Laccase-Mediated Catalyzed Fluorescent Reporter Deposition for Live-Cell Imaging

Catalyzed reporter deposition (CARD) is a widely established method for labeling biological samples analyzed using microscopy. Horseradish peroxidase, commonly used in CARD to amplify reporter signals, requires the addition of hydrogen peroxide, which may perturb samples used in live-cell microscopy. Herein we describe an alternative method of performing CARD using a laccase enzyme, which does not require exogenous hydrogen peroxide. Laccase is an oxidative enzyme which can carry out single-electron oxidations of phenols and related compounds by reducing molecular oxygen. We demonstrate proof-of-concept for this technique through the nontargeted covalent labeling of bovine serum albumin using a fluorescently labeled ferulic acid derivative as the laccase reporter substrate. We further demonstrate the viability of this approach by performing live-cell CARD with an antibody-conjugated laccase against a surface-bound target. CARD using laccase produces an amplified fluorescence signal by labeling cells without the need for exogenous hydrogen peroxide.     

Comparing Agent-Based Delivery of DNA and PNA Forced Intercalation (FIT) Probes for Multicolor mRNA Imaging

Fluorogenic oligonucleotide probes allow mRNA imaging in living cells. A key challenge is the cellular delivery of probes. Most delivery agents, such as cell-penetrating peptides (CPPs) and pore-forming proteins, require interactions with the membrane. Charges play an important role. To explore the influence of charge on fluorogenic properties and delivery efficiency, we compared peptide nucleic acid (PNA)- with DNA-based forced intercalation (FIT) probes. Perhaps counterintuitively, fluorescence signaling by charged DNA FIT probes proved tolerant to CPP conjugation, whereas CPP–FIT PNA conjugates were affected. Live-cell imaging was performed with a genetically engineered HEK293 cell line to allow the inducible expression of a specific mRNA target. Blob-like features and high background were recurring nuisances of the tested CPP and lipid conjugates. By contrast, delivery by streptolysin-O provided high enhancements of the fluorescence of the FIT probe upon target induction. Notably, DNA-based FIT probes were brighter and more responsive than PNA-based FIT probes. Optimized conditions enabled live-cell multicolor imaging of three different mRNA target sequences.     

Potential of Single-Molecule Live-Cell Imaging for Chemical Translational Biology

Single-molecule live-cell imaging is the most direct approach for monitoring the motility of molecules in living cells. Considering the relationship between the motility of molecules and their function, information obtained from single-molecule imaging involves essential clues for understanding the regulatory mechanisms of the processes of target molecules, and translation to applied sciences such as drug discovery. In this Concept, examples of single-molecule imaging studies on G protein-coupled receptors (GPCRs) are mainly introduced, and recent techniques of single-molecule imaging for overcoming the limitation of single-molecule live-cell imaging are discussed. Based on these studies, the prospects of single-molecule imaging will be outlined.     

Biomedical Applications of Fluorescent and Magnetic Resonance Imaging Dual-Modality Probes

Molecular imaging plays a critical role in biomedical research. The combination of different modalities can generate complementary information and provide synergistic advantages over single modality alone. Noninvasive and nonradioactive fluorescent imaging (FI)/magnetic resonance imaging (MRI) dualmodality probes fuse the high sensitivity of FI and the high temporal and spatial resolution and deep-tissue penetration of MRI, and their increasing applications have been reported in biomedical research and clinical practices, including cell labeling, enzyme activity measurement, tumor diagnosis and therapy, and anatomical localization and real-time assessment during surgery.     

Peptide-Conjugated Phosphorodiamidate Morpholino Oligomers for In Situ Live-Cell Molecular Imaging of Dengue Virus Replication

Current methods to detect and monitor pathogens in biological systems are largely limited by the tradeoffs between spatial context and temporal detail. A new generation of molecular tracking that provides both information simultaneously involves in situ detection coupled with non-invasive imaging. An example is antisense imaging that uses antisense oligonucleotide probes complementary to a target nucleotide sequence. In this study, we explored the potential of repurposing antisense oligonucleotides initially developed as antiviral therapeutics as molecular probes for imaging of viral infections in vitro and in vivo. We employed nuclease-resistant phosphorodiamidate synthetic oligonucleotides conjugated with cell-penetrating peptides (i.e., PPMOs) previously established as antivirals for dengue virus serotype-2 (DENV2). As proof of concept, and before further development for preclinical testing, we evaluated its validity as in situ molecular imaging probe for tracking cellular DENV2 infection using live-cell fluorescence imaging. Although the PPMO was designed to specifically target the DENV2 genome, it was unsuitable as in situ molecular imaging probe. This study details our evaluation of the PPMOs to assess specific and sensitive molecular imaging of DENV2 infection and tells a cautionary tale for those exploring antisense oligonucleotides as probes for non-invasive imaging and monitoring of pathogen infections in experimental animal models.     

Detection and Imaging of Active Substances in Early Atherosclerotic Lesions Using Fluorescent Probes

Atherosclerosis (AS) is a vascular disease caused by chronic inflammation and lipids that is the main cause of myocardial infarction, stroke and other cardiovascular diseases. Atherosclerosis is often difficult to detect in its early stages due to the absence of clinically significant vascular stenosis. This is not conducive to early intervention or treatment of the disease. Over the past decade, researchers have developed various imaging methods for the detection and imaging of atherosclerosis. At the same time, more and more biomarkers are being found that can be used as targets for detecting atherosclerosis. Therefore, the development of a variety of imaging methods and a variety of targeted imaging probes is an important project to achieve early assessment and treatment of atherosclerosis. This paper provides a comprehensive review of the optical probes used to detect and target atherosclerosis imaging in recent years, and describes the current challenges and future development directions.     

Fluorescent Probes for HOCl Imaging

Small-molecule fluorescent probes for hypochlorous acid (HOCl), one of the poorly understood reactive oxygen species (ROS), help to unveil HOCl functions in health and disease. Numerous small-molecule HOCl fluorescent probes have been developed in the past decade. Nevertheless, only a portion of them demonstrated their practical applications in biomedical research because of common problems in selectivity, sensitivity, chemostability, and photostability, etc. The problems could be addressed by a combination of rational probe design and careful selection of fluorophore templates. In this review, we describe several classes of representative HOCl fluorescent probes based on their fluorophore templates, and we discuss their design strategies, photophysical properties, and biological applications. A comprehension of their strengths, weaknesses, and common uses will facilitate the development of ideal HOCl assays and the discovery of novel biological processes.     

Fluorescent Phase-Changing Perfluorocarbon Nanodroplets as Activatable Near-Infrared Probes

The sensitivity of fluorescence imaging is limited by the high optical scattering of tissue. One approach to improve sensitivity to small signals is to use a contrast agent with a signal that can be externally modulated. In this work, we present a new phase-changing perfluorocarbon nanodroplet contrast agent loaded with DiR dye. The nanodroplets undergo a liquid-to-gas phase transition when exposed to an externally applied laser pulse. This results in the unquenching of the encapsulated dye, thus increasing the fluorescent signal, a phenomenon that can be characterized by an ON/OFF ratio between the fluorescence of activated and nonactivated nanodroplets, respectively. We investigate and optimize the quenching/unquenching of DiR upon nanodroplets’ vaporization in suspension, tissue-mimicking phantoms and a subcutaneous injection mouse model. We also demonstrate that the vaporized nanodroplets produce ultrasound contrast, enabling multimodal imaging. This work shows that these nanodroplets could be applied to imaging applications where high sensitivity is required.     

Reaction-based AIE-active Fluorescent Probes for Selective Detection and Imaging

Fluorescent probes have been widely investigated for their features of rapid response, easy operation and high sensitivity. Among them, reaction-based fluorescent probes, for their unique reaction-based nature, guarantee them with excellent selectivity, effectively avoiding the possible interference from other chemical and biological species in physiological environment. Conventional reaction-based fluorescent probes are aggregation-caused quenching (ACQ) fluorophores. The application of these kinds of probes are limited for their poor photostability and narrow Stokes shifts. Compared with ACQ fluorophores, aggregation-induced emission (AIE) fluorophores become emissive in aggregation states with higher signal-to-noise ratio, better photostability and larger Stokes shifts. In this review, we summarize the latest developed reaction-based AIE-active probes, including the design principle and application in various sensing systems and give an outlook for the future development of this kind of promising fluorescent probes.     

A Large Stokes Shift Fluorescent Protein Constructed from the Fusion of Red Fluorescent mCherry and Far-Red Fluorescent BDFP1.6

Phycobiliproteins are constituents of phycobilisomes that can harvest orange, red, and far-red light for photosynthesis in cyanobacteria and red algae. Phycobiliproteins in the phycobilisome cores, such as allophycocyanins, absorb far-red light to funnel energy to the reaction centers. Therefore, allophycocyanin subunits have been engineered as far-red fluorescent proteins, such as BDFP1.6. However, most current fluorescent probes have small Stokes shifts, which limit their applications in multicolor bioimaging. mCherry is an excellent fluorescent protein that has maximal emittance in the red spectral range and a high fluorescence quantum yield, and thus, can be used as a donor for energy transfer to a far-red acceptor, such as BDFP1.6, by FRET. In this study, mCherry was fused with BDFP1.6, which resulted in a highly bright far-red fluorescent protein, BDFP2.0, with a large Stokes shift (≈79 nm). The excitation energy was absorbed maximally at 587 nm by mCherry and transferred to BDFP1.6 efficiently; thus emitting strong far-red fluorescence maximally at 666 nm. The effective brightness of BDFP2.0 in mammalian cells was 4.2-fold higher than that of iRFP670, which has been reported as the brightest far-red fluorescent protein. The large Stokes shift of BDFP2.0 facilitates multicolor bioimaging. Therefore, BDFP2.0 not only biolabels mammalian cells, including human cells, but also biolabels various intracellular components in dual-color imaging.     

Carbonate-Based Fluorescent Chemical Tool for Uncovering Carboxylesterase 1 (CES1) Activity Variations in Live Cells

Carboxylesterase 1 (CES1) plays a key role in the metabolism of endogenous biomolecules and xenobiotics including a variety of pharmaceuticals. Despite the established importance of CES1 in drug metabolism, methods to study factors that can vary CES1 activity are limited with only a few suitable for use in live cells. Herein, we report the development of FCP1, a new CES1 specific fluorescent probe with a unique carbonate substrate constructed from commercially available reagents. We show that FCP-1 can specifically report on endogenous CES1 activity with a robust fluorescence response in live HepG2 cells through studies with inhibitors and genetic knockdowns. Subsequently, we deployed FCP-1 to develop a live cell fluorescence microscopy-based approach to identify activity differences between CES1 isoforms. To the best of our knowledge, this is the first application of a fluorescent probe to measure the activity of CES1 sequence variants in live cells.     

Small BODIPY Probes for Combined Dual 19F MRI and Fluorescence Imaging

The combination of the two complementary imaging modalities ¹⁹F magnetic resonance imaging (MRI) and fluorescence imaging (FLI) possesses high potential for biological and medical applications. Herein we report the first design, synthesis, dual detection validation, and cytotoxic testing of four promising BODIPY dyes for dual ¹⁹F MRI–fluorescence detection. Using straightforward Steglich reactions, small fluorinated alcohols were easily covalently tethered to a BODIPY dye in high yields, leaving its fluorescence properties unaffected. The synthesized compounds were analyzed with various techniques to demonstrate their potential utility in dual imaging. As expected, the chemically and magnetically equivalent trifluoromethyl groups of the agents exhibited a single NMR signal. The determined longitudinal relaxation times T₁ and the transverse relaxation times T₂, both in the lower second range, enabled the imaging of four compounds in vitro. The most auspicious dual ¹⁹F MRI–fluorescence agent was also successfully imaged in a mouse post‐mortem within a 9.4 T small‐animal tomograph. Toxicological assays with human cells (primary HUVEC and HepG2 cell line) also indicated the possibility for animal testing.     

Live-Cell Copper-Induced Fluorescence Quenching of the Flavin-Binding Fluorescent Protein CreiLOV

CreiLOV is a flavin-binding fluorescent protein derived from the blue-light photoreceptor protein family that contains light-oxygen-voltage (LOV) sensing domains. Flavin-binding fluorescent proteins represent a promising foundation for new fluorescent reporters and biosensors that can address limitations of the well-established green fluorescent protein (GFP) family. Flavin-binding fluorescent proteins are smaller than GFPs, are stable over a wider pH range, offer rapid chromophore incorporation, and are oxygen-independent so can be applied to live anaerobic organisms. Among the flavin-binding fluorescent proteins, CreiLOV has a high quantum yield and excellent photophysical properties, making it promising for cellular applications. Here, we investigated the suitability of CreiLOV as an intensity- and fluorescence-lifetime-based metal sensor. CreiLOV selectively binds copper(II) over other biologically relevant metals with low-micromolar affinity, resulting in fluorescence quenching and a decrease in the fluorescence lifetime that can be observed in cuvettes and live bacterial cells.     

Design and Expeditious Synthesis of Quinoline-Pyrene-Based Ratiometric Fluorescent Probes for Targeting Lysosomal pH

Intracellular pH plays a significant role in many pathological and physiological processes. A series of quinoline-pyrene probes were synthesized in one-step fashion through an oxonium-ion-triggered alkyne carboamination sequence involving C−C, C−O and C−N bond formation for intracellular pH sensing. The quinoline-pyrenes showed significant red shifts at low pH. Fluorescence lifetime decay measurements of the probes showed decreases in lifetime at pH 4. The probes showed excellent selectivity in the presence of various potential interfering agents such as amino acids and cations/anions. Furthermore, the probes were found to show completely reversible emission behaviour in the window between pH 4 and 7. A morpholine-substituted quinoline-pyrene probe efficiently stained lysosomes with high Pearson correlation coefficients (0.86) with Lysotracker Deep Red DND-99 as a reference. A co-localization study of the probe with Lysotracker DND-99 showed selective intracellular targeting and a shift in fluorescence emission due to acidic lysosomal pH.     

Crenolanib-Derived Probes Suitable for Cell- and Tissue-Based Protein Profiling and Single-Cell Imaging

Crenolanib (CP-868,596), a potent inhibitor of FLT3 and PDGFRα/β, is currently under phase III clinical investigation for the treatment of acute myeloid leukemia. However, the protein targets of Crenolanib in cancer cells remain obscure, which results in difficulties in understanding the mechanism of actions and side effects. To alleviate this issue, in this study, a photoaffinity probe and two fluorescent probes were created based on Crenolanib, followed by competitive protein profiling and bioimaging studies, with the aim of characterizing the cellular targets. A series of unknown protein hits, such as MAPK1, SHMT2, SLC25A11, and HIGD1A, were successfully identified by means of pull-down/LC-MS/MS; these might provide valuable clues for understanding drug action and potential toxicities. Moreover, the fluorescent probes are suitable for imaging drug distribution at the single-cell level.     

新型喹啉类Zn2+探针的合成及细胞成像研究

以2-甲酰基-8-羟基喹啉为原料,与苯肼进行缩合反应合成了荧光分子探针HSZn,采用FTIR、NMR、HRMS对其结构进行了表征。紫外可见光谱和荧光光谱分析结果表明:该探针在水相中对Zn~(2+)具有较好的荧光增强效应,而其他金属离子对其干扰较小;HSZn的荧光强度与Zn~(2+)浓度(1.0×10–5~1.0×10–4 mol/L)具有良好的线性关系,检测限低至4.0×10–8 mol/L;Job’s曲线和LC-MS分析表明:Zn~(2+)与HSZn的络合比为1∶2,且探针在pH=5~13内对Zn~(2+)都有较灵敏的荧光响应。细胞成像实验表明:该探针可在Hela活细胞中实现对Zn~(2+)的荧光成像。     

DNA Primer Extension with Cyclopropenylated 7‐Deaza‐2′‐deoxyadenosine and Efficient Bioorthogonal Labeling in Vitro and in Living Cells

A deoxyadenosine triphosphate (dATP) analogue for DNA labeling was synthesized with the 1‐methylcyclopropene (1MCP) group at the 7‐position of 7‐deaza‐2′‐deoxyadenosine and applied for primer extension experiments. The real‐time kinetic data reveals that this 1MCP‐modified dATP analogue is incorporated into DNA much faster than that of the similarly 1MCP‐modified deoxyuridine triphosphate (dUTP) analogue. The postsynthetic fluorescent labeling of these oligonucleotides works efficiently according to PAGE analysis, and can be applied for immobilization of a functional antibody on a surface. Site‐specific labeling at two different positions in DNA was achieved and the bioorthogonality of the postsynthetic fluorescent labeling was demonstrated in living HeLa cells.     

Mitochondrion-Targeting Identification of a Fluorescent Apoptosis-Triggering Molecule by Mass Spectrometry Elucidates Drug Tracking

The real-time tracking of localization and dynamics of small molecules in organelles helps to understand their function and identification of their potential targets at subcellular resolution. To identify the mitochondrion-targeting effects of small molecules (NA-17 and NA-2a) in cancer cells, we used mass spectrometry to study their distribution and accumulation in mitochondria and in the surrounding cytoplasm thus enabling tracing of action processes of therapeutic compounds. Colocalization analysis with the aid of imaging agents suggests that both NA-17 and NA-2a display mitochondrion-targeting effects. However, MS analysis reveals that only NA-2a displays both a mitochondrion-targeting effect and an accumulation effect, whereas NA-17 only distributes in the surrounding cytoplasm. A combination of mitochondrion imaging, immunoblotting, and MS analysis in mitochondria indicated that NA-17 neither has the ability to enter mitochondria directly nor displays any mitochondrion-targeting effect. Further studies revealed that NA-17 could not enter into mitochondria even when the mitochondrial permeability in cells changed after NA-17 treatment, as was evident from reactive oxygen species (ROS) generation and cytochrome c release. In the process of cellular metabolism, NA-17 itself is firmly restricted to the cytoplasm during the metabolic process, but its metabolites containing fluorophores could accumulate in mitochondria for cell imaging. Our studies have furnished new insights into the drug metabolism processes.     

荧光小分子pH探针的最新研究进展

细胞内的H+在生理及病理过程中发挥着至关重要的作用。因此,研究细胞内的H+具有重要的理论和实践意义。荧光法由于其非破坏性,高的灵敏度和选择性,以及广泛可用的荧光染料在测定pH时比其他方法更具优越性。文章以不同的荧光团分类:香豆素、荧光素、罗丹明、BODIPY、花菁、双光子染料等,按pKa从小到大的顺序排列,综述了近十年来,荧光小分子pH探针的研究进展。