Chemical & Biomedical Imaging最新文献

The aggregation of fusion in sarcoma (FUS) in the cytoplasm and nucleus is a pathological feature of Amyotrophic lateral sclerosis (ALS) and Frontotemporal Dementia (FTD). Genetic mutations, abnormal protein synthesis, environmental stress, and aging have all been implicated as causative factors in this process. Salt ions are essential to many physiological processes in the body, and the imbalance of them is an important environmental stress factor in cells. However, their effect on liquid–liquid phase separation (LLPS) of FUS proteins in living cells is not well understood. Here, we map the various salt-induced LLPS of FUS in living cells by genetically coding and self-labeling FUS with organic dyes. The brightness and photostability of the dyes enable long-term imaging to track the mechanism of the assembly and disappearance of FUS phase separation. The FUS protein showed a better phase separation tendency under 0.3 M salt stimulation, and there was a large amount of FUS shuttling from the nucleus to the cytoplasm. At this concentration, various salt solutions displayed different effects on the phase separation of FUS protein, following the Hofmeister effects. We further observed that the assembly of FUS droplets underwent a process of rapid formation of small droplets, plateaus, and mutual fusion. Strikingly, The CsCl-stimulated FUS droplets were not completely reversible after washing, and some solid-like granules remained in the nucleus. Taken together, these results help broaden our understanding of the LLPS of FUS proteins in cellular stress responses.

Single-molecule fluorescence microscopy enables the direct observation of individual reaction events at the surface of a catalyst. It has become a powerful tool to image in real time both intra- and interparticle heterogeneity among different nanoscale catalyst particles. Single-molecule fluorescence microscopy of heterogeneous catalysts relies on the detection of chemically activated fluorogenic probes that are converted from a nonfluorescent state into a highly fluorescent state through a reaction mediated at the catalyst surface. This review article describes challenges and opportunities in using such fluorogenic probes as proxies to develop structure–activity relationships in nanoscale electrocatalysts and photocatalysts. We compare single-molecule fluorescence microscopy to other microscopies for imaging catalysis in situ to highlight the distinct advantages and limitations of this technique. We describe correlative imaging between super-resolution activity maps obtained from multiple fluorogenic probes to understand the chemical origins behind spatial variations in activity that are frequently observed for nanoscale catalysts. Fluorogenic probes, originally developed for biological imaging, are introduced that can detect products such as carbon monoxide, nitrite, and ammonia, which are generated by electro- and photocatalysts for fuel production and environmental remediation. We conclude by describing how single-molecule imaging can provide mechanistic insights for a broader scope of catalytic systems, such as single-atom catalysts.

Pseudomonas aeruginosa is an opportunistic human pathogen capable of causing a wide range of diseases in immunocompromised patients. In order to better understand P. aeruginosa behavior and virulence and to advance drug therapies to combat infection, it would be beneficial to understand how P. aeruginosa cells survive stressful conditions, especially environmental stressors. Here, we report on a strategy that measures potential-dependent fluorescence of individual P. aeruginosa cells, as a sentinel, for cellular response to starvation, hunger, and oxidative stress. This is accomplished using a micropore electrode array capable of trapping large numbers of isolated, vertically oriented cells at well-defined spatial positions in order to study large arrays of single cells in parallel. We find that conditions promoting either starvation or oxidative stress produce discernible changes in the fluorescence response, demonstrated by an increase in the prevalence of fluorescence transients, one of three canonical spectroelectrochemical behaviors exhibited by single P. aeruginosa cells. In contrast, more modest nutrient limitations have little to no effect on the spectroelectrochemical response when compared to healthy cells in the stationary phase. These findings demonstrate the capabilities of micropore electrode arrays for studying the behavior of single microbial cells under conditions where the intercellular spacing, orientation, and chemical environment of the cells are controlled. Realizing single-cell studies under such well-defined conditions makes it possible to study fundamental stress responses with unprecedented control.

The endoplasmic reticulum (ER) is an important targeting organelle for metal-based immunogenic cell death (ICD) inducers. Metal complexes can induce ER stress by causing protein misfolding, which can be reflected by alternations in microenvironmental parameters, including viscosity. We present here a theranostic Re(I) complex (Re1) that shows viscosity-dependent emission intensity and lifetime. Re1 can trigger immunogenic cell death (ICD) in MDA-MB-231 cells by localizing in the ER and causing ER stress. We demonstrate that Re1 can simultaneously induce and monitor the gradual increase in the ER viscosity quantitatively.

Fluorescence nanoscopy has become increasingly powerful for biomedical research, but it has historically afforded a small field-of-view (FOV) of around 50 μm × 50 μm at once and more recently up to ∼200 μm × 200 μm. Efforts to further increase the FOV in fluorescence nanoscopy have thus far relied on the use of fabricated waveguide substrates, adding cost and sample constraints to the applications. Here we report PRism-Illumination and Microfluidics-Enhanced DNA-PAINT (PRIME-PAINT) for multiplexed fluorescence nanoscopy across millimeter-scale FOVs. Built upon the well-established prism-type total internal reflection microscopy, PRIME-PAINT achieves robust single-molecule localization with up to ∼520 μm × 520 μm single FOVs and 25–40 nm lateral resolutions. Through stitching, nanoscopic imaging over mm² sample areas can be completed in as little as 40 min per target. An on-stage microfluidics chamber facilitates probe exchange for multiplexing and enhances image quality, particularly for formalin-fixed paraffin-embedded (FFPE) tissue sections. We demonstrate the utility of PRIME-PAINT by analyzing ∼10⁶ caveolae structures in ∼1,000 cells and imaging entire pancreatic cancer lesions from patient tissue biopsies. By imaging from nanometers to millimeters with multiplexity and broad sample compatibility, PRIME-PAINT will be useful for building multiscale, Google-Earth-like views of biological systems.

Electrogenerated chemiluminescence (ECL) imaging is gaining increasing attention in various fields because of its high sensitivity, low background, and good temporal and spatial resolution. However, ECL imaging of microsized objects at the opaque electrode via top-view configuration is challenged with the reactants’ diffusion and light propagation. Here, we imaged and numerically simulated ruthenium derivative coating polystyrene microbeads (Ru1-PS@MB) at the glassy carbon electrode (GCE) via top-view configuration by ECL imaging. The ruthenium derivative (bis(2,2′-bipyridine)-4′-methyl-4-carboxybipyridine-ruthenium N-succinimidyl ester-bis (hexafluorophosphate), Ru1), a typical ECL reagent, was covalently linked onto the surface of aminated PS@MBs via the amide reaction. “Strong emission in edge and weak emission in center” phenomena for fluorescence (FL) and ECL emissions were obtained from Ru1-PS@MB on GCE. Z-Stack imaging of the microsized Ru1-PS@MB luminescence was performed on GCE in the presence of tri-n-propylamine (TPA). It is found that the clear luminescence range of Ru1-PS@MB perpendicular to the electrode surface in ECL image is slightly smaller than that in the FL image. The bigger was the diameter of the microbeads (from 5 to 18 μm), the larger was the ECL luminescence range of Ru1-PS@MB perpendicular to the electrode surface (from 5 to 7 μm). Our findings, which are also supported by numerical simulation, provide insights into the ECL imaging of microsized objects at the electrode surface, which will raise promising ECL applications in bioassays and cell imaging at the microscale level.

Abnormal accumulation of intracellular and extracellular β-amyloid (Aβ) aggregates is closely related to the pathogenesis of Alzheimer’s disease (AD). In this work, we use quinolinium derivatives with electron-rich aniline substituents as the skeletons to develop a set of spontaneous blinking fluorophores by the formation of long-lived radicals. These probes can target Aβ_1–40 aggregates and exhibit strong deep-red emission upon binding to Aβ_1–40 aggregates. More importantly, at the single-molecule level, these probes display spontaneous blinking, low duty cycle, and high photon output, which are suitable for the nanoscopic imaging of Aβ aggregates in living cells. The assembly process of the Aβ aggregates was then tracked with nanoscopic imaging. The elongation rate on the cell membrane was noticeably fast over that in the solution. This work provides a feasible strategy for the design of spontaneous blinking fluorophores for Aβ aggregates.

Fluorescence imaging is an emerging strategy for preoperative diagnosis and intraoperative resection. In particular, owing to their outstanding spatial resolution and deep-tissue penetration, imaging agents in the near-infrared (NIR)-II window (1000–1700 nm) have received intensive interest for biomedical applications. However, NIR II-based imaging agents for targeted visualization of hepatocellular carcinoma (HCC) have barely been barely developed. Here, we report the construction of structurally uniform, biocompatible human serum albumin (HSA)-based particles orthogonally modified with two functional peptides as a carrier for the delivery of NIR-II imaging agents to HCC cell-derived solid tumor in vivo. Cysteine conjugation combined with host–guest chemistry enables the orthogonal introduction of two functionally independent peptides to HSA-based nanoparticles. One of these peptides targets glypican-3 (GPC-3), a specific biomarker of HCC, and the other facilitates the escape of the nanoparticles from macrophagic phagocytosis. Series of cellular and in vivo assays were carried out to demonstrate the efficacy of the dual-peptide-functionalized HSA nanoparticles for targeted NIR-II fluorescence imaging of HCC.

The direct relationship between facilitative glucose transporters (GLUTs) and metabolic diseases opens new avenues for sensing metabolic deregulations and drives the development of molecular probes for GLUT-targeted detection of metabolic diseases. Radiotracer-based molecular imaging probes have been effectively utilized in reporting alterations in sugar uptake as an indication of metabolic deregulations, cancer development, or inflammation. Progress in developing fluorophore-based tools facilitated GLUT-specific analyses using more accessible fluorescence-based instrumentation. However, restrictions on the emission range of fluorophores and the requirement for substantial post-treatments to reduce background fluorescence have brought to light the critical directions for improvement of the technology for broader use in screening applications. Here we present turn-on GLUT activity reporters activated upon cells’ internalization. We demonstrate a specific delivery of a sizable rhodamine B fluorophore through GLUT5 and showcase a stringent requirement in conjugate structure for maintaining a GLUT-specific uptake. With the turn-on GLUT probes, we demonstrate the feasibility of high-throughput fluorescence microscopy and flow cytometry-based GLUT activity screening in live cells and the probes’ applicability for assessing sugar uptake alterations in vivo.