Atovaquone enhances antitumor efficacy of TCR-T therapy by augmentation of ROS-induced ferroptosis in hepatocellular carcinoma.

T-cell receptor (TCR) engineered T-cell therapy has recently emerged as a promising adoptive immunotherapy approach for tumor treatment, yet hindered by tumor immune evasion resulting in poor therapeutic efficacy. The introduction of ferroptosis-targeted inducers offers a potential solution, as they empower T cells to induce ferroptosis and exert influence over the tumor microenvironment. Atovaquone (ATO) stands as a prospective pharmaceutical candidate with the potential to target ferroptosis, effectively provoking an excessive generation and accumulation of reactive oxygen species (ROS). In this study, we evaluated the effectiveness of a combination therapy comprising ATO and TCR-T cells against hepatocellular carcinoma (HCC), both in vitro and in vivo. The results of lactate dehydrogenase and cytokine assays demonstrated that ATO enhanced cytotoxicity mediated by AFP-specific TCR-T cells and promoted the release of IFN-γ in vitro. Additionally, in an established HCC xenograft mouse model, the combined therapy with low-dose ATO and TCR-T cells exhibited heightened efficacy in suppressing tumor growth, with no apparent adverse effects, comparable to the results achieved through monotherapy. The RNA-seq data unveiled a significant activation of the ferroptosis-related pathway in the combination therapy group in comparison to the TCR-T cells group. Mechanistically, the synergy between ATO and TCR-T cells augmented the release of IFN-γ by TCR-T cells, while concurrently elevating the intracellular and mitochondrial levels of ROS, expanding the labile iron pool, and impairing the integrity of the mitochondrial membrane in HepG2 cells. This multifaceted interaction culminated in the potentiation of ferroptosis within the tumor, primarily induced by an excess of ROS. In summary, the co-administration of ATO and TCR-T cells in HCC exhibited heightened vulnerability to ferroptosis. This heightened susceptibility led to the inhibition of tumor growth and the stimulation of an anti-tumor immune response. These findings suggest that repurposing atovaquone for adoptive cell therapy combination therapy holds the potential to enhance treatment outcomes in HCC.

Strong Electron Transfer in Covalently Integrating Cu(I)-Organic Frameworks Enabling Effective Radionuclide Capture.

Rational construction of strong electron-transfer materials remains a challenging task. Herein, we show a design rule for the construction of strong electron-transfer materials through covalently integrating electron-donoring Cu(I) clusters and electron-withdrawing triazine monomers together. As expected, Cu-CTF-1 (Cu(I)-triazine framework) was found to enable strong electron transfer up to 0.46|e| from each Cu(I) metal center to each adjacent triazine fragment. This finally leads to good spatial separation in both photogenerated electron-hole pairs and function units for photocatalytic uranium reduction under ambience and no sacrificial agent and to good charge separation of [I+][I5-] for I2 immobilization under extremely rigorous conditions. The results have not only opened up a structural design principle to access electron-transfer materials but also solved several challenging tasks in the field of radionuclide capture and CTFs.

Neutral MOF Anion Receptor: Radical-Promoted Precise Anion Recognition.

Precise ion recognition plays a key role in the anionic decontamination in water. However, the established anionic recognition based on neutral or cationic anion receptor is still restricted by the inherent limitation, such as narrow application scope in organic solvent rather than water for neutral anion receptor and poor selectivity due to non-directional electrostatic interaction for cationic anion receptor. Herein, for the first time, a neutral metal-organic framework (MOF) anion receptor is shown, enabling precise anion recognition, for example, the presence of a variety of 1000-fold competitive anions does not affect the selective adsorption of the target anion at all. A radical-dominating anion-recognition mechanism is proposed for rationalizing the efficacy of the neutral MOF.

Unveiling the Surface Structure of ZnO Nanorods and H2 Activation Mechanisms with 17O NMR Spectroscopy.

ZnO plays a very important role in many catalytic processes involving H2, yet the details on their interactions and H2 activation mechanism are still missing, owing to the lack of a characterization method that provides resolution at the atomic scale and follows the fate of oxide surface species. Here, we apply 17O solid-state NMR spectroscopy in combination with DFT calculations to unravel the surface structure of ZnO nanorods and explore the H2 activation process. We show that six different types of oxygen ions in the surface and subsurface of ZnO can be distinguished. H2 undergoes heterolytic dissociation on three-coordinated surface zinc and oxygen ions, while the formed hydride species migrate to nearby oxygen species, generating a second hydroxyl site. When oxygen vacancies are present, homolytic dissociation of H2 occurs and zinc hydride species form from the vacancies. Reaction mechanisms on oxide surfaces can be explored in a similar manner.

Generalized Excess Spectroscopy.

With a clear enhancement of the apparent resolution of experimentally determined spectra, excess spectroscopy has been developed as a powerful tool to study solution structures and molecular interactions. In the standard procedure of the method, excess spectra are calculated based on the ideal spectra constructed using two pure compounds. This limits the applications of the method when the pure compounds are unstable or their physical state is different from that of the mixtures. To overcome the problem or to extend the application, we propose generalized excess spectroscopy in this work, where the ideal spectrum is evaluated from the spectra of reference mixtures. After deducing the working equations, we performed digital simulation and then applied the novel approach to a binary system consisting of tert-butanol and carbon tetrachloride. Both results illustrated the feasibility and universality of the method.

Exosomal LncRNA RP5-977B1 as a novel minimally invasive biomarker for diagnosis and prognosis in non-small cell lung cancer.

BACKGROUND: Lung cancer is the leading cause of cancer-related deaths in the world. Non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancer cases. For lack of conveniently sensitive and specific biomarkers, the majority of patients are in the late stage at initial diagnosis. Long non-coding RNAs (LncRNAs), a novel type of non-coding RNA, have recently been recognized as critical factors in tumor initiation and progression, but the role of exosomal LncRNAs has not been thoroughly excavated in NSCLC yet. METHODS: We isolated exosomes from the serum of patients with NSCLC and healthy controls. Exosome RNA deep sequencing was subsequently performed to detect differentially expressed exosomal LncRNAs. qRT-PCR assay was then utilized to validate dysregulated LncRNAs in both testing and multicentric validation cohort. Receiver operating characteristic (ROC) curve was used to detect the diagnostic capability of exosomal biomarkers. Furthermore, Kaplan-Meier analysis was applied to evaluate the prognostic values of these molecules. RESULTS: On the basis of analysis, we found that novel exosomal LncRNA RP5-977B1 exhibited higher levels in NSCLC than that in the healthy controls. The area under the curve (AUC) value of exosomal RP5-977B1 was 0.8899 and superior to conventional biomarkers CEA and CYFRA21-1 both in testing and multicentric validation cohort. Interestingly, the diagnostic capability of exosomal RP5-977B1 was also validated in early-stage patients with NSCLC. Furthermore, high expression of exosomal RP5-977B1was closely related with worse prognosis in NSCLC (P = 0.036). CONCLUSIONS: Our results suggested that exosomal RP5-977B1 might serve as a novel "liquid biopsy" diagnostic and prognostic biomarker to monitor NSCLC and improve possible therapy.

Abnormalities of the Halogen Bonds in the Complexes between Y2CTe (Y = H, F, CH3) and XF (X = F, Cl, Br, I).

In this work, the hydrogen bonds and halogen bonds in the complexes between Y2CTe (Y = H, F, CH3) and XF (X = F, Cl, Br, I) have been studied by quantum chemical calculations. We found three interesting abnormalities regarding the interactions. Firstly, the strength of halogen bonds increases in the order of IF < BrF < ClF < F2. Secondly, the halogen bonds formed by F2 are very strong, with an interaction energy in the range between −199.8 and −233.1 kJ/mol. Thirdly, all the halogen bonds are stronger than the hydrogen bonds in the systems we examined. All these results are against the general understanding of halogen bonds. These apparent abnormal properties are reconciled with the high polarizability of the Te atom and the strong inducing effect of F on the Te atom of Y2CTe. These findings provide a new perspective on halogen bonds. Additionally, we also proposed bonding distance-based methods to compare the strength of halogen/hydrogen bonds formed between different donor atoms and the same acceptor atom.

Tracking the Micro-Heterogeneity and Hydrogen-Bonding Interactions in Hydroxyl-Functionalized Ionic Liquid Solutions: A Combined Experimental and Computational Study.

Ionic liquids (ILs) are an important class of media that are usually used in combination with polar solvents to reduce costs and tune their physicochemical properties. In this regard, it is essential to understand the influence of adding solvents on the properties of ILs. In this work, the micro-heterogeneity and H-bonding interactions between a hydroxyl-functionalized IL, [HOEmim][TFSI], and acetonitrile (ACN) were investigated by attenuated total reflection Fourier transform infrared spectroscopy and molecular simulations. All studied IL-ACN mixtures were found to deviate from the ideal mixtures. The degree of deviations reaches the maximum at about x(ACN)=0.7 with the presence of both homogeneous clusters of pure IL/ACN and heterogeneous clusters of IL-ACN. With the addition of ACN to IL, the mixtures undergo the transformation from "ACN solvated in [HOEmim][TFSI]" to "[HOEmim][TFSI] solvated in ACN". It is found that the newly formed H-bonding interactions between the IL and ACN is the main factor that contributes to the red shifts of O-H, C2 -H, C4,5 -H, and Calkyl -H of [HOEmim]+ cation, and the blue shifts of C-D, C≡N of ACN, and C-F, S=O of [TFSI]- anion. These in-depth studies on the mixtures of hydroxyl-functionalized IL and acetonitrile would help to understand the micro-heterogeneity and H-bonding interactions of miscible solutions and shed light on exploring their applications.

The structural properties of a ZnCl2-ethylene glycol binary system and the peculiarities at the eutectic composition.

ATR-FTIR spectroscopy was performed on a series of ZnCl2-ethylene glycol (EG) mixtures with a wide-range of compositions (1 : 1.5-1 : 14 in molar ratios), involving the stable ZnCl2-4EG deep-eutectic solvent (DES) composition, to explore the spectral variations, structural heterogeneity, and hydrogen bonding (H-bonding) properties. To enhance the resolution of the spectra, excess absorption and two-dimensional correlation spectroscopies were employed. In the initial IR spectra, a quasi-isosbestic point was identified, signaling that the major disturbance on EG microstructures by adding ZnCl2 is to form a distinct complex. Further analysis uncovered the main transformation process to be from the EG tetramer to the ZnCl2-4EG complex. It was also found that as the EG content increases, negative charge increasingly transfers to ZnCl2, resulting in the strengthening of the Zn ← O coordination bonds and the weakening and finally dissociation of Zn-Cl bonds. Regarding the ZnCl2-4EG DES, several incomparable specificities were observed. It was found that ZnCl2 destructed the H-bonding network of pure EG to the largest extent, resulting in the highest production of the dimer and trimer of EG. Moreover, in comparison with other compositions, the ZnCl2-4EG DES showed abrupt increases in the negative charge of the salt, the length of the Zn-Cl bond, and the strength of the Zn ← O coordination bond. All these imply the strongest intermolecular interactions and the highest solvation of ZnCl2 in EG at the eutectic composition compared to those of other mixtures, resulting in a super-stable liquid mixture. The work provides physical insights into the structural and interactive properties of deep-eutectic solvents.

The distinct effects of two imidazolium-based ionic liquids, [C4mim][OAc] and [C6mim][OAc], on the phase behaviours of DPPC.

Ionic liquids (ILs) are potential green solvents with very broad application prospects. Their toxicity and other biological effects are largely related to their hydrophobic properties. In this work, the effects of two imidazolium-based ILs with either a butyl or a hexyl chain, [C4mim][OAc] or [C6mim][OAc], on the phase behaviours of a representative phospholipid, dipalmitoylphosphatidylcholine (DPPC), were examined using synchrotron small- and wide-angle X-ray scattering and differential scanning calorimetry techniques. A series of samples with a lipid : IL molar ratio ranging from 1 : 0 to 1 : 4/1 : 5 were prepared as aqueous dispersions in the form of multi-lamellar vesicles. The two ILs were found to have distinct effects on the phase behaviours of DPPC. For [C4mim][OAc], its effect is very limited. In contrast, for [C6mim][OAc], it could eliminate the pre-transition of DPPC, markedly affect the main phase transition of the lipid, and insert into the DPPC bilayer at gel state to form an interdigitated gel phase. The findings increased our understanding on the biological effects of imidazolium-based ILs and might shed light on the design of novel IL-based antimicrobials.

The Structures of ZnCl2-Ethanol Mixtures, a Spectroscopic and Quantum Chemical Calculation Study.

We report in this article the structural properties, spectral behavior and heterogeneity of ZnCl2-ethanol (EtOH) mixtures in a wide-composition range (1:3 to 1:14 in molar ratios), using ATR-FTIR spectroscopy and quantum chemical calculations. To improve the resolution of the initial IR spectra, excess spectroscopy and two-dimensional correlation spectroscopy were employed. The transformation process was suggested to be from EtOH trimer and EtOH tetramer to EtOH monomer, EtOH dimer and ZnCl2-3EtOH complex upon mixing. The theoretical findings showed that increasing the content of EtOH was accompanied with the flow of negative charge to ZnCl2. This led to reinforcement of the Zn←O coordination bonds, increase of the ionic character of Zn‒Cl bond and weakening and even dissociation of the Zn‒Cl bond. It was found that in some of the ZnCl2-EtOH complexes optimized at the gas phase or under the solvent effect, there existed hydroxyls with a very special interactive array in the form of Cl‒Zn+←O‒H…Cl-, which incredibly red-shifted to wavenumbers <3000 cm-1. This in-depth study shows the physical insights of the respective electrolyte alcoholic solutions, particularly the solvation process of the salt, help to rationalize the reported experimental results, and may shed light on understanding the properties of the deep eutectic solvents formed from ZnCl2 and an alcohol.

Influence of Hydration on the Structure and Interactions of Ethaline Deep-Eutectic Solvent: A Spectroscopic and Computational Study.

Deep-eutectic solvents (DESs) are regarded as alternative green solvents to ionic liquids. In this work we report the structural properties and hydrogen bonding (H-bonding) interactions of an aqueous DES system. The used DES, ethaline (ETH), is composed of choline chloride and ethylene glycol (EG) in 1 : 2 molar ratio. The investigations were carried out by FTIR spectroscopy combined with quantum chemical calculations. Excess spectroscopy and two-dimensional correlation spectroscopy (2D-COS) were used to explore the data in detail. The results showed that, upon mixing, ETH transforms to EG dimers and trimers and D2 O clusters transform to various ETH-D2 O complexes. Theoretical calculations show that water molecules insert between the anion and cation of ETH, break the strong doubly ionic Cl-… H-OCh+ H-bond, share charges of the ions and form H-bond with them, thus modulate the interaction properties of ETH. This study deepens our molecular-level understanding of the system and would shed light on its applications.

Fabrication of Asymmetric Phosphatidylserine-Containing Lipid Vesicles: A Study on the Effects of Size, Temperature, and Lipid Composition.

The asymmetric distribution of lipids in plasma membranes is closely related to the physiological functions of cells. To improve our previous approach in fabricating asymmetric vesicles, we defined a parameter, asymmetric degree, in this work and investigated the effects of vesicle size, incubation temperature, and lipid composition on the formation process of asymmetric phosphatidylserine (PS)-containing lipid vesicles. The results indicate that all of the three factors have marked but different effects on the time-dependent asymmetric degree of the vesicles as well as the flip and flop rate constants of the PS lipids. However, only vesicle size and PS content show significant influence on the maximal asymmetric degree of the vesicles, while the incubation temperature exhibits negligible effect. This work not only deepens our understanding on the packing property of PS molecules in self-assembled membranes and the formation mechanism of asymmetric vesicles but also practically provides a solution to regulate the asymmetric degree of the PS-containing vesicles using the established kinetic equation. In addition, the method would facilitate researches related to asymmetric vesicles or reconstruction of biological membranes.

The effect of introducing an ether group into an imidazolium-based ionic liquid in binary mixtures with DMSO.

Ether-functionalized ionic liquids (ILs) have successfully been employed in diverse applications, but their interactions with other solvents are not understood well. In this work, mixtures of 1-methoxyethyl-3-methylimidazolium bis(fluorosulfonyl)imide (EOMIMFSI) and dimethylsulfoxide (DMSO) are studied in terms of their solution structure and hydrogen bonding interactions. The corresponding alkyl-substituted IL 1-ethyl-3-methylimidazolium bis(fluorosulfonyl)imide (EMIMFSI) is analyzed for comparison. A combination of FTIR spectroscopy, excess spectroscopy, and quantum chemical calculations is employed for this purpose. The datasets allow drawing a number of conclusions as follows: (1) the ether group forms intramolecular hydrogen bonds that compete with anions and DMSO; hence, introducing ether groups into the imidazolium-based IL leads to the weakening of hydrogen bonds in the mixtures. (2) With the help of excess spectra and quantum chemical calculations, some complexes such as ion clusters, ion pairs, and individual ions were identified and assigned in the two systems. The solution structures at different concentrations were examined by analyzing the excess spectra of ν(C2-H) and ν(C-D) in the two IL-DMSO-d6 systems. (3) The introduced ether groups result in changes of the main interaction sites, which were found to be concentration-dependent. In the EOMIMFSI-DMSO system, when isolated ions are the main existing form, the C2-Hs are still the main sites interacting with DMSO. However, when ion pairs or larger ion clusters are the main existing species, the C4-Hs are the main sites interacting with DMSO.

Is the Fourier Transform Infrared Free-OH Band of t-Butanol Only from Free OHs? Case Studies on the Binary Systems of the Alcohol with CCl4 and CHCl3.

Attenuated total reflection-Fourier transform infrared spectroscopy and quantum chemical calculations were performed on tert-butyl alcohol (t-BuOH) and its binary solutions with CCl4 and CHCl3. The study was focused on the free-OH stretching bands. Two resolution-enhancing methods, excess spectroscopy and two-dimensional correlation spectroscopy, were employed to examine the structural heterogeneity and search for the detailed contributors to the free-OH bands. Unexpectedly, CCl4 was found not to be an inert solvent and, similar to CHCl3, formed hydrogen/halogen bonds (H-/X-bond) with t-BuOH. It was observed that the free-OH band in the t-BuOH-CHCl3 system is larger and more red-shifted than that in the t-BuOH-CCl4 system, indicating the stronger intermolecular interactions in the former system. Furthermore, in the t-BuOH-CHCl3 system, the H-bonds are stronger than the X-bonds, while in the t-BuOH-CCl4 system, both interactions are similar in strength. To assign the free-OH bands, it was found that they are not only from the free OH of the t-BuOH monomer, but they are also contributed by the quasi-free OH with the oxygen bonded to H or Cl and even the weakly H-bonded OH of t-BuOH molecules. Finally, all the identified species increased simultaneously via cosolvent addition, suggestive of the destabilization of the highly associated t-BuOH clusters.

Evaluation of small dense low-density lipoprotein concentration for predicting the risk of acute coronary syndrome in Chinese population.

BACKGROUND: Acute coronary syndrome (ACS) is the leading cause of death in developing and developed countries, yet assessing the risk of its development remains challenging. Several lines of evidence indicate that small, dense low-density lipoproteins (sd-LDL) are associated with increased cardiovascular disease risk. We aim to evaluate sd-LDL concentration for predicting the risk of ACS in Chinese population. METHODS: Baseline characteristics of 121 patients with ACS and 172 healthy controls were obtained. Plasma sd-LDL-C was measured using homogeneous assay, and the proportion of sd-LDL-C in LDL-C was detected. RESULTS: There was gender and age effect on the sd-LDL-C concentration and sd-LDL-C/LDL-C ratio among healthy subjects. Elevated sd-LDL-C concentrations and sd-LDL-C/LDL-C ratio were observed in ACS patients with unstable angina pectoris (UAP), non-ST-segment elevation myocardial infarction (STEMI), and ST-segment elevation myocardial infarction (NSTEMI) compared with healthy controls (P < .05); however, there were no differences among ACS groups. According to Pearson's correlation coefficient analyses, sd-LDL-C concentration and sd-LDL-C/LDL-C ratio were positively correlated with triglyceride (TG) and LDL-C concentrations (P < .05) and negatively correlated with high-density lipoprotein (HDL) concentration (P < .05). Based on the receiver operating characteristic (ROC) curves, the cutoff values of sd-LDL-C and sd-LDL-C/LDL-C ratio for the prediction of ACS were 1.06 mmol/L and 34.55%, respectively. Multivariate logistic regression analysis demonstrated that the sd-LDL-C/LDL-C ratio, but not sd-LDL-C concentration, was significantly associated with ACS events [OR (95% CI): 1.24, 1.11-1.38, P < .001]. CONCLUSIONS: The sd-LDL-C/LDL-C ratio may be associated with an increased risk of developing ACS in Chinese population.

Dual Graphitic-N Doping in a Six-Membered C-Ring of Graphene-Analogous Particles Enables an Efficient Electrocatalyst for the Hydrogen Evolution Reaction.

Graphene-based materials still exhibit poor electrocatalytic activities for the hydrogen evolution reaction (HER) although they are considered to be the most promising electrocatalysts. We fabricated a graphene-analogous material displaying exceptional activity towards the HER under acidic conditions with an overpotential (57 mV at 10 mA cm-2 ) and Tafel slope (44.6 mV dec-1 ) superior to previously reported graphene-based materials, and even comparable to the state-of-the art Pt/C catalyst. X-ray absorption near-edge structure (XANES) and solid-state NMR studies reveal that the distinct feature of its structure is dual graphitic-N doping in a six-membered carbon ring. Density functional theory (DFT) calculations show that the unique doped structure is beneficial for the activation of C-H bonds and to make the carbon atom bonded to two graphitic N atoms an active site for the HER.

Identifying Different Halogen-/Hydrogen-Bonding Interaction Modes in Binary Systems that Contain an Acetate Ionic Liquid and Various Halobenzenes.

Elucidation of the nature of noncovalent interactions between ionic liquids (ILs) and halogenated molecules is of particular importance for both fundamental research and drug development. Herein, the noncovalent interactions between 1-butyl-3-methyl-imidazolium acetate and three halobenzenes C6 F5 X (X=I, Br, H) were investigated. The iodine derivative shows the strongest interaction with the IL, followed by C6 F5 Br and C6 F5 H. As indicated by the positive/negative peaks and "multi/two-state" phenomena in the excess IR spectra, combined with DFT calculations, various interaction modes were differentiated. Three complexes, namely anion-C6 F5 I, anion-2 C6 F5 I, and ion-pair-C6 F5 I in the IL-C6 F5 I system were identified, whereas only ion-pair-C6 F5 Br/C6 F5 H complexes, together with self-associates, were found in the other two systems. A possible reason for the behavior of the IL-C6 F5 I system could be that the iodine-based halogen-bonding interactions in the system are strong enough to break interactions between the IL cations and anions. This might make C6 F5 I a good co-solvent to regulate the properties of acetate-based ILs.

Evidence that Acetonitrile is Sensitive to Different Interaction Sites of Ionic Liquids as Revealed by Excess Spectroscopy.

By studying the interactions between an ionic liquid, 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide, and a co-solvent acetonitrile, the C≡N stretching vibration is found to be sensitive to different interaction sites as shown by excess infrared spectroscopy. Four existing forms of acetonitrile molecules are identified and a detailed transformation process of the ionic liquid upon dilution is obtained. Such characteristics of the nitrile group are discussed from the viewpoint of its ability to form hydrogen bonds with proton donors. It is believed that this is due to the intermediate charge donating ability of the C≡N group as compared with other groups such as S=O, CH3 , and aromatic C-H.

Long-Time Plasma Membrane Imaging Based on a Two-Step Synergistic Cell Surface Modification Strategy.

Long-time stable plasma membrane imaging is difficult due to the fast cellular internalization of fluorescent dyes and the quick detachment of the dyes from the membrane. In this study, we developed a two-step synergistic cell surface modification and labeling strategy to realize long-time plasma membrane imaging. Initially, a multisite plasma membrane anchoring reagent, glycol chitosan-10% PEG2000 cholesterol-10% biotin (abbreviated as "GC-Chol-Biotin"), was incubated with cells to modify the plasma membranes with biotin groups with the assistance of the membrane anchoring ability of cholesterol moieties. Fluorescein isothiocyanate (FITC)-conjugated avidin was then introduced to achieve the fluorescence-labeled plasma membranes based on the supramolecular recognition between biotin and avidin. This strategy achieved stable plasma membrane imaging for up to 8 h without substantial internalization of the dyes, and avoided the quick fluorescence loss caused by the detachment of dyes from plasma membranes. We have also demonstrated that the imaging performance of our staining strategy far surpassed that of current commercial plasma membrane imaging reagents such as DiD and CellMask. Furthermore, the photodynamic damage of plasma membranes caused by a photosensitizer, Chlorin e6 (Ce6), was tracked in real time for 5 h during continuous laser irradiation. Plasma membrane behaviors including cell shrinkage, membrane blebbing, and plasma membrane vesiculation could be dynamically recorded. Therefore, the imaging strategy developed in this work may provide a novel platform to investigate plasma membrane behaviors over a relatively long time period.