Chemical & Biomedical Imaging最新文献

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.

For specific drug research and development, a drug-screening strategy (DSS) plays an indispensable role in the biomedical field. Unfortunately, traditional strategies are complicated and insufficiently accurate due to the widely used single-target screening method. Herein, a simple dual-target-based drug-screening strategy (dt-DSS) is proposed to screen highly effective drugs by fluorescence imaging. As a proof of concept, we utilized a dual-responsive fluorescence probe to screen drugs for diabetic cardiomyopathy (DCM). We first developed and took advantage of a dual-response probe HDB to detect reactive oxygen species (ROS) and mitophagy levels in cellular starvation and high glucose models. Based on this, HDB was utilized to study the effects of different drugs in the mitophagy process caused by the high-glucose cell model for DCM. Combined with Western blotting assays, we found that Drp-1 inhibitors could fundamentally reduce mitophagy caused by the high-glucose cells model. Compared with commercial single-target antioxidant drugs, the drugs with simultaneous antioxidant capacity and Drp-1 inhibition screened by dt-DSS, such as resveratrol and icariin, could treat DCM better. Therefore, HDB as an effective tool could accurately and quickly screen high-potency drugs for DCM. We believe that this work provides an attractive strategy to explore the pathogenesis of diabetic cardiomyopathy and precisely screen for highly effective drugs.

Cerenkov radiation-induced photodynamic therapy (CR-induced PDT) has shown the potential to overcome the light penetration limitation in conventional PDT. In addition, the tumor-associated antigens (TAAs) produced by PDT can initiate an antitumor immune process but only show a limited immunotherapeutic effect without the use of immunotherapeutic agents. Herein, a CR-induced PDT hydrogel (R837/⁸⁹Zr-HG-PpIX) has been developed by in situ formation of a hyaluronic acid (HA)-based hydrogel integrated with internal light source ⁸⁹Zr, photosensitizer protoporphyrin IX (PpIX), and immune adjuvant imiquimod (R837). The obtained R837/⁸⁹Zr-HG-PpIX hydrogel with long-term tumor retention and low radiation leakage can provide long-lasting photodynamic therapy without phototoxicity in normal tissues. In addition, the loaded R837 improves the immunogenicity of TAAs released after PDT, resulting in considerably enhanced immune responses. At relatively low radioactivity, R837/⁸⁹Zr-HG-PpIX shows significant inhibition in subcutaneous H22 tumor-bearing BALB/c mice and orthotopic VX2 liver tumor-bearing rabbits. Furthermore, the combination of such a CR-induced PDT hydrogel with anti-PD-L1 exhibits the abscopal effect to inhibit the growth of distant tumors. Therefore, the proposed in situ formed CR-induced PDT hydrogel with long-term photodynamic-immunotherapy provides an effective strategy for deep tumor therapy.

Molecular imaging was developed from basic molecular recognition. It can visualize not only the expression levels of specific molecules in a living system but also specific biological processes, thus providing guidance for early detection and treatment of diseases. As a noninvasive method, imaging agents are one of the foundations of high spatial resolution imaging, and their sensitivity and specificity can be improved by coupling targeting ligands to imaging probes. Among the various targeting ligands (antibodies, aptamers, etc.), targeting peptides are widely used in various modalities of molecular imaging due to their high affinities toward the molecular target and their excellent physicochemical properties. In this review, we summarize the design concepts and methods of targeting peptides in molecular imaging, introduce the combination of targeting peptides and imaging probes in different imaging modalities (e.g., fluorescence imaging, radionuclide imaging), and provide examples of their applications in bioimaging. Finally, the challenges and strategies for clinical translation and practical application of targeting peptide-based imaging reagents are briefly discussed.