microscopy is rapidly becoming an established method for chemical and biomedical imaging due to the combination of high spatial resolution and chemical information contained within the three-dimensional data set. Chemometric analysis techniques based on linear unmixing, or multivariate analysis, have become indispensable when visualizing hyperspectral data sets. The application of spectral phasor analysis has also been extremely fruitful in this regard, providing a convenient method to retrieve the spatial and chemical components of the data set. Here, we demonstrate the application of spectral phasor analysis for unmixing the overlapping spectral features within the cell-silent region of the SRS spectrum (2000-2300 cm^-1). In doing so, we show it is possible to identify specific Raman signals for DNA, proteins, and lipids following glucose-d₇ metabolism in dividing cells. In addition, we show that spectral phasor analysis is capable of distinguishing different bioorthogonal Raman signals including alkynes and carbon-deuterium (C-D) bonds. We demonstrate the application of spectral phasor analysis for multicomponent unmixing of bioorthogonal Raman groups for high-content cellular imaging applications.

Among various electrochemical imaging techniques, electrochemiluminescence microscopy (ECLM) stands out as a powerful approach to visualize electrochemical reactions by converting localized reactivity into optical signals. This study investigates ECL light emission spatial distribution in a confined space by using microelectrode arrays (MEAs) fabricated on glassy carbon (GC) and gold (Au) substrates via thermal nanoimprint lithography (TNIL). With the Ru(bpy)₃²⁺/TPrA system, ECL imaging revealed distinct emission profiles, with Au exhibiting a broader spatial distribution compared to GC under identical geometric conditions. The estimated thickness of the ECL emitting layer (TEL) was significantly larger on Au (∼7 μm) than on GC (∼4 μm), attributed to the interplay between the electrode material and dominant ECL mechanism. Decreasing Ru(bpy)₃²⁺ concentration resulted in minimal perturbation of the GC ECL profile, consistent with a predominant oxidative–reductive mechanism. In contrast, a significant narrowing of the ECL profile was observed on Au, indicative of a transition from a catalytic to an oxidative–reductive pathway. These observations were corroborated and rationalized by finite element simulations. Our findings demonstrate the capacity to fine-tune the Thickness of the Emission Layer (TEL) and modulate ECL emission through electrode material selection and luminophore concentration. Such precise control has significant implications for the development of highly sensitive and spatially resolved bioanalytical assays, particularly those employing bead-based detection methodologies.

Due to its exceptionally high sensitivity and specificity, surface-enhanced Raman scattering (SERS) is widely employed in diverse fields, including biomedicine, environmental monitoring, and food safety. Nonetheless, the lack of reproducibility and substrate uniformity has seriously hindered its application in quantitative detection, particularly at low analyte concentrations. Recently, the concept of digitization has been integrated into SERS, enabling quantitative and sensitive detection with promising applications (Bi et al. Nature2024, 628, 771-775). In this work, we further developed a wide-field digital SERS (WidiSERS) by employing wide-field microscopy for high throughput. Protein assembled gold nanorod dimers are used for largely enhancing the Raman signals. Reproducible quantification of a wide range of target molecules at extremely low concentrations is achievable through single-molecule measurements. Trace-level quantification of ciprofloxacin in a complex milk environment and phenylalanine in cell culture medium was also achieved, verifying the practicability and accuracy of this method. Meanwhile, the gold nanorod dimer substrate is both simple to prepare and reusable after UV/ozone cleaning. WidiSERS is expected to emerge as a preferred method for ultrafast and effective detection across various fields.

The acid–base properties of catalytic materials play a crucial role in facilitating chemical transformations. Nanoscale structural heterogeneities within these catalysts can significantly affect the distribution, type, and strength of their acid–base sites, thereby influencing both localized and overall catalytic reactivity. In this study, high spatial-resolution chemical imaging of basic sites on supported Mg–Al mixed oxide (MgAlO_x) particles, which serve as catalysts for aldol condensation reactions, was achieved using atomic force microscopy–infrared (AFM-IR) nanospectroscopy measurements while using formic acid as a chemical probe for surface basic sites detection. This approach enabled us to identify the distribution, geometry, and strength of basic sites with nanoscale precision. It was revealed that platelet MgAlO_x particles predominantly exhibit a uniform bidentate adsorption of formic acid, whereas aggregates display a heterogeneous distribution of both monodentate and bidentate adsorption modes, indicating differences in the distribution, geometry, and strength of the basic sites. Additionally, upon exposure to formic acid, smaller particles underwent phase reconstruction, transitioning into cubic-like structures characterized by distinct bidentate adsorption of formic acid. This transformation was attributed to the rehydration and intercalation of formate species. The insights gained by conducting high spatial resolution nanospectroscopy measurements highlight the correlation between flat surfaces, characterized by a low density of surface defects, and a homogeneous distribution of basic sites, with a dominant bidentate adsorption mode of formic acid. These results emphasize the critical role of high spatial resolution chemical imaging in unraveling the link between structural features and acid–base functionality in catalytic materials.