Lysosomal Imaging Research Articles

High-fidelity imaging probe for lysosomes and selective visualization of cancer cells and tissues

Lysosome is one of the most crucial organelles in living systems. Lysosomal viscosity is an important microenvironment parameter in lysosomes, which is closely related to the occurrence and development of many diseases, including cancer. To monitor the highly dynamic lysosomes and the lysosomal viscosity changes, we developed a versatile high-performance fluorescent probe DCMP in this work. DCMP shows stronger fluorescence under acidic conditions or in solutions with higher viscosity, especially with high sensitivity to the change of viscosity. More importantly, DCMP can use a low dose (100 nM) and can target lysosomes and image lysosomes with high signal-to-noise ratio and high fidelity. It can also track the lysosomal motility and viscosity changes in real-time, and effectively monitor the interaction between lysosomes and damaged organelles. We also found that DCMP can be successfully used to selectively and sensitively light up cancer cells and cancer tissues. All the results show that this new probe has great potential not only in lysosomal high-fidelity imaging, but also in cancer detection.

Highly emissive dimethylamino naphthalenyl phenylethene derivatives for visualization of latent fingerprints and imaging of lysosomes

Organic fluorescent materials have been widely used in various fields including visualization of latent fingerprints and imaging of lysosomes. High-performance organic fluorescent molecules based on simple molecular structures are still limited and highly demanded for high-definition and high-contrast imaging of latent fingerprints and lysosomes. In this work, two organic fluorescent molecules (DANPAN and DANBEN) were developed based on dimethylamino naphthalenyl phenylethene structure. These two molecules have quite different fluorescent properties, and the position of cyano group significantly affects the optical properties of these dimethylamino naphthalenyl phenylethene derivatives. Compound DANPAN shows strong yellow emission in solid state, while compound DANBEN has strong fluorescent emission in organic solutions. In addition to these differences, both compounds have large Stokes shifts and positive solvatochromic properties. Further studies showed that these compounds could be used to display latent fingerprints on various substrate surfaces. The obtained fingerprint images have excellent contrast and high resolution. It is also found that these molecules could specifically stain lysosomes in living cell within short incubation time. This work provides a series of new high-performance organic fluorescent materials with diverse applications.

Near-Infrared-Emitting Silver Nanoclusters as Fluorescent Probes for Super-resolution Radial Fluctuation Imaging of Lysosomes

Near-Infrared-Emitting Silver Nanoclusters as Fluorescent Probes for Super-resolution Radial Fluctuation Imaging of Lysosomes

Rational design of water-soluble supramolecular AIEgen with ultra-high quantum yield, and its application in α-amylase activity detection and wash-free lysosome imaging

A class of AIEgens with D-π-A structure and high quantum yield was designed and synthesized, in which the solid-state fluorescence quantum yield of TTAM was as high as 58.30% in the study. The supramolecular AIEgen TTAM β-CDs constructed by a simple method could be used for the detection of α-amylase activity, wash-free lysosomal imaging and lysosomal dynamic monitoring. The assay has the advantages of easy manipulation, high sensitivity (LOD = 0.007902 U·mL−1) and high specificity. TTAM β-CDs method was successfully applied to the determination of α-amylase activity in human oral saliva with high accuracy, which was almost error-free compared to commercial method. It was found that TTAM β-CDs, with a high lysosomal colocalization coefficient (0.91) in HeLa cells, was not only avoid the tedious water washing process but also reduce the interference of cell secretions on lysosomal imaging signals in the study of lysosomal imaging, and its precise monitoring of lysosomal translocation was of great significance for the study of tumor invasion and metastasis.

Quinolinium-based viscosity probes for lysosome imaging and tracing lysosomal viscosity changes in living cells

Lysosomal viscosity fluctuation is related to various diseases such as diabetes, neurodegenerative diseases, and cancer. We designed and synthesized four novel pH-insensitive fluorescent viscosity probes (Lyso-QAP1–4) using a quinolinium as the fluorophore and acceptor group while an internal amino benzene ring was incorporated as a donor group and a targeting group for monitoring the viscosity change of lysosomes. Lyso-QAP1–4 possessed a highly sensitive response toward viscosity change with red emission at around 625–640 nm (excited around 570–580 nm), excellent water solubility, good photostability, favorable membrane permeabilization, and moderate cytotoxicity. Colocalization study proved that Lyso-QAP1–4 probes exhibited fast lysosomal detection (within 15 min) without influencing effect from other microenvironments like pH, polarity, and interferent species but Lyso-QAP4 (without a phenyl ring on C2 of quinolinium) also localized in nucleus. Importantly, we demonstrated that a phenyl ring on C2 of quinolinium and an internal amino benzene played an important role in the lysosome specificity. Lyso-QAP1–4 probes can be applied for intracellular viscosity detection. Moreover, Lyso-QAP3 was successfully applied to living cell imaging for cellular and lysosomal viscosity changes. These results suggest that Lyso-QAP3 would provide new opportunities for biomedical diagnosis and imaging applications.

Three-dimensional structured illumination microscopy data of mitochondria and lysosomes in cardiomyoblasts under normal and galactose-adapted conditions

This three-dimensional structured illumination microscopy (3DSIM) dataset was generated to highlight the suitability of 3DSIM to investigate mitochondria-derived vesicles (MDVs) in H9c2 cardiomyoblasts in living or fixed cells. MDVs act as a mitochondria quality control mechanism. The cells were stably expressing the tandem-tag eGFP-mCherry-OMP25-TM (outer mitochondrial membrane) which can be used as a sensor for acidity. A part of the dataset is showing correlative imaging of lysosomes labeled using LysoTracker in fixed and living cells. The cells were cultivated in either normal or glucose-deprived medium containing galactose. The resulting 3DSIM data were of high quality and can be used to undertake a variety of studies. Interestingly, many dynamic tubules derived from mitochondria are visible in the 3DSIM videos under both glucose and galactose-adapted growth conditions. As the raw 3DSIM data, optical parameters, and reconstructed 3DSIM images are provided, the data is especially suitable for use in the development of SIM reconstruction algorithms, bioimage analysis methods, and for biological studies of mitochondria.

Accessing conjugated and twisted structures for efficient dual-state emission fluorophore and its sensitive lysosomal imaging

Herein, based on 1,8-naphtholactam, we have developed twisted and conjugated fluorophores, NPM-1 and NPM-2, which featured efficient emissions in both dispersion and aggregation states with quantum yields up to 0.76 and 0.84, respectively. From spectral and density functional theory (DFT) calculation, the luminescence mechanism of both compounds was intramolecular charge transfer (ICT) in this D-A type molecules, and the structural differences lead to differences in the intensity of ICT effects. Moreover, NPM-1 exhibited efficient and stable emissions in a variety of environments due to its rational structural design. It was a sensitive lysosome-targeting dye and retains its fluorescence intensity under continuous laser irradiation. All these features made it an unprecedented chemical tool for real-time monitoring of lysosomal status and changes, leading to a better understanding of physiological and pathological processes.

Superparamagnetic Iron Oxide Nanoparticles with Large Magnetic Saturation and High Particle Photon Counts for Super-Resolution Imaging of Lysosomes

Superparamagnetic Iron Oxide Nanoparticles with Large Magnetic Saturation and High Particle Photon Counts for Super-Resolution Imaging of Lysosomes

Covalent organic framework-based fluorescent nanoprobe for intracellular pH sensing and imaging

Lysosomes are the acidic organelles in the cells that play an important role in intracellular degradation and other various cellular functions. The pH disturbance of lysosomes will result in the lysosomal dysfunction and many lysosomal related diseases. In this work, we reported a methoxy-based covalent organic framework (TAPB-DMTP-COF) that a novel pH-responsive fluorescent probe for lysosomal pH imaging in cells. The prepared TAPB-DMTP-COF presented regular crystal structure, low toxicity and good pH responsive property. The rich imine structure in the material enabled pH-responsive properties of the TAPB-DMTP-COF and made it exhibited pH-dependent fluorescence response. Good detection linearity for pH measurements in aqueous solution was achieved by this probe. Moreover, the TAPB-DMTP-COF can be used for the selective lysosomal pH imaging. Confocal fluorescence imaging results demonstrated that the pH fluctuations (from 4.0 to 7.4) and the pH changes in lysosomes can be effectively monitored in situ by the developed probe. This study may provide a new avenue for the intracellular pH sensing, deep study and understanding about the mechanism of diseases related to abnormal lysosomal pH.

Three in One: Stimuli-Responsive Fluorescence, Solid-State Emission, and Dual-Organelle Imaging Using a Pyrene-Benzophenone Derivative.

Small organic luminogens, owing to their contrasting stimuli-responsive fluorescence in solution along with strong emission in aggregated and solidstates, have been employed in optoelectronic devices, sensors, and bioimaging. Pyrene derivatives usually exhibit strong fluorescence and concentration-dependent excimer/aggregate emission in solution. However, the impacts of microenvironments on the monomer and aggregate emission bands and their relative intensities in solution, solid, and supramolecular aggregates are intriguing. The present study delineates a trade-off between the monomer and aggregate emissions of a pyrene-benzophenone derivative (ABzPy) in solution, in the solid-state, and in nanoaggregates through a combined spectroscopic and microscopic approach. The impact of external stimuli (viscosity, pH) on the aggregate emission was demonstrated using steady-state and time-resolved spectroscopy, including fluorescence correlation spectroscopy and fluorescence anisotropy decay analysis. The aggregate formation was noticed at a higher concentration (>10 μM) in solution, at 77 K (5 μM), and in the solid-state due to the π-π stacking interactions (3.6 Å) between two ABzPy molecules. In contrast, no aggregate formation was observed in the viscous medium as well as in a micellar environment even at a higher concentration of ABzPy (50 μM). The crystal structure analysis further shed light on the intermolecular hydrogen-bonding-assisted solid-state emission, which was found to be highly sensitive toward external stimuli like pH and mechanical forces. The broad emission band comprising both monomer and aggregate in the aqueous dispersion of nanoaggregates was used for the specific cellular imaging of lysosomes and lipid droplets, respectively.

The fluorescence properties of 4′-Methoxychalcone derivates modified by substituents and investigation of lysosomal imaging

Three types of 4′-methoxychalcone derivatives Cha-1 ∼ Cha-9 with different electron-withdrawing or electron-donating substituents at different positions were synthesized and studied. When the A ring has a hydroxyl group at the 2-position, the strong electron-donating groups at the 4-position of the B ring was more conducive to the near-infrared and aggregation-enhanced emission (AEE) features of the compound than the electron withdrawing groups. The single-crystal analysis confirmed that the good planarity of the 4′-methoxychalcone derivatives favors their fluorescence quantum yields. Moreover, chalcone fluorophores with different fluorescence properties could be obtained by modulating substituent at the 2-position of A ring. In addition, Cha-9 was selected as the AEE luminogen to locate the lysosomes in HeLa cells. These results provided fundamental knowledge for the design and application of 4′-methoxychalcone derivatives of aggregation-induced emission (AIE) compounds.

Nitrogen-doped cyan-emissive carbon quantum dots for fluorescence tetracycline detection and lysosome imaging.

Tetracyclines (TCs) prevent the growth of peptide chains and the synthesis of proteins, and they are widely used to inhibit Gram-positive and -negative bacteria. For the detection of tetracyclines in cell and in vitro, a convenient and simple detection system based on nitrogen-doped cyan carbon quantum dots (C-CQDs) was developed. C-CQDs have excellent excitation-independent properties, the best optimal excitation peak is 360 nm and the best emission peak is 480 nm. Based on the inner filter effect (IFE), the fluorescence intensity of C-CQDs in solution decreases with the increase of tetracyclines. In the range of 0-100 μM, C-CQDs present a good linear relationship with three tetracyclines (CTC, TET, OCT), with R 2 all greater than 0.999. C-CQDs can detect tetracycline in milk samples with recovery in the range of 98.2-103.6%, which demonstrates their potential and broad application in real samples. Furthermore, C-CQDs exhibit excellent lysosomal targeting, as indicated by a Pearson's coefficient of 0.914 and an overlap of 0.985. The internalisation of C-CQDs was mainly affected by lipid raft-mediated endocytosis in endocytic pathway experiments. These experiments indicate that C-CQDs can be effectively used to detect TC content and target lysosomes as an alternative to commercial dyes.

A pH-activated fluorescent probe via transformation of azo and hydrazone forms for lysosomal pH imaging.

We developed a fluorescent probe Sth-NH by introducing a 6-hydroxypyridone skeleton. The presence of an active proton enables the probe to transform from a deprotonated azo form to a hydrazone form in a strongly acidic environment to realize fluorescence light-up behavior, thus monitoring the lower lysosomal pH of cancer cells and distinguishing them from normal cells.

Polarization modulation with optical lock-in detection reveals universal fluorescence anisotropy of subcellular structures in live cells

The orientation of fluorophores can reveal crucial information about the structure and dynamics of their associated subcellular organelles. Despite significant progress in super-resolution, fluorescence polarization microscopy remains limited to unique samples with relatively strong polarization modulation and not applicable to the weak polarization signals in samples due to the excessive background noise. Here we apply optical lock-in detection to amplify the weak polarization modulation with super-resolution. This novel technique, termed optical lock-in detection super-resolution dipole orientation mapping (OLID-SDOM), could achieve a maximum of 100 frames per second and rapid extraction of 2D orientation, and distinguish distance up to 50 nm, making it suitable for monitoring structural dynamics concerning orientation changes in vivo. OLID-SDOM was employed to explore the universal anisotropy of a large variety of GFP-tagged subcellular organelles, including mitochondria, lysosome, Golgi, endosome, etc. We found that OUF (Orientation Uniformity Factor) of OLID-SDOM can be specific for different subcellular organelles, indicating that the anisotropy was related to the function of the organelles, and OUF can potentially be an indicator to distinguish normal and abnormal cells (even cancer cells). Furthermore, dual-color super-resolution OLID-SDOM imaging of lysosomes and actins demonstrates its potential in studying dynamic molecular interactions. The subtle anisotropy changes of expanding and shrinking dendritic spines in live neurons were observed with real-time OLID-SDOM. Revealing previously unobservable fluorescence anisotropy in various samples and indicating their underlying dynamic molecular structural changes, OLID-SDOM expands the toolkit for live cell research.

Novel heterobimetallic Ir(III)-Re(I) complexes: design, synthesis and antitumor mechanism investigation.

The reasonable design of binuclear or multinuclear metal complexes has demonstrated their potential advantages in the anticancer field. Herein, three heterobimetallic Ir(III)-Re(I) complexes, [Ir(C^N)2LRe(CO)3DIP](PF6)2 (C^N = 2-phenylpyridine (ppy, in IrRe-1), 2-(2-thienyl)pyridine (thpy, in IrRe-2) and 2-(2,4-difluorophenyl)pyridine (dfppy, in IrRe-3); L = pyridylimidazo[4,5-f][1,10]phenanthroline; DIP = 4,7-diphenyl-1,10-phenanthroline), were designed and synthesized. The heterobimetallic IrRe-1-3 complexes show pH-sensitive emission properties, which can be used for specific imaging of lysosomes. Additionally, IrRe-1-3 display higher cytotoxicity against tested tumor cell lines than the clinical chemotherapeutic drug cisplatin. Further mechanisms indicate that IrRe-1-3 can induce apoptosis and autophagy, increase intracellular reactive oxygen species (ROS), depolarize the mitochondrial membrane (MMP), block the cell cycle at the G0/G1 phase and inhibit cell migration. To the best of our knowledge, this is the first example of the synthesis of heterobimetallic Ir(III)-Re(I) complexes with superior anticancer activities and evaluation of their anticancer mechanisms.

Quinolinium-Based Viscosity Probes for Lysosome Imaging and Tracing Lysosomal Viscosity Changes in Living Cells

Quinolinium-Based Viscosity Probes for Lysosome Imaging and Tracing Lysosomal Viscosity Changes in Living Cells

Rational Design of Water-Soluble Supramolecular Aiegen with Ultra-High Quantum Yield, and its Application in Α-Amylase Activity Detection and Wash-Free Lysosome Imaging

Rational Design of Water-Soluble Supramolecular Aiegen with Ultra-High Quantum Yield, and its Application in Α-Amylase Activity Detection and Wash-Free Lysosome Imaging

Multicolor Photoactivatable Raman Probes for Subcellular Imaging and Tracking by Cyclopropenone Caging.

Photoactivatable probes, with high-precision spatial and temporal control, have largely advanced bioimaging applications, particularly for fluorescence microscopy. While emerging Raman probes have recently pushed the frontiers of Raman microscopy for noninvasive small-molecule imaging and supermultiplex optical imaging with superb sensitivity and specificity, photoactivatable Raman probes remain less explored. Here, we report the first general design of multicolor photoactivatable alkyne Raman probes based on cyclopropenone caging for live-cell imaging and tracking. The fast photochemically generated alkynes from cyclopropenones enable background-free Raman imaging with desired photocontrollable features. We first synthesized and spectroscopically characterized a series of model cyclopropenones and identified the suitable light-activating scaffold. We further engineered the scaffold for enhanced chemical stability in a live-cell environment and improved Raman sensitivity. Organelle-targeting probes were then generated to achieve targeted imaging of mitochondria, lipid droplets, endoplasmic reticulum, and lysosomes. Multiplexed photoactivated imaging and tracking at both subcellular and single-cell levels was next demonstrated to monitor the dynamic migration and interactions of the cellular contents. We envision that this general design of multicolor photoactivatable Raman probes would open up new ways for spatial-temporal controlled profiling and interrogations in complex biological systems with high information throughput.

Real-Time and High-Fidelity Tracking of Lysosomal Dynamics with a Dicyanoisophorone-Based Fluorescent Probe.

The development of high-performance probes that can visualize and track the dynamic changes of lysosomes is very important for the in-depth study of lysosomes. Herein, we report that a dicyanoisophorone-based probe (named DCIP) can be used for high-fidelity imaging of lysosomes and lysosomal dynamics. DCIP can be easily prepared and shows strong far-red to near-infrared emissions centered at 653 nm in water with a huge Stokes shift (224 nm), high quantum yield (Φ = 0.15), high pKa value (∼8.79), and good biocompatibility. DCIP also shows good cell permeability and can label lysosomes rapidly with bright fluorescence without a time-consuming washing process before imaging. DCIP also possesses good photostability and negligible background, making it effective for long-term and high spatiotemporal resolution (0.44 s of exposure) imaging of lysosomes. Moreover, DCIP achieved high-fidelity tracking of lysosomal dynamics at an extremely low concentration (1 nM). Finally, we also demonstrated that DCIP could real-time track the interactions of lysosomes with other organelles (damaged mitochondria as a model) and image the drug-escape processes from lysosomes. All of the results show that DCIP holds broad prospects in lysosome-related research.

A simple hydrazone probe for recognition of Al3+ and PPi and its applicability in lysosomal imaging

A simple hydrazone probe (1) was designed and synthesized for the successive detection of Al3+ and pyrophosphate (PPi) in almost 100% buffer environment. The probe provided O2N donor set for chelation with Al3+, leading to a distinct fluorescence boost at 510 nm. The in-situ formed 1-Al3+ complex detected PPi with an “on-off” behavior. The detection limits for Al3+ and PPi were 35.7 nM and 76 nM, respectively. Benefiting from the existence of morpholine as lysosome-targeting group, probe 1 was successfully applied to the detection of Al3+ and PPi in lysosomes.