Visual Probe Research Articles

An ultra-photostable and leakage-free fluorescent probe for long-term and high-fidelity mitochondria visualization and tracking

Mitochondria are important morphologically diverse organelles, and each characteristic has specific physiological significance. Thus, mitochondrial long-term and high-fidelity visualization and tracking are valuable. As a consequence, an ultra-photostable and leakage-free fluorescent probe is desired. Herein, we report a cationic probe DQPI-12 with a long alkyl chain, which can exclusively and firmly target mitochondria independent of mitochondria membrane potential changes. In particular, even after the cells have been fixed and left in place for 12 days, filamentous fluorescence signals from mitochondria could still be observed clearly, indicating DQPI-12 possesses excellent leakage-free performance. At the same time, compared with commercial mitochondria probes and those reported by others, DQPI-12 also displays excellent photostability. Inherent characters of DQPI-12 enable it to long-term and high-fidelity visualize mitochondria within a special microscopic field under continuous or intermittent irradiation of laser. Benefit from these advantages, DQPI-12 is successfully applied to track mitochondrial dynamic processes in living HeLa cells and neurons, and monitor the occurrence of mitophagy real-time, in-situ, and long-term.

Intramolecular cyclization reaction enabling fluorescent probes for dual-color visualization of lysosomes and lipid droplets with prominent spectral shift

Fluorescent probes that enable labeling lipid droplets (LDs) and lysosomes in different emission colors are promising tools to reveal the interaction and interplay of the two organelles. However, currently such fluorescent probes displayed limited spectral difference (no larger than 150 nm) in the two organelles, and the crosstalk signals may hinder clearly visualizing their interplay. In this work, based on the pH-dependent reversible intramolecular cyclization reaction of spiropyran, two fluorescent probes were constructed to visualize LDs and lysosomes in blue and near-infrared (NIR) fluorescent colors with prominent spectral shift up to 290 nm. The probes were designed as simple D-π-A structure, and the change in both conjugated size and ICT effect should be responsible for the extremely large spectral differences. With the probe, the different regulation of LDs and lysosomes amount under two kinds of high-fat conditions was revealed, the dynamic interplays of the two organelles were in-situ monitored, and the distinctive changes of lysosomal pH and LDs amount during autophagy were for the first time revealed.

Tell me how you feel, I will tell you what you look at: Impact of mood and craving on alcohol attentional bias in binge drinking.

Alcohol-related attentional bias (AB) is thought to play a key role in the emergence and maintenance of excessive alcohol use. Recent models suggest that AB, classically considered as a permanent feature in alcohol use disorders, is rather modulated by temporary motivational states. We explored the influence of current mood and craving on AB in binge drinking, through a mood induction procedure combined with eye-tracking measures of AB. In Experiment 1, we measured AB (visual probe task with eye-tracking measures) among binge drinkers (n = 48) and light drinkers (n = 32) following positive, negative and neutral mood inductions. Participants reported subjective craving and mood before/after induction. In Experiment 2, we measured AB among the same binge drinkers compared with 29 moderate drinkers following alcohol-related negative, non-alcohol-related negative and neutral mood inductions. In Experiment 1, induced negative mood and group positively predicted subjective craving, which was positively associated with AB. We found no effect of induced positive mood nor a direct mood-AB association. In Experiment 2, the relationships AB presented with both induced negative mood and group were again mediated by craving. Inducing alcohol-related negative mood did not modify the mood-craving association. Alcohol-related AB is not a stable binge drinking characteristic but rather varies according to transient motivational (i.e., craving) and emotional (i.e., negative mood) states. This study provides important insights to better understand AB in subclinical populations and emphasizes the importance of considering motivational and affective states as intercorrelated, to offer multiple ways to reduce excessive alcohol use.

Construction of an intelligent fluorescent probe that can accurately track β-galactosidase activity in fruits and living organisms

As a hydrolytic enzyme, β-galactosidase (β-Gal) is widely present in various fruits and organisms. It was found that β-Gal activity was positively correlated with fruit ripening and was also a biomarker for some cancers. Therefore, the construction of a highly sensitive and visual β-Gal fluorescent probe is of great significance for fruit processing and preservation and early diagnosis of cancer. Here, a proportional fluorescent probe CB-FR that can be used to detect β-Gal activity ultra-rapidly by smartphone was developed. This method requires low volume (20 μL) and only takes 3 min for each measurement. The spectral data showed that the detection limit of β-Gal was very low and had a fast response (<15 s). We hope that the probe can be used as a powerful tool for real-time and rapid detection of β-Gal activity in fruits and organisms, which will have positive significance for further understanding of physiological and pathological processes in animals and plants.

Impact of cannabidiol on reward- and stress-related neurocognitive processes among individuals with opioid use disorder: A pilot, double-blind, placebo-controlled, randomized cross-over trial.

Opioid use disorder (OUD) continues to be a significant public health concern. Medications for OUD (MOUD) such as buprenorphine reduce overdose mortality, but relapses occur often, leading to adverse outcomes. Preliminary data suggest that cannabidiol (CBD) may be a potential adjunctive treatment to MOUD by attenuating cue-reactivity. This pilot study sought to evaluate the impact of a single dose of CBD on reward- and stress-related neurocognitive processes implicated in relapse among those with OUD. The study was a pilot, double-blind, placebo-controlled, randomized cross-over trial aimed at assessing the effects of a single dose of CBD (Epidiolex®) 600 mg or matching placebo administered to participants with OUD receiving either buprenorphine or methadone. Vital signs, mood states, pain, opioid withdrawal, cue-induced craving, attentional bias, decision-making, delayed discount, distress tolerance, and stress-reactivity were examined at each testing session on two separate testing days at least 1 week apart. Ten participants completed all study procedures. Receipt of CBD was associated with a significant decrease in cue-induced craving (0.2 vs. 1.3, p = 0.040), as well as reduced attentional bias toward drug-related cues as measured by the visual probe task (-80.4 vs. 100.3, p = 0.041). No differences were found among all the other outcomes examined. CBD may have promise as an adjunct to MOUD treatment by attenuating the brain response to drug-related cues, which, in turn, may reduce the risk of relapse and overdoses. Further research is warranted to evaluate the potential for CBD as an adjunctive therapy for individuals in treatment for OUD. https://clinicaltrials.gov/ct2/show/NCT04982029.

Boron Dopants in Red-Emitting B and N Co-Doped Carbon Quantum Dots Enable Targeted Imaging of Lysosomes.

Lysosomes are of great significance to cell growth, metabolism, and survival, as they independently maintain acidity and regulate various balances in cells. Therefore, it is essential to develop advanced probes for lysosome visualization and live tracking. Herein, a type of lysosome-targeting probe based on boron (B) and nitrogen (N) co-doped carbon quantum dots (B/N-CQDs) is presented, which exhibits red emission at 618 nm, high quantum yield (28%), and excellent fluorescence stability (97% at 1 h). These B/N-CQDs are prepared by a novel and green solid-state reaction and purified using a simple extraction process without additional chemical modifications. It is found that the boron dopants in the structure play a crucial role in the resultant lysosome-specific targeting property through borate esterification between boronic acid groups in the sample and diol structures in glycoproteins. This can be applied as a powerful tool for cell apoptosis, necrosis, and endosomal escape tracking. This work not only offers a new concept for targeted subcellular probe designs via chemical doping but also demonstrates the feasibility of these tools for analyzing complex cellular physiological activities.

Development of near-infrared lysosomal pH-activatable fluorescent probe for real-time visualization of autophagy progression

As the central role in maintaining physiological homeostasis, tracking the autophagy in living cells is highly desired in biological research and pharmacologic development. Herein, a lysosome-targeted pH-activatable probe (Lyso-QL) with emission at near-infrared region (NIR) was developed for monitoring the progression of autophagy. During autophagy progress, the double-membraned autophagosomes are engulfed into lysosomes to provide autolysosomes, exhibiting significantly enhanced acidification. Based on the characteristic, the real-time monitoring of autophagic flux were realized by evaluating the pH changes in these acidic compartments with Lyso-QL under nutrient-deprivation conditions as well as pharmacological induction. Importantly, through the NIR changes, the modulation of tamoxifen on autophagic levels in estrogen receptor-negative cancer cells was disclosed. Further, the elevated hepatic autophagy in mice during acetaminophen-induced liver injury was revealed, and the N-acetylcysteine pretreatment was demonstrated with a certain protective effect to restore the autophagic levels. The study demonstrates probe Lyso-QL would provide an edge for mechanistic insights into autophagy and evaluating the activities of autophagy modulators.

Mitochondria immobilized fluorescent probe for dual-emissive monitoring mitophagy based on aggregation/monomer principle

Mitophagy plays essential roles in mitochondrial quality control, metabolism regulation, and other crucial biological procedures, and abnormal regulation of mitophagy was directly related to serious diseases. Fluorescent probes are important tools for the visualization and evaluation of mitophagy, but the presented probes were primary based on pH-responsive mechanism, which were significantly interfered by the pH variation in lysosomes and mitochondria. To overcome this, herein, we have for the first time constructed a mitochondria-immobilized probe for dual-emissive visualization of mitophagy based on aggregation/monomer principle. The robust probe was enriched in mitochondria and immobilized via the reaction with thiol group of mitochondrial proteins, and formed aggregates with near-infrared (NIR) fluorescence. During mitophagy, the probe molecules were delivered into autophagolysosomes together with mitochondria, which switched to monomers to give green emission. In this manner, the mitophagy procedure was visualized in dual-emissive mode. With the robust probe, the mitophagy induced by starvation, oxidative stress, and hypoxia has been successfully visualized. In particular, the unique probe enabled monitoring mitophagy on non-pH-responsive mechanism, which can serve as the important complement for the researches on mitophagy.

Molecular engineering of benzenesulfonyl analogs for visual hydrogen polysulfide fluorescent probes based on Nile red skeleton

Hydrogen polysulfide (H2Sn, n > 1) has a valuable function in various aspects of biological regulation. Therefore, it is of great significance to realize the visual monitoring of H2Sn levels in vivo. Herein, a series of fluorescent probes NR-BS were constructed by changing types and positions of substituents on the benzene ring of benzenesulfonyl. Among them, probe NR-BS4 was optimized due to its wide linear range (0 ∼ 350 μM) and little interference from biothiols. In addition, NR-BS4 has a broad pH tolerance range (pH = 4 ∼ 10) and high sensitivity (0.140 μM). In addition, the PET mechanism of probe NR-BS4 and H2Sn was demonstrated by DFT calculations and LC-MS. The intracellular imaging studies indicate that NR-BS4 can be successfully devoted to monitor the levels of exogenous and endogenous H2Sn in vivo.

Rapid imaging of thymoma and thymic carcinoma with a fluorogenic probe targeting γ-glutamyltranspeptidase

In recent years, thoracoscopic and robotic surgical procedures have increasingly replaced median sternotomy for thymoma and thymic carcinoma. In cases of partial thymectomy, the prognosis is greatly improved by ensuring a sufficient margin from the tumor, and therefore intraoperative fluorescent imaging of the tumor is especially valuable in thoracoscopic and robotic surgery, where tactile information is not available. γ-Glutamyl hydroxymethyl rhodamine green (gGlu-HMRG) has been applied for fluorescence imaging of some types of tumors in the resected tissues, and here we aimed to examine its validity for the imaging of thymoma and thymic carcinoma. 22 patients with thymoma or thymic carcinoma who underwent surgery between February 2013 and January 2021 were included in the study. Ex vivo imaging of specimens was performed, and the sensitivity and specificity of gGlu-HMRG were 77.3% and 100%, respectively. Immunohistochemistry (IHC) staining was performed to confirm expression of gGlu-HMRG's target enzyme, γ-glutamyltranspeptidase (GGT). IHC revealed high GGT expression in thymoma and thymic carcinoma in contrast to absent or low expression in normal thymic parenchyma and fat tissue. These results suggest the utility of gGlu-HMRG as a fluorescence probe for intraoperative visualization of thymomas and thymic carcinomas.

Post-interval potentials in temporal judgements

Research suggests that post-stimulus positive deflections could be associated with timing. We compared offset-locked potentials N1, P2, N1P2, and late positive component (LPC) in temporal generalization and temporal bisection—with visual probe intervals. In both tasks, the LPC amplitude decreased with the duration of the current probe interval. A larger LPC was found after shorter intervals, whereas other ERP amplitudes did not change between tasks or across durations. We also found that the LPC for different responses indicates subjective time. We discussed our findings in relation to theories of human timing.

Fabrication of Eu3+-dipicolinic acid complex functionalized nanoSiO2 composites and their Langmuir-Blodgett films as visual fluorescence probe for tetracycline and oxytetracycline

Luminescent lanthanide complexes have attracted growing attention in the visual probe for detection of toxic and dangerous materials. Here, we report an interfacial self-assembly of Eu3+-dipicolinic acid complex functionalized nanoSiO2 composites (nanoSiO2PyTAEu) and their Langmuir-Blodgett (LB) films as well as the luminescent probes to tetracycline (TC) and oxytetracycline (OTC). Organic linkers and Eu3+ ions are anchored on the hydrophilic nanoSiO2 particles’ surface via the surface silanization, substitution and coordination reactions, so they exhibit high stability and well distribution in the solutions and LB films. Both the nanocomposites of nanoSiO2PyTAEu and their LB films show strong and enhanced red fluorescence emissions of Eu3+ ions when they are immersed in the aqueous TC solution, with the detection limit to 3.3 and 0.91 nmol/L, respectively. The present nanocomposites and LB films have the advantages of high sensitivity and stability.

Food word processing in Chinese reading: A study of restrained eaters.

Food-related attentional bias refers that individuals typically prioritize rewarding food-related cues (e.g. food words and food images) compared with non-food stimuli; however, the findings are inconsistent for restrained eaters. Traditional paradigms used to test food-related attentional bias, such as visual probe tasks and visual search tasks, may not directly and accurately enough to reflect individuals' food-word processing at different cognitive stages. In this study, we introduced the boundary paradigm to investigate food-word attentional bias for both restrained and unrestrained eaters. Eye movements were recorded when they performed a naturalistic sentence-reading task. The results of later-stage analyses showed that food words were fixated on for less time than non-food words, which indicated a superiority of foveal food-word processing for both restrained and unrestrained eaters. The results of early-stage analyses showed that restrained eaters spent more time on pre-target regions in the food-word valid preview conditions, which indicated a parafoveal food-word processing superiority for restrained eaters (i.e. the parafoveal-on-foveal effect). The superiority of foveal food-word processing provides new insights into explaining food-related attentional bias in general groups. Additionally, the enhanced food-word attentional bias in parafoveal processing for restrained eaters illustrates the importance of individual characteristics in studying word recognition.

A glutathione-activated NIR-II fluorescent probe for precise localization of micrometastases

Accurate detection and localization of micrometastases are extremely important for tumor diagnosis and comprehensive treatment. However, it is clinically difficult to finely localize micrometastases, especially focal lesions smaller than 1 mm. To that purpose, this work designed a tumor microenvironment factor (glutathione, GSH)-activated second near-infrared transparency window (NIR-II) fluorescent probe for visualization of micrometastases. It is constructed by complexing copper ions with 3-mercaptopropionic acid-modified silver sulfide (Ag2S-MPA) quantum dots. The experimental results showed that the probe has good and stable GSH activation properties and can achieve tumor-specific NIR-II fluorescence imaging. Further, in vivo imaging experiments demonstrated that the probe can precisely localize melanomas with a diameter of < 0.1 mm in lung micrometastases. This work provides a potential strategy for the development of novel tools for localization and visualization of micrometastases.

Development of a novel visual servoing probe test method for fault diagnosis of printed circuit boards

Printed circuit board (PCB) measurement and repair is a challenging task that requires experience and expertise to perform. PCB diagnosis and repair shops employ skilled operators to carry out the corresponding measurement tasks using measuring instruments (e.g., oscilloscopes, multimeters) in order to uncover the condition of a particular product. However, these tasks are often repetitive and meticulous, and additionally, the results need to be collected and carefully documented so that the gathered experience regarding the product can be re-used when the next product of the same type arrives into the shop. Nevertheless, the diagnosis of used PCBs is less researched and current flexible automation possibilities are limited. In this paper, a novel visual servoing probe test method and measurement tool are proposed to provide a flexible solution for PCB diagnosis with a higher level of automation. The aim of the approach is to reduce the burden on the operators by carrying out the repetitive measurement tasks and automatically storing the results while leaving the responsibility of measurement profile setup to the human expert. The proposed visual servo system uses manually teached-in measurement points, where template patterns are recorded using cameras, and it is capable of compensating positioning errors in the range of a couple of millimeters. The proof of concept of the proposed method is presented through motherboard measuring experiments, with a 99.7% success rate.

Visual Probing: Cognitive Framework for Explaining Self-Supervised Image Representations

Recently introduced self-supervised methods for image representation learning provide on par or superior results to their fully supervised competitors, yet the corresponding efforts to explain the self-supervised approaches lag behind. Motivated by this observation, we introduce a novel visual probing framework for explaining the self-supervised models by leveraging probing tasks employed previously in natural language processing. The probing tasks require knowledge about semantic relationships between image parts. Hence, we propose a systematic approach to obtain analogs of natural language in vision, such as visual words, context, and taxonomy. Our proposal is grounded in Marr's computational theory of vision and concerns features like textures, shapes, and lines. We show the effectiveness and applicability of those analogs in the context of explaining self-supervised representations. Our key findings emphasize that relations between language and vision can serve as an effective yet intuitive tool for discovering how machine learning models work, independently of data modality. Our work opens a plethora of research pathways towards more explainable and transparent AI.

Computational assessment of amino acid-coupled benzanthrone 2-aminoacetamides as molecular probes for insulin amyloid fibril visualization

Fluorescent proteins are currently the most extensively studied fluorescent markers used in bio-imaging, and many such fluorescent markers are used in research to label amino acids and proteins.

A chemical probe for proteomic analysis and visualization of intracellular localization of lysine-succinylated proteins.

Protein acylation is a vital post-translational modification that regulates various protein functions. In particular, protein succinylation has attracted significant attention because of its potential relationship with various biological events and diseases. In this report, we establish a new method for the comprehensive detection and analysis of potentially succinylated proteins using a chemical tagging technology. The newly synthesized alkyne-containing succinyl substrate successfully labeled lysine residues of proteins through intracellular metabolic labeling independent of other acylation pathways such as protein malonylation. Furthermore, reporter molecules such as biotin moieties and fluorescent dyes were conjugated to alkyne-tagged succinylated proteins via Click reactions, permitting enrichment for proteomic analysis and fluorescence imaging of the labeled proteins. We successfully analyzed and identified numerous potential succinylated proteins associated with various biological processes using gel electrophoresis, proteomic and bioinformatic analyses, and their visualization in cells.

Visual and ratiometric fluorescent probe via an intramolecular charge transfer for detection of a nerve agent simulant in solutions and in the gas phase

A simple two-step synthesis was done to make a probe PPID for rapid detection of DCP in the solution and vapor phase. The mechanism involved in the sensing was enhanced ICT.

Late-stage Attentional Bias towards Food Cues Varies According to Weight Status.

The Current food environment has become increasingly obesogenic, with rates of obesity and related conditions continually rising. Advertisements for energy-dense foods are abundant and promote unhealthy eating behaviors by capitalizing on one's attentional bias towards food cues, a cognitive process resulting from the sensitization of highly reinforcing food. A heightened awareness towards food cues may promote overconsumption of energy-dense foods. The current study employed novel eye-tracking methodology to capture sustained, or late-stage, attentional bias towards food cues. Late-stage attentional bias is the aspect of attentional bias under conscious control and likely more prone to modification compared to initial/ early-stage attentional bias, which reflects automatic processes. The present study hypothesized late-stage attentional bias towards food cues is greater among individuals classified as overweight/obese than those classified as normal weight. Thirty (30) participants classified as overweight/obese (BMI ≥25) and 47 classified as normal weight (BMI <25) were assessed for late-stage attentional bias towards food cues, conceptualized as the percentage of time fixated on food cues when both food and neutral images were presented during a food-specific visual probe procedure task. Percentage of time fixated on food cues was 51.25 ± 1.27 (mean + SE) among individuals classified as overweight to obese while those classified as normal weight had a percent fixation of 47.26 ± 0.87 (P=0.03). In conclusion, individuals classified as overweight to obese have greater late-stage attentional bias towards food cues. This establishes an important factor influencing energy intake that may be modified in future clinical trials.