Chemical & Biomedical Imaging最新文献

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.

Characterizing and identifying cells in multicellular in vitro models remain a substantial challenge. Here, we utilize hyperspectral confocal Raman microscopy and principal component analysis coupled with linear discriminant analysis to form a label-free, noninvasive approach for classifying bone cells and osteosarcoma cells. Through the development of a library of hyperspectral Raman images of the K7M2-wt osteosarcoma cell lines, 7F2 osteoblast cell lines, RAW 264.7 macrophage cell line, and osteoclasts induced from RAW 264.7 macrophages, we built a linear discriminant model capable of correctly identifying each of these cell types. The model was cross-validated using a k-fold cross validation scheme. The results show a minimum of 72% accuracy in predicting cell type. We also utilize the model to reconstruct the spectra of K7M2 and 7F2 to determine whether osteosarcoma cancer cells and normal osteoblasts have any prominent differences that can be captured by Raman. We find that the main differences between these two cell types are the prominence of the β-sheet protein secondary structure in K7M2 versus the α-helix protein secondary structure in 7F2. Additionally, differences in the CH₂ deformation Raman feature highlight that the membrane lipid structure is different between these cells, which may affect the overall signaling and functional contrasts. Overall, we show that hyperspectral confocal Raman microscopy can serve as an effective tool for label-free, nondestructive cellular classification and that the spectral reconstructions can be used to gain deeper insight into the differences that drive different functional outcomes of different cells.

Chemiluminescence has emerged as a vital tool for bioimaging in vivo. The red shift emission of chemiluminophores is extremely useful for in vivo bioimaging. In this work, the conjugation system of the luminol was extended to achieve a red-shifted emission (591 nm) along with excellent water solubility. The probe (HM-ASPH-PF) has a molecular weight of only 396.42, which contains a benzothiazole and a cyclic phthalhydrazide structure. The probe has been used for in vivo luminescence imaging of neutrophil-mediated acute liver injury, including alcoholic liver injury (ALI) and acute liver failure (ALF) in mice, by exploiting myeloperoxidase (MPO) as a biomarker. The activated neutrophils were specifically imaged by HM-ASPH-PF. HM-ASPH-PF was also successfully applied to monitor the neutrophils in livers in mouse models of ALI and ALF. Consequently, HM-ASPH-PF, as an effective luminescent small molecule that possesses a red-shift emission near 600 nm, has been applied for the detection of MPO in living cells and neutrophil-mediated acute liver injury. This work also demonstrates the applied potential of the luminescent probe for the diagnosis of other neutrophil-associated liver diseases.

The coronavirus disease 2019 (COVID-19) has impacted health globally. Cumulative evidence points to long-term effects of COVID-19 such as cardiovascular and cognitive disorders, diagnosed in patients even after the recovery period. In particular, micrometer-sized blood clots and hyperactivated platelets have been identified as potential indicators of long COVID. Here, we resolve microclot structures in the plasma of patients with different subphenotypes of COVID-19 in a label-free manner, using 3D digital holo-tomographic microscopy (DHTM). Based on 3D refractive index (RI) tomograms, the size, dry mass, and prevalence of microclot composites were quantified and then parametrically differentiated from fibrin-rich microclots and platelet aggregates in the plasma of COVID-19 patients. Importantly, fewer microclots and platelet aggregates were detected in the plasma of healthy controls compared to COVID-19 patients. Our imaging and analysis workflow is built around a commercially available DHT microscope capable of operation in clinical settings with a 2 h time period from sample preparation and data acquisition to results.

Fluorogenic probes have shown great potential in imaging biological species as well as in diagnosing diseases, especially cancers. However, the fluorogenic mechanisms are largely limited to a few photophysical processes to date, typically including photoinduced electron transfer (PeT), fluorescence resonant energy transfer (FRET), and intramolecular charge transfer (ICT). Herein, by calculations and experiments, we set forth that the inhibition of the excited-state π-conjugation in meso-ester Si-rhodamine SiR-COOM or the de-π-conjugation in meso-ester cyanine 5 Cy5-COOM via the “ester-to-carboxylate” conversion can operate as a general fluorogenic mechanism to fabricate fluorogenic probes. Based on the mechanism and considering the higher chemical stability of Cy5-COOM than that of SiR-COOM, we developed, as a proof-of-concept, three fluorogenic probes Cy5-APN, Cy5-GGT, and Cy5-NTR on the basis of the Cy5-COOM platform for sensing cancer biomarkers aminopeptidase N (APN), γ-glutamyltranspeptidase (GGT), and nitroreductase (NTR), respectively, and demonstrated their outstanding performances in distinguishing between cancerous and normal tissues with the high tumor-to-normal tissue ratios in the range of 9–14.

Blood viscosity changes and blood clots are high-impact diseases, but the pathogenic mechanisms and detection methods are still limited. Due to the complexity of the cellular microenvironment, viscosity is a key factor in regulating the behavior of mitochondria and lysosomes in cells. Conventional fluorescence probes are highly restrictive for complex viscosity detection in live animals. Therefore, we developed two near-infrared fluorescence probes, QL1 and QL2, with dual responses to the pH and viscosity. Notably, QL2 has two maximum fluorescence emissions at 680 and 750 nm, when excitation by 580 and 700 nm, respectively. QL2 exhibited both a pH and viscosity switchable fluorescence response. The two emission peaks exhibited a reverse change trend: the fluorescence at 680 nm decreased by 90%, and the fluorescence at 750 nm increased by about 5-fold with pH from 2 to 10. Meanwhile, both emission peaks show remarkable fluorescence enhancement toward viscosity change, with 185 and 32 times enhancement, respectively. The sensing mechanism and spectral changes are confirmed by DFT calculations. QL2 was further used for viscosity imaging in live cells, zebrafish, and live animals. Most importantly, QL2 is able to successfully track changes in blood clots in live mice and organs, thus enabling the study of blood clots in cerebral strokes and the underlying pathological mechanisms.

With high efficiency, mild conditions, and rapid reaction rate, click reactions have garnered much attention in the field of bioimaging since proposed by Sharpless et al. in 2001 ( Angew. Chem., Int. Ed. 2001, 40, 2004−2021). Inspired by the regenerative pathway of d-luciferin in fireflies, Liang et al. ( Nat. Chem. 2010, 2, 54−60) raised a 2-cyanobenzothiazole (CBT)-cysteine (Cys) click condensation reaction in 2010, which exhibits a higher second-order reaction rate (9.19 M^–1 s^–1) and superior biocompatibility. As it has been developed in the past decade, remarkable progress has been made in the construction of enzyme-instructed CBT-Cys-based bioimaging probes. This review introduces the concept of the CBT-Cys click reaction, elucidates the mechanism of the CBT-Cys click reaction, and concerns the development progress of CBT-Cys reaction and its derived reactions [i.e., 2-cyano-6-hydroxyquinoline (CHQ)-Cys reaction and 2-pyrimidinecarbonitrile (PMN)-Cys reaction]. Furthermore, we give a comprehensive and up-to-date review of enzyme-instructed CBT-Cys-like click reaction-based probes with significantly enhanced imaging signal and contrast for various bioimaging modes, including fluorescence imaging, photoacoustic imaging, magnetic resonance imaging, and positron emission tomography. In the end, we discuss the possible challenges and opportunities that may arise in the future.

Extracellular vesicles (EVs) are small, membrane-bound structures released by various cell types into the extracellular environment, which play a crucial role in intercellular communication and the transfer of biomolecules between cells. Given their functional significance, there are intense research interests to use EVs as disease markers and drug carriers. However, EVs characterization is greatly hindered by the small size, the low biomolecule payload, and the high level of heterogeneity. To address these challenges, researchers have adopted sensitive microscopic methods such as single-molecule fluorescence imaging, single-particle dark-field imaging, surface-enhanced Raman scattering, and surface plasmon resonance imaging for single EV analysis. These techniques can detect signals from individual EVs, enabling a detailed study of the heterogeneity. Analysis of EVs cargo has provided insights into the protein/nucleic acid expression and enabled subgroup differentiation. Superresolution mapping has visualized EVs structures, and single EV tracking has offered insights into their release and uptake mechanisms. In this review, we will summarize the recent advances in optical imaging of single EVs, including the biomarkers used for EV labeling, the performance of the reported microscopic methods, and their biological findings. Finally, we will address the limitations of the existing methods and outline prospects for future development in this field.