Chemical & Biomedical Imaging最新文献

The ribosome, a 2.6 megadalton biomolecule measuring approximately 20 nm in diameter, coordinates numerous ligands, factors, and regulators to translate proteins with high fidelity and speed. Understanding its complex functions necessitates multiperspective observations. We developed a dual-FRET single-molecule Förste Resonance Energy Transfer method (dual-smFRET), allowing simultaneous observation and correlation of tRNA dynamics and Elongation Factor G (EF-G) conformations in the same complex, in a 10 s time window. By synchronizing laser shutters and motorized filter sets, two FRET signals are captured in consecutive 5 s intervals with a time gap of 50–100 ms. We observed distinct fluorescent emissions from single-, double-, and quadruple-labeled ribosome complexes. Through comprehensive spectrum analysis and correction, we distinguish and correlate conformational changes in two parts of the ribosome, offering additional perspectives on its coordination and timing during translocation. Our setup’s versatility, accommodating up to six FRET pairs, suggests broader applications in studying large biomolecules and various biological systems.

The application of X-ray spectro-microscopy to image changes in the chemical state in application areas such as catalysis, environmental science, or biological samples can be limited by factors such as the speed of measurement, the presence of dilute concentrations, radiation damage, and thermal drift during the measurement. We have adapted a reduced-order model approach, known as the discrete empirical interpolation method, which identifies how to optimally subsample the spectroscopic information, accounting for background variations in the signal, to provide an accurate approximation of an equivalent full spectroscopic measurement from the sampled material. This approach uses readily available prior information to guide and significantly reduce the sampling requirements impacting both the total X-ray dose and the acquisition time. The reduced-order model approach can be adapted more broadly to any spectral or spectro-microscopy measurement where a low-rank approximation can be made from prior information on the possible states of a system, and examples of the approach are presented.

Medium depolarization imaging by catheter-based polarization-sensitive optical coherence tomography (PS-OCT) can provide valuable insight into significant features of lipid, macrophages, and cholesterol crystals in atherosclerotic vulnerable plaques. In this paper, we demonstrate a method to achieve an accurate estimation of the medium depolarization index (EMDI) with noise immunity in catheter-based PS-OCT. EMDI is calculated by an iterative approximation based on Lu–Chipman matrix decomposition and Frobenius norm judgment of incoherent averaging of Mueller matrices. Monte Carlo simulation results verify that the medium depolarization measurement by EMDI is 3.3 times more accurate compared with those of the depolarization index (DI) and degree of polarization uniformity (DOPU). In experiments, we design a microsphere suspension with various concentrations and measure EMDI under different additive noise. Consistently, the measurement accuracy by EMDI is increased 2.85 times compared to those by DI and DOPU. For vascular plaques detection, we use protein and cholesterol gel as plaque phantoms. Based on PS-OCT images of plaque phantom in vitro and in ex vivo porcine coronary artery, the recognition rate of plaque by EMDI is 2.99 to 4.65 times higher than those by DI and DOPU evaluated by spatial response of the Laplacian operator (SRLO).

The Dual Imaging and Diffraction (DIAD) beamline at Diamond Light Source (Didcot, U.K.) implements a correlative approach to the dynamic study of materials based on concurrent analysis of identical sample locations using complementary X-ray modalities to reveal structural detail at various length scales. Namely, the underlying beamline principle and its practical implementation allow the collocation of chosen regions within the sample and their interrogation using real-space imaging (radiography and tomography) and reciprocal space scattering (diffraction). The switching between the two principal modes is made smooth and rapid by design, so that the data collected is interlaced to obtain near-simultaneous multimodal characterization. Different specific photon energies are used for each mode, and the interlacing of acquisition steps allows conducting static and dynamic experiments. Building on the demonstrated realization of this state-of-the-art approach requires further refining of the experimental practice, namely, the methods for gauge volume collocation under different modes of beam–sample interaction. To address this challenge, experiments were conducted at DIAD devoted to the study of human dental enamel, a hierarchical structure composed of hydroxyapatite mineral nanocrystals, as a static sample previously affected by dental caries (tooth decay) as well as under dynamic conditions simulating the process of acid demineralization. Collocation and correlation were achieved between WAXS (wide-angle X-ray scattering), 2D (radiographic), and 3D (tomographic) imaging. While X-ray imaging in 2D or 3D modes reveals real-space details of the sample microstructure, X-ray scattering data for each gauge volume provided statistical nanoscale and ultrastructural polycrystal reciprocal-space information such as phase and preferred orientation (texture). Careful registration of the gauge volume positions recorded during the scans allowed direct covisualization of the data from two modalities. Diffraction gauge volumes were identified and visualized within the tomographic data sets, revealing the underlying local information to support the interpretation of the diffraction patterns. The present implementation of the 4D microscopy paradigm allowed following the progression of demineralization and its correlation with time-dependent WAXS pattern evolution in an approach that is transferable to other material systems.

Due to the biological importance of cysteine (Cys), the development of organic fluorescence probes for Cys has been a wide, potent, and outstanding research field in most recent years. It has been used as a biomarker in treating various diseases; therefore, developing a sensing mechanism for detecting Cys is very important. In this Review, we focus on and summarize the specific results of recent exciting literature regarding the sensing mechanism of Cys-specific fluorescence probes and their applications in Cys recognition. Moreover, a design strategy of the sensing mechanism of Cys can be classified into seven reaction mechanisms, including the aromatic substitution rearrangement reaction, cyclization of aldehyde, Michael addition reaction, Se–N or S–S or bond cleavage reaction, addition cyclization of acrylate, metal complex reaction, and nucleophilic substitution reaction. In all sections, discussions have corresponded to Cys-specific sensing mechanisms, which consist of emission, color changes, and detection limits and deal with the application and recognition sites of molecules. Future directions and challenges have been proposed for the preparation of Cys-specific probes.

Hydrogen peroxide and polarity are closely related to many physiological activities and pathological processes. However, near-infrared fluorescent probes that are sensitive to both H₂O₂ and polarity are still scarce. Herein, we developed the first dual-channel near-infrared fluorescent probe NBO, with an AIE effect, enabling simultaneous monitoring of H₂O₂ and polarity. The probe presented high sensitivity, high selectivity, and low detection limit for H₂O₂. It also had high sensitivity to polarity, independent of pH and viscosity, with large Stokes shifts, good photostability, and low cytotoxicity. Moreover, NBO was able to detect both endogenous and exogenous H₂O₂ as well as polarity fluctuations in vivo as a method to effectively differentiate between cancer cells and normal cells. Importantly, it also could monitor the therapeutic effects of drugs in inflammation and iron-dead cells and mice. Based on NIR emission, NBO could be used as an imaging tool and a way to evaluate the therapeutic effect of drugs for inflammation and ferroptosis.

High-resolution spatial and temporal analysis and 3D visualization of time-dependent processes, such as human dental enamel acid demineralization, often present a challenging task. Overcoming this challenge often requires the development of special methods. Dental caries remains one of the most important oral diseases that involves the demineralization of hard dental tissues as a consequence of acid production by oral bacteria. Enamel has a hierarchically organized architecture that extends down to the nanostructural level and requires high resolution to study its evolution in detail. Enamel demineralization is a dynamic process that is best investigated with the help of in situ experiments. In previous studies, synchrotron tomography was applied to study the 3D enamel structure at certain time points (time-lapse tomography). Here, another distinct approach to time-evolving tomography studies is presented, whereby the sample image is reconstructed as it undergoes continuous rotation over a virtually unlimited angular range. The resulting (single) data set contains the data for multiple (potentially overlapping) intermediate tomograms that can be extracted and analyzed as desired using time-stepping selection of data subsets from the continuous fly-scan recording. One of the advantages of this approach is that it reduces the amount of time required to collect an equivalent number of single tomograms. Another advantage is that the nominal time step between successive reconstructions can be significantly reduced. We applied this approach to the study of acidic enamel demineralization and observed the progression of demineralization over time steps significantly smaller than the total acquisition time of a single tomogram, with a voxel size smaller than 0.5 μm. It is expected that the approach presented in this paper can be useful for high-resolution studies of other dynamic processes and for assessing small structural modifications in evolving hierarchical materials.

Enzymes are an important tool used for signal amplification in biosensing. However, traditional amplification methods based on enzymes are always dependent on their catalytic activities, so their signals fluctuate with the change of microenvironment (e.g., pH and temperature). In this work, we communicate an activity-independent enzyme-powered (AIEP) amplification strategy for biosensing to improve signal stability and fidelity. To verify this hypothesis, the monitoring of oxidative stress during drug-induced liver injury was carried out. Carboxylesterase (CEs), highly expressed in hepatic tissue, was selected as the amplification tool. A CEs configuration-matching fluorophore (CMF) was designed and screened, and a nanobeacon was fabricated by loading CMF within an O₂^•–-responsive polymeric micelle. Since the degradation of the nanobeacon was triggered by O₂^•–, CMF was released to bind with CEs, and the fluorescence was lit by CEs-CMF configuration matching but not catalytic reaction. Results demonstrated that the oxidative stress during drug-induced liver injury could be successfully monitored, and the hepatoprotective effects of repair drugs could be evaluated by cell and in vivo imaging. This strategy is flexible for bioactive molecules by altering the responsive unit and generally accessible for pharmacological evaluation.

Viscosity and polarity are crucial microenvironmental parameters within cells, intimately linked to the physiological activities of organisms. We constructed and synthesized an innovative dual-functional fluorescent probe, DHBP. In the green channel, the fluorescence signal notably intensifies with decreasing environmental polarity, while in the red channel, fluorescence signal amplification occurs due to the collaborative effects of viscosity and polarity, resulting in more pronounced changes. Additionally, DHBP demonstrates high sensitivity in detecting changes in polarity and viscosity induced by drug-induced inflammation in cells and mice. Importantly, DHBP has been effectively utilized to monitor alterations in viscosity and polarity in the liver injury induced by diabetes in vivo in mice and further employed to assess the therapeutic efficacy of drugs. Therefore, DHBP holds promise for advancing research on viscosity and polarity in future studies of physiological and pathological processes.