Fluorescence Imaging Of Cells Research Articles

Clog P-Guided Development of Multi-Colored Buffering Fluorescent Probes for Super-Resolution Imaging of Lipid Droplet Dynamics.

Super-resolution fluorescence imaging of live cells increasingly demands fluorescent probes capable of multi-color and long-term dynamic imaging. Understanding the mechanisms of probe-target recognition is essential for the engineered development of such probes. In this study, it is discovered that the molecular lipid solubility parameter, Clog P, determines the staining performance of fluorescent dyes on lipid droplets (LDs). Fluorescent dyes with Clog P values between 2.5 and 4 can form buffering pools outside LDs, replacing photobleached dyes within LDs to maintain constant fluorescence intensity in LDs, thereby enabling dynamic super-resolution imaging of LDs. Guided by Clog P, four different colored buffering LD probes spanning the visible light spectrum have been developed. Using Structured Illumination Microscopy (SIM), the role of LD dynamics have been tracked during cellular ferroptosis with the secretion, storage, and degradation of overexpressed ACSL3 proteins. It is found that LDs serve as storage sites for these proteins through membrane fusion, and further degrade overexpressed proteins via interactions with organelles like lysosomes or through lipophagy, thereby maintaining cellular homeostasis.

A Large Model-Derived Algorithm for Complex Organoids with Internal Morphogenesis and Digital Marker Derivation.

Automated segmentation and evaluation algorithms have been demonstrated to enhance the simplicity and translational utility of organoid technology. However, there is a pressing need for the development of complex organoids that possess epithelium environmental elements, dense regional cell aggregation, and intraorganoid morphologies. Nevertheless, there has been limited progress, including both the construction of data sets and the development of algorithms, in the use of user-friendly microscopy to address such complex organoids. In this study, a data set of bright-field and living cell fluorescence images in paired forms and with temporal variance was constructed using droplet-engineered lung organoids. Additionally, a large model-based algorithm was developed. Both the organoid contours and intraorganoid morphologies were included in the data set, and their physical parameters were included and screened to form multiplex digital markers for organoid evaluation. The algorithm has been demonstrated to outperform existing methods and is therefore suitable for the evaluation of complex organoids. It is expected that the algorithm will facilitate the successful demonstration of AI in organoid evaluation and decision-making regarding their status.

A Novel Rhodamine‐Tetraphenylporphyrin Fluorescence Probe for Imaging Mercury In Vitro and In Vivo

ABSTRACTA highly selective fluorescent sensor Por‐RBO for determination of Hg2+ was designed and synthesized with Rhodamine B and Tetraphenylporphyrin. The sensor can determine the Hg2+ of a solution within the pH 5.0–8.0, free from interference of other metal ions. When detected in buffer solution, the fluorescence was the strongest when five times the concentration of Hg2+ ion was added. Calculated by fluorescence titration method, the detection limit of sensor Por‐RBO for Hg2+ ion was as low as 0.12 μmol/L. The geometries of Por‐RBO and Por‐RBO‐Hg2+ were optimized at the B3LYP/6‐31G** level by density functional theory. The charge distribution, orbital interactions, and bonding characteristics were analyzed and compared in detail to discuss the recognition mechanism and structure–fluorescence property relationships. The results of cell fluorescence imaging and CCK‐8 experiments indicate that the sensor Por‐RBO exhibits lower cytotoxicity. Por‐RBO can be used for fluorescence distribution imaging of Hg2+ in living cells.

Synthesis of a novel water-soluble pyridine dicarboxylate and its application in fluorescence cell imaging

Abstract A novel water-soluble substituted pyridine dicarboxylate containing a quaternary ammonium ion with potential applications in fluorescence cell imaging was synthesized and characterized. Remarkably, this compound exhibited water solubility in the absence of additional solubilizers, enabling direct dissolution in phosphate-buffered saline for cell studies. No obvious cytotoxicity was observed in 4T1 cells (mouse breast cancer cells) within the concentration range of 0.2–25 μmol L−1, with a cell survival rate above 89%. Furthermore, the compound exhibited a maximum two-photon absorption cross-section of 86 GM at an excitation wavelength of 780 nm, demonstrating high cell permeability, effective distribution in the cytoplasm, and preferential targeting of organelles. Single- and two-photon fluorescence imaging of cells revealed a distinctive blue emission and strong bright green emission, respectively. The newly synthesized substituted pyridine dicarboxylate exhibited low cytotoxicity and strong fluorescence imaging properties, demonstrating its potential in cell imaging applications.

A pH visual sensing platform based on dual-emission chiral carbon dots for discrimination of normal/cancer cells and monitoring food freshness

Accurate detection of pH is important in pathological processes and food freshness. Developing sensors of sensitive response and visualization for pH is highly demanded. In this work, Chiral carbon dots(CCDs) was synthesized via one-pot hydrothermal process using o-phenylenediamine and L-Tryptophan, which displayed circular dichroism (CD) signals at 200–255 nm and 255–300 nm. The CCDs exhibited dual-emission peaks with blue and red emission bands when excited at 360 nm. The ratiometric signals of UV–vis absorption and fluorescence intensity of L-CDs were responsive over the pH range of 2.0 to 11.0 with significant color changes in solution. Fluorescence imaging of live cells displayed different signals related to pH in both the blue and red channels, allowing accurate measurement of the pH of the cellular environment. Furthermore, the pH test paper based on L-CDs enabled monitoring the freshness of shrimp and pork under 365 nm UV light. Therefore, L-CDs provided a multifunctional visual pH sensing platform for environmental monitoring and biosensing.

Dual-color fluorescent multi-component hybrid materials for copper ion detection, NADH regeneration and cell imaging

Gold nanoclusters (AuNCs) exhibit unique physical, chemical and photoelectric properties with various advantages. On the other hand, small organic fluorescent molecules show alternative specialized characteristics. To overcome their weaknesses and integrate strengths, we propose here a workable platform to fabricate protein-gold-dye multifunctional hybrid materials. Briefly, protein skeleton bovine serum albumin (BSA), AuNCs, and organic molecule dye Thioflavin T (ThT) are co-assembled mutually to form a dual-color fluorescent material BSA-AuNCs@ThT. This approach is simple, cost-effective, green and environmental friendly. To tackle their biological application, we coat BSA-AuNCs@ThT with cationic polyethyleneimine (PEI). The prepared BSA-AuNCs@ThT@PEI hybrid materials can be used to fluorescence imaging of microbial cells as well as HeLa cells. Due to the unique background of BSA-AuNCs@ThT@PEI, the hybrid materials work well in differentiate heavy metal ions such as Hg2+ and Cu2+ ions. Moreover, this system is also suitable to do the photocatalytic transformation and regeneration of coenzyme NADH. Therefore, we propose this platform to build new nano-dye hybrid materials for copper ion detection, NADH regeneration and cell imaging, or potentially other applications.

BODIPY Photosensitizers Functionalized with Mannose for Fluorescence Cell-imaging and Photodynamic Therapy

The synthesis of four water-soluble mannose-tethering BODIPY-based photosensitizers (PSs) with varying substituents (-H, -OMe, -NO2 and -I) in the para-phenyl moiety attached to the meso-position of the BODIPY core, offering tumor-targeting and photodynamic therapeutic (PDT) potentials, is reported. The PDT capability of the synthesized PSs was investigated using the cervical cancer cell line HeLa. Owing to the heavy atom effect, BODIPY I, enhanced the intersystem crossing (ISC) to produce singlet oxygen (1O2) and maintained a high fluorescence quantum yield (ΦF) of 0.71. In contrast, the electron-donating methoxy substituent (BODIPY OMe) caused a notable perturbation in the occupied frontier molecular orbitals, subsequently achieving a higher 1O2 generation capability with a high ΦF of 0.85. Conversely, substitution with the electron-withdrawing nitro group (BODIPY NO2) resulted in a perturbation of unoccupied frontier molecular orbitals and generated a forbidden dark S1 state, negatively affecting both fluorescence and 1O2 generation efficiencies. The cytotoxicity of the synthesized water-soluble BODIPY PSs in the dark and under LED irradiation against the HeLa cell line was also assessed, with BODIPY I, OMe and H showing favorable PDT activity. Collectively, the performance of the mannose-conjugated BODIPY PSs in this study contributed promising results for the development of photodynamic therapeutic agents for cancer treatment.

Rosmarinic acid attenuates glioblastoma cells and spheroids’ growth and EMT/stem-like state by PTEN/PI3K/AKT downregulation and ERK-induced apoptosis

BackgroundGlioblastoma (GB) is a highly malignant type of brain cancer with a poor prognosis. Therapeutic strategies for GB are still limited. Rosmarinic acid (RA), a polyphenolic compound, is a promising experimental anticancer agent, but its specific protein targets for GB remain unclear. PurposeThis study aimed to elucidate the anticancer effects of RA in 2D- and 3D-GB cells and the underlying mechanisms. Methods3D-tumor spheroids (mimics in vivo tumors) were obtained by the hanging-drop/agarose method. RA's anti-glioma activity on U-87MG (p53-wt/PTEN-mt) and LN229 (p53-mt/PTEN-wt) cells was evaluated through cell viability, colony-formation, migration/invasion/angiogenesis assays, fluorescence imaging, and spheroid growth analysis. The underlying mechanism of the anticancer effects of RA was investigated by Western blot and immunofluorescence analysis. The MEK inhibitor U0126 was used to block ERK phosphorylation. ResultsRA treatments exerted anti-proliferative and pro-apoptotic effects on human GB cells. RA dose-dependently reduced angiogenesis and intracellular ROS levels, suppressed glioma growth, and migration/invasion in 2D-culture and cancer stem cell (CSC)-like 3D-spheroid culture (SPC). Repeated therapy in SPC was more effective by leading to disrupted structure than a single treatment. Treatments in SPC also suppressed epithelial-mesenchymal transition (EMT) and CSC-like properties. Strikingly, RA downregulated the SIRT1/FOXO1/NF-κB axis independently of p53 or PTEN function in both gliomas. Immunofluorescence labeling revealed decreased SIRT1 and NF-κB-p65 and increased FOXO1 and GAPDH proteins in nuclear location (associated with apoptosis). Surprisingly, RA increased p-ERK1/2 levels, but priming with U0126 abolished RA-mediated p-ERK upregulation; thus, autophagy and apoptosis induction in GB cells were prevented, and the growth of GB spheroids accelerated. Specifically, RA also inhibited the PTEN/PI3K/AKT pathway in U-87MG cells. Due to genetic differences in cells, U-87MG cells were more sensitive to RA treatments than LN229 cells. Meanwhile, our positive control drug trial results with FDA-approved temozolomide (TMZ) used in GB treatment showed that our test compound rosmarinic acid exhibited higher therapeutic effects than TMZ at lower doses. ConclusionSuppression of EMT, downregulation of SIRT1/FOXO1/NF-κB axis, inhibition of PTEN/PI3K/AKT signaling pathway, and ERK-induced apoptosis and autophagy were determined to be involved in stopping glioma progression. Our findings for the first time, revealed that RA may have potential therapeutic use by having multiple targets in human brain cancer with further clinical studies.

Bilayer MOF nanomachine for precision breast cancer cell fluorescent imaging and therapy.

Abilayer MOF reporter (ZIF-67@FAM-mRNA@ZIF-8) was synthesized, and the ZIF-67 was used as a carrier and fluorescent quencher to connect the FAM reporter through electrostatic adsorption and coordination effect. The ZIF-8 covering the outer layer can improve the stability of the probe and cell permeability, which helps the FAM reporter effectively release. After entering the cancer cells, the acidic environment in the cells induced the decomposition of ZIF-8. The excess ATP in the tumor cells competitively binds ZIF-67, causing the FAM reporter to shed and restore fluorescence. The shed FAM reporter was specifically bound to the overexpressed miRNA-21 in breast cancer cells to achieve fluorescence imaging and therapy of breast cancer. The results of specific imaging and apoptosis experiments of breast cancer cells indicate that bilayer MOF nanomachine provides an effective nanotherapy platform for accurate fluorescence imaging.

The scaffolding protein IQGAP1 enhances EGFR signaling by promoting oligomerization and preventing degradation

IQGAP1 is a large, multi-domain scaffold that connects and modulates different signaling networks including the one initiated by epidermal growth factor (EGF). In this study, we have used live cell fluorescence imaging methods along with other biochemical techniques to follow the mechanisms used by IQGAP1 to enhance EGF signaling. We show that IQGAP1 enhances EGF signaling by promoting the oligomerization of its receptor, EGFR, upon EGF addition along with concurrent IQGAP oligomerization. Using cellular markers, we find that IQGAP1 promotes the plasma membrane localization of EGFR and promotes association to one of its phosphoinositide lipid pathway ligands, PI(3,4,5)P3. Additionally, we find that binding of EGFR to IQGAP1 protects EGFR from lysosomal degradation. Taken together, our results show that IQGAP1 enhances EGF-mediated pathway progression through mechanisms that augment simple scaffolding activities.

Preclinical Evaluation of a PSMA Aptamer-Based Bifunctional PET and Fluorescent Probe.

Prostate cancer is the most prevalent malignant tumor affecting male individuals worldwide. The accurate early detection of prostate cancer is crucial to preventing unnecessary diagnosis and subsequent excessive treatment. Prostate-specific membrane antigen (PSMA) has emerged as a promising biomarker for the diagnosis of prostate cancer. In this study, a dual-modality imaging probe utilizing aptamer technology was developed for positron emission tomography/near-infrared fluorescence (PET/NIRF) imaging, and the specificity and sensitivity of the probe toward PSMA were evaluated both in vitro and in vivo. The probe precursor NOTA-PSMA-Cy5 was synthesized via automated solid-phase oligonucleotide synthesis. Subsequently, the PET/NIRF dual-modality probe [68Ga]Ga-NOTA-PSMA-Cy5 was successfully prepared and exhibited favorable fluorescence properties and stability in vitro. The binding specificity of [68Ga]Ga-NOTA-PSMA-Cy5 to PSMA was assessed through flow cytometry, fluorescence imaging, and cellular uptake experiments in LNCaP cells and PC-3 cells. In vivo PET/NIRF imaging studies demonstrated the sensitive and specific binding of [68Ga]Ga-NOTA-PSMA-Cy5 to PSMA. Overall, the PET/NIRF dual-modality probe [68Ga]Ga-NOTA-PSMA-Cy5 shows promise for the diagnosis of prostate cancer and for the fluorescence-guided identification of PSMA-positive cancer lesions during surgical procedures.

Synthesis and optical properties of tyramine-functionalized boron dipyrromethene dyes for cell bioimaging

Tyramine signaling amplification (TSA) technology is generally applied in immunofluorescence, enzyme-linked immunoassays, in situ hybridization techniques, etc. Successful amplification of fluoresence signals cannot be achieved without excellent fluorescent dyes. BODIPY fluorophore is an ideal probe for cell fluorescence imaging, but pristine BODIPY cannot be direct used in the TSA system. In the paper, the new red-shifted tyramide-conjugated BODIPY (BDP-B/C/D) was synthesized via the Knoevenagel condensation reaction, which based on the tyramide-conjugated BODIPY (BDP-A). The synthesized dyes were combined with tyramine to obtain which could be used as a fluorescent substrate for enzymatic reaction of TSA. By using the selected substrate (BDP-C) in TSA, we found it to be more sensitive than the commercial dye 594 styramide for the detection of low-abundance antigen proteins.

Single-detector multiplex imaging flow cytometry for cancer cell classification with deep learning.

Imaging flow cytometry, which combines the advantages of flow cytometry and microscopy, has emerged as a powerful tool for cell analysis in various biomedical fields such as cancer detection. In this study, we develop multiplex imaging flow cytometry (mIFC) by employing a spatial wavelength division multiplexing technique. Our mIFC can simultaneously obtain brightfield and multi-color fluorescence images of individual cells in flow, which are excited by a metal halide lamp and measured by a single detector. Statistical analysis results of multiplex imaging experiments with resolution test lens, magnification test lens, and fluorescent microspheres validate the operation of the mIFC with good imaging channel consistency and micron-scale differentiation capabilities. A deep learning method is designed for multiplex image processing that consists of three deep learning networks (U-net, very deep super resolution, and visual geometry group 19). It is demonstrated that the cluster of differentiation 24 (CD24) imaging channel is more sensitive than the brightfield, nucleus, or cancer antigen 125 (CA125) imaging channel in classifying the three types of ovarian cell lines (IOSE80 normal cell, A2780, and OVCAR3 cancer cells). An average accuracy rate of 97.1% is achieved for the classification of these three types of cells by deep learning analysis when all four imaging channels are considered. Our single-detector mIFC is promising for the development of future imaging flow cytometers and for the automatic single-cell analysis with deep learning in various biomedical fields.

Bodipy-Based highly sensitive hydrogen sulfide fluorescent probe and its fluorescence imaging in cells and zebrafish

Hydrogen sulfide (H2S) is a crucial endogenous gasotransmitter that plays a role in various physiological and pathological processes. Therefore, accurate and rapid monitoring of H2S in organisms is highly significant for understanding the underlying pathological mechanisms and facilitating early diagnosis of related diseases. In this study, we developed a novel fluorescent probe, B-CHO-NO2, based on a bodipy fluorophore, which exhibits excellent sensitivity and selectivity towards H2S. The design of the probe exploits the nucleophilicity of H2S by introducing a formyl group as the ortho-participating moiety, significantly enhancing the reaction rate with H2S. In cellular and zebrafish models, the probe B-CHO-NO2 successfully achieved fluorescence imaging of endogenous and exogenous H2S. The development of probe B-CHO-NO2 provides a powerful tool for biological studies of H2S and diagnosis of related diseases.

Facile Light-Driven Synthesis of Highly Luminous Sulfur Quantum Dots for Fluorescence Sensing and Cell Imaging.

Sulfur quantum dots (SQDs) are emerging fluorescent nanomaterials, whereas most of the methods for synthesizing SQDs are limited to thermal synthesis. In this study, we report the first case of a light-driven strategy for facile synthesis of SQDs and further applied the SQDs for fluorescence cell imaging. The light-driven synthesis strategy only utilized Na2S as the sulfur source and nano-TiO2 as the photosensitizer. Under ultraviolet illumination, the nano-TiO2 photosensitizer generated a large number of •O2- and •OH to oxidize S2- to Sx2- and further to elemental sulfur, which could be obtained as monodispersed SQDs after etching by H2O2. The prepared SQDs exhibit excellent tunable photoluminescence properties, superior stability, and a uniform small size, with particle diameters in the range of 0.5-4 nm, and the fluorescence absolute quantum yield is as high as 27.8%. Meanwhile, the prepared SQDs also exhibited extreme biocompatibility and stability, and we further applied it for intracellular imaging and Hg2+ sensing with satisfactory results. In comparison to the widely reported thermal synthesis, the light-driven synthesis method is greener and simpler, opening a new way for the preparation of biocompatible SQDs.

Selective Cellular Uptake and Druggability Efficacy through Functionalized Chitosan-Conjugated Polyamidoamine (PAMAM) Dendrimers.

Nanotechnology has ushered in significant advancements in drug design, revolutionizing the prevention, diagnosis, and treatment of various diseases. The strategic utilization of nanotechnology to enhance drug loading, delivery, and release has garnered increasing attention, leveraging the enhanced physical and chemical properties offered by these systems. Polyamidoamine (PAMAM) dendrimers have been pivotal in drug delivery, yet there is room for further enhancement. In this study, we conjugated PAMAM dendrimers with chitosan (CS) to augment cellular internalization in tumor cells. Specifically, doxorubicin (DOX) was initially loaded into PAMAM dendrimers to form DOX-loaded PAMAM (DOX@PAMAM) complexes via intermolecular forces. Subsequently, CS was linked onto the DOX-loaded PAMAM dendrimers to yield CS-conjugated PAMAM loaded with DOX (DOX@CS@PAMAM) through glutaraldehyde crosslinking via the Schiff base reaction. The resultant DOX@CS@PAMAM complexes were comprehensively characterized using Fourier-transform infrared (FTIR) spectroscopy, transmission electron microscopy (TEM), and dynamic light scattering (DLS). Notably, while the drug release profile of DOX@CS@PAMAM in acidic environments was inferior to that of DOX@PAMAM, DOX@CS@PAMAM demonstrated effective acid-responsive drug release, with a cumulative release of 70% within 25 h attributed to the imine linkage. Most importantly, DOX@CS@PAMAM exhibited significant selective cellular internalization rates and antitumor efficacy compared to DOX@PAMAM, as validated through cell viability assays, fluorescence imaging, and flow cytometry analysis. In summary, DOX@CS@PAMAM demonstrated superior antitumor effects compared to unconjugated PAMAM dendrimers, thereby broadening the scope of dendrimer-based nanomedicines with enhanced therapeutic efficacy and promising applications in cancer therapy.

Dicyanisophorone-based viscosity fluorescent probe and its fluorescence imaging in cells and zebrafish

Cellular viscosity is a critical parameter for maintaining normal cellular functions, and its abnormalities are associated with the occurrence and development of various diseases. Therefore, real-time monitoring of cellular viscosity is of significant importance for understanding viscosity-related biological functions and revealing the pathological mechanisms of diseases. In this study, we developed a novel fluorescence probe (IP-HBO) by hybridizing dicyanisophorone with 2-(2-hydroxyphenyl)benzoxazole for monitoring of cellular microenvironment and viscosity changes in zebrafish. The probe IP-HBO combines the principles of molecular rotors and enhanced solid-state fluorescence properties, demonstrating highly specific responsiveness to viscosity, long-wavelength emission, large Stokes shift, and excellent photostability. It has been successfully employed for fluorescence imaging of viscosity in live HeLa cells and zebrafish. The construction of this probe provides a new tool for gaining insights into viscosity-related biological processes and disease mechanisms within cells.

Benzothiazole-Quinoline-Based Fluorescent Probe for Fe3+ and its Applications in Environmental and Biological Samples.

Due to the its high abundance, iron ion contamination and toxicity is one of the most challenging issue for living beings. Although, iron is extremenly important for several body functions, excess amount of iron in the body can also be fatal. In last century, rapid industrialization, iron extraction and mismanagement of industrial waste disposal leads to iron contamination in water bodies. Therefore, versatile iron sensors needs to be develop which can be employed for detection in biological as well as real water samples. 8-hydroxyquinoline is well-known for its strong affinity towards transition metals including Fe3+. In this regard, we have synthesised benzothiazole-quinoline derived 1,2,3- triazole (4HBTHQTz), in which 4-(benzo[d]thiazol-2-yl)phenolic (4-HBT) group acts as a fluorophore. 4HBTHQTz showed high fluorescence and induced a selective decrease in fluorescence with Fe3+ at 380nm (λex. = 320nm). The detection limit of 4HBTHQTz with Fe3+ is calculated as 0.64μM, which is lower than the WHO recommended limit in drinking water. 4HBTHQTz works over the 5-8 pH range and has shown promising results for quantitative detection of Fe3+ in water samples collected from tap, river and seawater. 4HBTHQTz can also detect the Fe3+ in biological samples which is confirmed by fluorescence cell imaging using L929 mouse fibroblast cells. Overall, 4HBTHQTz showed advantages such as high selectivity, quick detection, and good limit of detection (LOD) for Fe3+.

Novel pyrene-based Schiff base ligand as a fluorescent sensor for Al3+ and colorimetric sensor for Ni2+ ions detection: Synthesis, photo-physical and cell imaging studies

A simple fluorescent and colorimetric pyrene-based ((Z)-4-hydroxy-3-((pyren-1-ylmethylene)amino)benzoic acid (HPMB) chemosensor was synthesized through the condensation reaction between pyrene-1-carboxaldehyde and 3-amino-4-hydroxybenzoic acid. The structure of HPMB was elucidated using 1H NMR, MS, and UV–Visible spectroscopic techniques. The receptor HPMB displayed a detectable, colorimetric colour shift in the presence of Ni2+ and the colour of the solution was observed to be light brown. On the other hand, it also exhibited a fluorescent “turn on” response when Al3+ ions were added in DMSO solution (HEPES 0.01 M, pH = 7.4) at room temperature. Job’s plot showed that HPMB binding ratio to Ni2+ and Al3+ ions was in 1:1 ratio. The detection limit for Ni2+ and Al3+ ions was found to be 0.33 × 10-6 M and 0.26 × 10-6 M, respectively. In addition, HPMB was utilised to create a molecular logic gate, and the fluorescence imaging of HeLa cells was investigated because of HPMB’s low cytotoxicity, indicating that it could be used to detect Al3+ and Ni2+ in living cells.

Extracellular vesicles involved in the crosstalk between apoptosis and ferroptosis revealed via a viscosity-sensitive fluorescent probe targeting lysosomes and nucleoli

Advancements in cancer therapy have emerged through the combination of multiple signaling pathways, minimizing drug resistance and off-target effects. Therefore, diverse drug design strategies have been developed, including hybrid apoptosis/ferroptosis pathways. However, not much is known regarding their crosstalk, particularly regarding the details of intracellular and intercellular communication. Extracellular vesicles (EVs) play a crucial role in signal transduction and matter exchange among cells and are categorized into ectosomes, exosomes, and apoptotic bodies of varying sizes. The biogenesis and microenvironment of EVs are crucial for understanding their biological functions; however, they are still under debate. Herein, we used a fluorescent probe with viscosity sensitivity that targets lysosomes and nucleoli to detect viscosity alterations during apoptosis and ferroptosis, resulting in triggered green emission. Further fluorescent imaging of cells revealed crosstalk between apoptosis and ferroptosis, detecting apoptotic bodies originating from lysosomes and condensed nucleoli as EVs in hybrid apoptosis/ferroptosis pathways.