Molecular probes for super-resolution imaging of drug dynamics.

Super-resolution molecular probes (SRMPs) are essential tools for visualizing drug dynamics within cells, transcending the resolution limits of conventional microscopy. In this review, we provide an overview of the principles and design strategies of SRMPs, emphasizing their role in accurately tracking drug molecules. By illuminating the intricate processes of drug distribution, diffusion, uptake, and metabolism at a subcellular and molecular level, SRMPs offer crucial insights into therapeutic interventions. Additionally, we explore the practical applications of super-resolution imaging in disease treatment, highlighting the significance of SRMPs in advancing our understanding of drug action. Finally, we discuss future perspectives, envisioning potential advancements and innovations in this field. Overall, this review serves to inform and practitioners about the utility of SRMPs in driving innovation and progress in pharmacology, providing valuable insights for drug development and optimization.

Fluorescence and Lifetime Imaging of Endoplasmic Reticulum Polarity Change During Ferroptosis.

As a new form of regulated cell death, ferroptosis is closely related to various diseases. Tracing ferroptosis related biological behavior is helpful to better understand this process and its related biology. Considering that ferroptosis is featured with remarkable lipid peroxidation which can easily change the membranes' compositions and structures, it is potential to detect intracellular environmental changes for direct assessment of ferroptosis. In view of the close relationship between endoplasmic reticulum (ER) and ferroptosis, we designed an ER-targeted and polarity-sensitive fluorescent probe SBD-CH, which has superior photostability and can respond to polarity with high selectivity without the affection of viscosity. SBD-CH can monitor the trend of ER polarity during ferroptosis by confocal laser scanning microscopy (CLSM), and analyze the distribution of polarity in ferroptosis by fluorescence lifetime imaging microscopy (FLIM). During Erastin induced ferroptosis, the polarity of ER in HT-1080 cells increased and the polarity distribution in ER was more dispersed. Our work provides an effective strategy for evaluating the process of ferroptosis by monitoring the changes of ER polarity.

A NIR-II Photoacoustic Probe for High Spatial Quantitative Imaging of Tumor Nitric Oxide in Vivo.

Nitric oxide (NO) exhibits both pro- and anti-tumor effects. Therefore, real-time in vivo imaging and quantification of tumor NO dynamics are essential for understanding the conflicting roles of NO played in pathophysiology. The current molecular probes, however, cannot provide high-resolution imaging in deep tissues, making them unsuitable for these purposes. Herein, we designed a photoacoustic probe with an absorption maximum beyond 1000 nm for high spatial quantitative imaging of in vivo tumor NO dynamics. The probe exhibits remarkable sensitivity, selective ratiometric response behavior, and good tumor-targeting abilities, facilitating ratiometric imaging of tumor NO throughout tumor progression in a micron-resolution level. Using the probe as the imaging agent, we successfully quantified NO dynamics in tumor, liver and kidney. We have pinpointed an essential concentration threshold of around 80 nmol/cm3 for NO, which plays a crucial role in the "double-edged-sword" function of NO in tumors. Furthermore, we revealed a reciprocal relationship between the NO concentration in tumors and that in the liver, providing initial insights into the possible NO-mediated communication between tumor and the liver. We believe that the probe will help resolve conflicting aspects of NO biology and guide the design of imaging agents for tumor diagnosis and anti-cancer drug screening.

An AIE-based monofunctional Pt(ii) complex for photodynamic therapy through synergism of necroptosis-ferroptosis.

Side effects and drug resistance are among the major problems of platinum-based anticancer chemotherapies. Photodynamic therapy could show improved tumor targeting ability and better anticancer effect by region-selective light irradiation. Here, we report an aggregation-induced emission (AIE)-based monofunctional Pt(ii) complex (TTC-Pt), which shows enhanced singlet oxygen production by introduction of a Pt atom to elevate the intersystem crossing (ISC) rate. Moreover, TTC-Pt exhibits decent capacity of inhibition on tumor cell growth upon light irradiation, with negligible dark toxicity compared to the commonly used chemodrug cisplatin. Mechanistic study suggests that TTC-Pt enters HeLa cells via the endocytosis pathway and locates mainly in lysosomes, causing FSP1 down-regulation and intracellular lipid peroxidation accumulation under irradiation, finally leading to ferroptosis and necroptosis. The synergistic dual cell death pathways could help to kill apoptosis-resistant tumor cells. Therefore, TTC-Pt could serve as a potent antitumor photosensitizer, which overcomes the drug resistance with minimum side effects.

Bibliometric analysis of biochar-based organic fertilizers in the past 15 years: Focus on ammonia volatilization and greenhouse gas emissions during composting.

Biochar-based organic fertilizer is a new type of ecological fertilizer formulated with organic fertilizers using biochar as the primary conditioning agent, which has received wide attention and application in recent years. This study conducted a comprehensive bibliometric analysis of the main hot spots and research trends in the field of biochar-based organic fertilizer research by collecting indicators (publication year, number, prominent authors, and research institutions) in the Web of Science database. The results showed that the research in biochar-based organic fertilizer has been in a rapid development stage since 2015, with exponential growth in publications number; the main institution with the highest publications number was Northwest Agriculture & Forestry University; the researchers with the highest number of publications was Mukesh Kumar Awasthi; the most publications country is China by Dec 30, 2022. The hot spots of biochar-based organic fertilizer research have been nitrogen utilization, greenhouse gas emission, composting product quality and soil fertility. Biochar reduces ammonia volatilization and greenhouse gas emissions from compost mainly through adsorption. The results showed that adding 10% biochar was an effective measure to achieve co-emission reduction of ammonia and greenhouse gases in composting process. In addition, biochar modification or combination with other additives should be the focus of future research to mitigate ammonia and greenhouse gas emissions from composting processes.

Different crystalline manganese dioxide and biochar co-conditioning aerobic composting: Reduced ammonia volatilization and improved organic fertilizer quality.

Aerobic composting is a sustainable and effective waste disposal method. However, it can generate massive amounts of ammonia (NH3) via volatilization. Effectively reducing NH3 volatilization is vital for advancing aerobic composting and protecting the ecological environment. Herein, two crystal types of MnO2 (α-MnO2 and δ-MnO2) are combined with biochar (hydrochar (WHC) and pyrochar (WPC), respectively) and used as conditioners for the aerobic composting of chicken manure. Results reveal that α-MnO2 (34.6%) can more effectively reduce NH3 accumulation than δ-MnO2 (27.1%). Moreover, the combination of WHC and MnO2 better reduces NH3 volatilization (48.5-58.9%) than the combination of WPC and MnO2 (15.8-40.1%). The highest NH3 volatilization reduction effect (58.9%) is achieved using the combination of WHC and δ-MnO2. Because the added WHC and δ-MnO2 promote the humification of the compost, the humic acid to fulvic acid ratio (HA/FA ratio) dramatically increases. The combination of WHC and δ-MnO2 doubled the HA/FA ratio and resulted in a net economic benefit of 130.0 RMB/t. Therefore, WHC and δ-MnO2 co-conditioning can promote compost decomposition, improving the quality of organic fertilizers and substantially reducing NH3 volatilization.

NCOA4 requires a [3Fe-4S] to sense and maintain the iron homeostasis.

NCOA4 is a selective cargo receptor for ferritinophagy, the autophagic turnover of ferritin (FTH), a process critical for regulating intracellular iron bioavailability. However, how ferritinophagy flux is controlled through NCOA4 in iron-dependent processes needs to be better understood. Here, we show that the C-terminal FTH-binding domain of NCOA4 harbors a [3Fe-4S]-binding site with a stoichiometry of approximately one labile [3Fe-4S] cluster per NCOA4 monomer. By analyzing the interaction between NCOA4 and HERC2 ubiquitin ligase or NCOA4 and FTH, we demonstrate that NCOA4 regulates ferritinophagy by sensing the intracellular iron-sulfur cluster levels. Under iron-repletion conditions, HERC2 recognizes and recruits holo-NCOA4 as a substrate for polyubiquitination and degradation, favoring ferritin iron storage. Under iron-depletion conditions, NCOA4 exists in the form of apo-protein and binds ferritin to promote the occurrence of ferritinophagy and release iron. Thus, we identify an iron-sulfur cluster [3Fe-4S] as a critical cofactor in determining the fate of NCOA4 in favoring iron storage in ferritin or iron release via ferritinophagy and provide a dual mechanism for selective interaction between HERC2 and [3Fe-4S]-NCOA4 for proteasomal degradation or between ferritin and apo-NCOA4 for ferritinophagy in the control of iron homeostasis.

Recent advances in Zn2+ imaging: From organelles to in vivo applications.

Zn2+ is involved in various physiological and pathological processes in living systems. Monitoring the dynamic spatiotemporal changes of Zn2+ levels in organelles, cells, and in vivo is of great importance for the investigation of the physiological and pathological functions of Zn2+. However, this task is quite challenging since Zn2+ in living systems is present at low concentrations and undergoes rapid dynamic changes. In this review, we summarize the design and application of fluorescent probes for Zn2+ imaging in organelles, cells, and live organisms reported over the past two years. We aim to provide inspiration for the design of novel Zn2+ probes for multi-level monitoring and deepen the understanding of Zn2+ biology.

Surface acidic sites strengthened core-shell HC@MnO2 for enhanced gaseous ammonia adsorption.

As a common gaseous pollutant in atmospheric environment, ammonia (NH3) not only contributes to the formation of haze, but also disturb the nitrogen balance in ecosystem through atmospheric nitrogen deposition. Therefore, the control of NH3 emission has important environmental significance. Adsorption is the most commonly used technology for NH3 purification in practice, and efficient adsorbents are the key to adsorption method. Herein, a core-shell structured HC@MnO2 adsorbent was constructed by in-situ growth of layered δ-MnO2 on hydrochar (HC) surface, and its surface acidic sites were further strengthened. The enhancement of surface acidic sites significantly improved the adsorption performance of HC@MnO2 for NH3, reaching 34.49 mg NH3/g, which was superior to commercial carbon-based materials (whose adsorption capacity was 8.47 times that of Coal-based activated carbon, 14.25 times that of Coconut shell activated carbon, and 12.77 times that of Bamboo charcoal). Moreover, the operating parameters and adsorption kinetics were detailly investigated. The adsorption of HC@MnO2 on NH3 was in accordance with pseudo-second-order adsorption kinetics model. Large surface area of core-shell structure and abundant surface acidic sites of δ-MnO2 are the decisive reasons for the excellent adsorption performance of HC@MnO2. Importantly, the enhancement of surface stronger Brønsted acidic sites is the key to improve NH3 adsorption performance of HC@MnO2. Finally, the thermal regeneration and recycling performance of HC@MnO2-H were also investigated. This study provides a suggestive for further research on low-cost composite materials with excellent NH3 adsorption performance.

A Near-Infrared Fluorescent and Photoacoustic Probe for Visualizing Biothiols Dynamics in Tumor and Liver.

Biothiols, including glutathione (GSH), homocysteine (Hcy) and cysteine (Cys), play crucial roles in various physiological processes. Though an array of fluorescent probes have been designed to visualize biothiols in living organisms, few one-for-all imaging agents for sensing biothiols with fluorescence and photoacoustic imaging capabilities have been reported, since instructions for synchronously enabling and balancing every optical imaging efficacy are deficient. Herein, a new near-infrared thioxanthene-hemicyanine dye (Cy-DNBS) has been constructed for fluorescence and photoacoustic imaging of biothiols in vitro and in vivo. Upon treatment with biothiols, the absorption peak of Cy-DNBS shifted from 592 nm to 726 nm, resulting in a strong NIR absorption as well as a subsequent turn-on PA signal. Meanwhile, the fluorescence intensity increased instantaneously at 762 nm. Then, Cy-DNBS was successfully utilized for imaging endogenous and exogenous biothiols in HepG2 cells and mice. In particular, Cy-DNBS was employed for tracking biothiols upregulation in the liver of mice triggered by S-adenosyl methionine by means of fluorescent and photoacoustic imaging methods. We expect that Cy-DNBS serves as an appealing candidate for deciphering biothiols-related physiological and pathological processes.

BODIPY-Based Multifunctional Nanoparticles for Dual Mode Imaging-Guided Tumor Photothermal and Photodynamic Therapy.

Multifunctional nanoparticles integrating accurate multi-diagnosis and efficient therapy hold great prospects in tumor theranostics. However, it is still a challenging task to develop multifunctional nanoparticles for imaging-guided effective eradication of tumors. Herein, we developed a near-infrared (NIR) organic agent Aza/I-BDP by coupling 2,6-diiodo-dipyrromethene (2,6-diiodo-BODIPY) with aza-boron-dipyrromethene (Aza-BODIPY). Through encapsulating with an amphiphilic biocompatible copolymer DSPE-mPEG5000, well-distributed Aza/I-BDP nanoparticles (NPs) were developed, which exhibited high 1O2 generation, high photothermal conversion efficiency, and excellent photo-stability. Notably, coassembly of Aza/I-BDP and DSPE-mPEG5000 effectively inhibits H-aggregation of Aza/I-BDP in aqueous solution and enhances the brightness simultaneously up to 31-fold. More importantly, in vivo experiments demonstrated that Aza/I-BDP NPs might be used for NIR fluorescent and photoacoustic imaging-guided photodynamic and photothermal therapy.

Blood-Brain Barrier Permeable Photoacoustic Probe for High-Resolution Imaging of Nitric Oxide in the Living Mouse Brain.

Alternations in the brain nitric oxide (NO) homeostasis are associated with a variety of neurodegeneration diseases; therefore, high-resolution imaging of NO in the brain is essential for understanding pathophysiological processes. However, currently available NO probes are unsuitable for this purpose due to their poor ability to cross the blood-brain barrier (BBB) or to image in deep tissues with spatial resolution. Herein, we developed a photoacoustic (PA) probe with BBB crossing ability to overcome this obstacle. The probe shows a highly selective ratiometric response toward NO, which enables the probe to image NO with micron resolution in the whole brain of living mice. Using three-dimensional PA imaging, we demonstrated that the probe could be used to visualize the detailed NO distribution in varying depth cross-sections (0-8 mm) of the living Parkinson's disease (PD) mouse brain. We also investigated the therapeutic properties of natural polyphenols in the PD mouse brain using the probe as an imaging agent and suggested the potential of the probe for screening therapeutic agents. This study provides a promising imaging agent for imaging of NO in the mouse brain with high resolution. We anticipate that these findings may open up new possibilities for understanding the biological functions of NO in the brain and the development of new imaging agents for the diagnosis and treatment of brain diseases.

Single-atom engineering of hemicyanine and its amphiphilic derivative for optimized near infrared phototheranostics.

Near-infrared (NIR) dyes are widely used in the field of in vivo phototheranostics. Hemicyanine dyes (HDs) have recently received tremendous attention due to their easy synthesis and excellent NIR features. However, HDs can easily form non-fluorescent aggregates and their potential for phototherapy still needs further exploration due to their poor ability to generate reactive oxygen species (ROS). Herein, a series of hemicyanine dyes with different chalcogen atom (O, S, Se) substitutions were constructed to achieve optimized potential for phototheranostics. By replacing O with the heavy atom Se in the xanthene skeleton, CySe-NEt2 showed much more favourable features such as extended NIR absorption/emission wavelength, boosted 1O2 generation rate and higher photothermal effect. In addition, a poly(ethylene glycol) (PEG) group was introduced into the scaffold and yielded a nanotheranostic agent CySe-mPEG5K, which easily formed nanoparticles with appealing features such as excellent photostability, effective prevention of unpleasant H-aggregation, fast/selective tumor accumulation and minimum dark toxicity. Solid tumor growth was significantly suppressed through combined photodynamic therapy (PDT) and photothermal therapy (PTT) guided by NIR fluorescence (NIRF) and photoacoustic (PA) imaging. This study not only presents the first example of selenium-substituted hemicyanine dyes, but also offers a reliable design strategy for the development of potent NIR phototheranostic agents with multi-mode imaging-guided combination therapeutic ability.

Manipulating the Subcellular Localization and Anticancer Effects of Benzophenothiaziniums by Minor Alterations of N-Alkylation.

Cationic, water-soluble benzophenothiaziniums have been recognized as effective type I photosensitizers (PSs) against hypoxic tumor cells. However, the study of the structure-property relationship of this type of PS is still worth further exploration to achieve optimized photodynamic effects and minimize the potential side effects. Herein, we synthesized a series of benzophenothiazine derivatives with minor N-alkyl alteration to study the effects on the structure-property relationships. The cellular uptake, subcellular organelle localization, reactive oxygen species (ROS) generation, and photocytotoxicity performances were systematically investigated. NH2NBS and EtNBS specifically localized in lysosomes and exhibited high toxicity under light with a moderate phototoxicity index (PI) due to the undesirable dark toxicity. However, NMe2NBS with two methyl substitutions accumulated more in mitochondria and displayed an excellent PI value with moderate light toxicity and negligible dark toxicity. Without light irradiation, NH2NBS and EtNBS could induce lysosomal membrane permeabilization (LMP), while NMe2NBS showed no obvious damage to lysosomes. After irradiation, NH2NBS and EtNBS were released from lysosomes and relocated into mitochondria. All compounds could induce mitochondria membrane potential (MMP) loss and nicotinamide adenine dinucleotide phosphate (NADPH) consumption under light to cause cell death. NMe2NBS exhibited remarkable in vivo photodynamic therapy (PDT) efficacy in a xenograft mouse tumor (inhibition rate, 89%) with no obvious side effects. This work provides a valuable methodology to investigate the structure-property relationships of benzophenothiazine dyes, which is of great importance in the practical application of PDT against hypoxia tumor cells.

Fluorescent Probes for Biological Species and Microenvironments: from Rational Design to Bioimaging Applications.

Some important biological species and microenvironments maintain a complex and delicate dynamic balance in life systems, participating in the regulation of various physiological processes and playing indispensable roles in maintaining the healthy development of living bodies. Disruption of their homeostasis in living organisms can cause various diseases and even death. Therefore, real time monitoring of these biological species and microenvironments during different physiological and pathological processes is of great significance. Fluorescent-probe-based techniques have been recognized as one of the most powerful tools for real time imaging in biological samples. In this Account, we introduce the representative works from our group in the field of fluorescent probes for biological imaging capable of detecting metal ions, small bioactive molecules, and the microenvironment. The design strategies of small molecule fluorescent probes and their applications in biological imaging will be discussed. By regulating the design strategy and mechanism (e.g., ICT, PeT, and FRET) of the electronic and spectral characteristics of the fluorescent platforms, these chemical probes show high selectivity and diverse functions, which can be used for imaging of various physiological and pathological processes. Through the exploration of the rational response mechanism and design strategy, combined with a variety of imaging techniques, such as super-resolution imaging, photoacoustic (PA) imaging, etc., we have realized multimode imaging of the important biological analytes from the subcellular level to the in vivo level, which provides powerful means to study the physiological and pathological functions of these species and microenvironments. This Account aims to offer insights and inspiration for the development of novel fluorescent probes for biological imaging, which could provide powerful tools for the study of chemical biology. Overall, we represent a series of turn-on/turn-off/ratiometric fluorescent/PA probes to visually and dynamically trace biological species and microenvironments in cells and even in vivo that seek higher resolution and depth molecular imaging to improve diagnostic methods and clarify new discoveries related to chemical biology. Our future efforts will be devoted to developing multiorganelle targeted fluorescent probes to study the mechanism of subcellular organelle interaction and employing various dual-mode probes of NIR II and PA imaging to investigate the development of related diseases and treat the related diseases at subcellular and in vivo levels.

An ER-targeted "reserve-release" fluorogen for topological quantification of reticulophagy.

The endoplasmic reticulum's (ER) dynamic nature, essential for maintaining cellular homeostasis, can be influenced by stress-induced damage, which can be assessed by examining the morphology of ER dynamics and, more locally, ER properties such as hydrophobicity, viscosity, and polarity. Although numerous ER-specific chemical probes have been developed to monitor the ER's physical and chemical parameters, the quantitative detection and super-resolution imaging of its local hydrophobicity have yet to be explored. Here, we describe a photostable ER-targeted probe with high signal-to-noise ratio for super-resolution imaging that can specifically respond to changes in ER hydrophobicity under stress based on a "reserve-release" mechanism. The probe shows an excellent ability to target ER over commercial ER dyes and can be used to track local changes of hydrophobicity by fluorescence intensity and morphology during the selective autophagy of ER (i.e., reticulophagy). By correlating the level and location of ER damage with the distribution of fluorescence intensity, we were able to assess reticulophagy at the subcellular level. Beyond that, we developed a topological analytical tool adaptable to any ER probe for detecting structural changes in ER and thus quantitatively identifying reticulophagy. The algorithm-assisted tool can also be adapted to a wide range of molecular probes and organelles. Altogether, the new probe and analytical strategy described here show promise for the quantitative detection and analysis of subtle ER damage and stress.

Super-Resolution Imaging of Mitochondrial HClO during Cell Ferroptosis Using a Near-Infrared Fluorescent Probe.

Ferroptosis is of great importance in physiological and pathological processes, which is associated with various inflammation-related diseases, cardiovascular diseases, and even cancer. Ferroptosis can cause abnormal change of reactive oxygen species (ROS) in mitochondria. Hypochlorous acid (HClO) acts as a typical ROS. Therefore, it is needed to study the relationship between mitochondrial morphology and HClO changes during ferroptosis at the subcellular level. To this end, a near-infrared-excitation/emission fluorescent probe, HD-Br-1, for rapid detection of mitochondrial HClO was developed based on the specific oxidative cleavage of the N,N-dimethylthiocarbamate moiety. The fluctuation in mitochondrial HClO content and the change in mitochondrial morphology during ferroptosis were monitored in real time by super-resolution imaging. In addition, HD-Br-1 was successfully applied to monitor exogenous and endogenous mitochondrial HClO during cell ferroptosis and visualize tumor to discriminate from healthy tissues. Therefore, we believe that HD-Br-1 could provide a valuable approach for the detection of mitochondrial HClO in cancer cells as well as for understanding the ferroptosis mechanism and early diagnosis of cancers associated with ferroptosis for future research.

Aggregation-Induced Emission Luminogens for Enhanced Photodynamic Therapy: From Organelle Targeting to Tumor Targeting.

Photodynamic therapy (PDT) has attracted much attention in the field of anticancer treatment. However, PDT has to face challenges, such as aggregation caused by quenching of reactive oxygen species (ROS), and short 1O2 lifetime, which lead to unsatisfactory therapeutic effect. Aggregation-induced emission luminogen (AIEgens)-based photosensitizers (PSs) showed enhanced ROS generation upon aggregation, which showed great potential for hypoxic tumor treatment with enhanced PDT effect. In this review, we summarized the design strategies and applications of AIEgen-based PSs with improved PDT efficacy since 2019. Firstly, we introduce the research background and some basic knowledge in the related field. Secondly, the recent approaches of AIEgen-based PSs for enhanced PDT are summarized in two categories: (1) organelle-targeting PSs that could cause direct damage to organelles to enhance PDT effects, and (2) PSs with tumor-targeting abilities to selectively suppress tumor growth and reduce side effects. Finally, current challenges and future opportunities are discussed. We hope this review can offer new insights and inspirations for the development of AIEgen-based PSs for better PDT effect.

An Endoplasmic Reticulum Targeting Type I Photosensitizer for Effective Photodynamic Therapy against Hypoxic Tumor Cells.

Organelle-targeted type I photodynamic therapy (PDT) shows great potential to overcome the hypoxic microenvironment in solid tumors. The endoplasmic reticulum (ER) is an indispensable organelle in cells with important biological functions. When the ER is damaged due to the production of reactive oxygen species (ROS), the accumulation of misfolded proteins will interfere with ER homeostasis, resulting in ER stress. Here, an ER-targeted benzophenothiazine-based photosensitizer NBS-ER was presented. ER targeting modification significantly reduced the dark toxicity and improved phototoxicity index (PI). NBS-ER could effectively produce O2 - ⋅ with near-infrared irradiation, making its phototoxicity under hypoxia close to that under normoxia. Meanwhile, the photoinduced ROS triggered ER stress and induced apoptosis. In addition, NBS-ER possessed excellent photodynamic therapeutic effect in 4T1-tumor-bearing mice.

A ratiometric pH probe for acidification tracking in dysfunctional mitochondria and tumour tissue in vivo.

Cellular dysregulated pH and mitochondrial metabolism are commonly two central factors for solid tumour progression. pH regulation and long-term mitochondrial tracking provide a great opportunity for tumour treatment. pH probes with suitable pKa and satisfactory mitochondria-immobilizing character are demanded for tumour recognition and therapy. Here, we report a ratiometric fluorescent probe, CouDa, for pH imaging in mitochondria and tumour tissue. CouDa displays an obvious blue-shifted emission (about 160 nm) in alkaline environment, with a pKa around 7.4 suitable for monitoring mitochondrial pH change. NMR and MS data confirmed an addition reaction mechanism of OH- upon the positively charged conjugated structure of hemicyanine fluorophore. Mitochondrial immobilization and acidification monitoring were realized by CouDa in cells treated with a mitochondrial uncoupler. Moreover, CouDa could distinguish acidified tumour tissue in living mice. Comparing with its analogue, the pH-sensitivity and mitochondria-immobilizing property are attributed to a hydrophobic long alkyl chain on indolium N atom. This work provides an effective strategy to track nucleophilic substances in dysfunctional mitochondria.