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Search Results (501)

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Keywords = fluorescence live-cell imaging

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13 pages, 1484 KiB  
Article
A Long-Wavelength Fluorescent Probe for Efficient Dual-Color Imaging of Boronic-Acid-Containing Agents in Living Cells
by Shinya Takada, Honghuo Du, Naoya Kondo, Anna Miyazaki, Fumiko Hara, Shizuyo Horiyama, Takashi Temma and Masayori Hagimori
Chemosensors 2025, 13(8), 283; https://doi.org/10.3390/chemosensors13080283 - 4 Aug 2025
Abstract
In boron neutron capture therapy (BNCT), the intracellular localization and concentration of boron-10 atoms significantly influence therapeutic efficacy. Although various boronic-acid-targeted fluorescent probes have been developed to evaluate BNCT agents, most of these probes emit at short wavelengths and are, therefore, incompatible with [...] Read more.
In boron neutron capture therapy (BNCT), the intracellular localization and concentration of boron-10 atoms significantly influence therapeutic efficacy. Although various boronic-acid-targeted fluorescent probes have been developed to evaluate BNCT agents, most of these probes emit at short wavelengths and are, therefore, incompatible with common nuclear-staining reagents such as Hoechst 33342 and 4′,6-diamidino-2-phenylindole (DAPI). While our previously reported probe, BS-631, emitted fluorescence above 500 nm, it exhibited limitations in terms of reaction rate and fluorescence intensity. To address these issues, we developed a boronic-acid-targeted fluorescent probe with a longer emission wavelength, rapid reactivity, and strong fluorescence intensity. Herein, we designed and synthesized BTTQ, a probe based on a 2-(2-hydroxyphenyl)benzothiazole core structure. BTTQ exhibited immediate fluorescence upon reaction with 4-borono-L-phenylalanine (BPA), with an emission wavelength of 567 nm and a sufficiently high fluorescence quantum yield for detection. BTTQ quantitatively detected BPA with high sensitivity (quantification limit of 10.27 µM), suitable for evaluating BNCT agents. In addition, BTTQ exhibited selective fluorescence for BPA over metal cations. Importantly, BTTQ enabled fluorescence microscopic imaging of intracellular BPA distribution in living cells co-stained with Hoechst 33342. These results suggest that BTTQ is a promising fluorescent probe for the evaluation of future BNCT agents. Full article
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17 pages, 1571 KiB  
Review
Super-Resolution Microscopy in the Structural Analysis and Assembly Dynamics of HIV
by Aiden Jurcenko, Olesia Gololobova and Kenneth W. Witwer
Appl. Nano 2025, 6(3), 13; https://doi.org/10.3390/applnano6030013 - 31 Jul 2025
Viewed by 132
Abstract
Super-resolution microscopy (SRM) has revolutionized our understanding of subcellular structures, including cell organelles and viruses. For human immunodeficiency virus (HIV), SRM has significantly advanced knowledge of viral structural biology and assembly dynamics. This review analyzes how SRM techniques (particularly PALM, STORM, STED, and [...] Read more.
Super-resolution microscopy (SRM) has revolutionized our understanding of subcellular structures, including cell organelles and viruses. For human immunodeficiency virus (HIV), SRM has significantly advanced knowledge of viral structural biology and assembly dynamics. This review analyzes how SRM techniques (particularly PALM, STORM, STED, and SIM) have been applied over the past decade to study HIV structural components and assembly. By categorizing and comparing studies based on SRM methods, HIV components, and labeling strategies, we assess the strengths and limitations of each approach. Our analysis shows that PALM is most commonly used for live-cell imaging of HIV Gag, while STED is primarily used to study the viral envelope (Env). STORM and SIM have been applied to visualize various components, including Env, capsid, and matrix. Antibody labeling is prevalent in PALM and STORM studies, targeting Env and capsid, whereas fluorescent protein labeling is mainly associated with PALM and focused on Gag. A recent emphasis on Gag and Env points to deeper investigation into HIV assembly and viral membrane dynamics. Insights from SRM studies of HIV not only enhance virological understanding but also inform future research in therapeutic strategies and delivery systems, including extracellular vesicles. Full article
(This article belongs to the Collection Review Papers for Applied Nano Science and Technology)
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36 pages, 1807 KiB  
Review
Thriving or Withering? Plant Molecular Cytogenetics in the First Quarter of the 21st Century
by Elzbieta Wolny, Luis A. J. Mur, Nobuko Ohmido, Zujun Yin, Kai Wang and Robert Hasterok
Int. J. Mol. Sci. 2025, 26(14), 7013; https://doi.org/10.3390/ijms26147013 - 21 Jul 2025
Viewed by 351
Abstract
Nearly four decades have passed since fluorescence in situ hybridisation was first applied in plants to support molecular cytogenetic analyses across a wide range of species. Subsequent advances in DNA sequencing, bioinformatic analysis, and microscopy, together with the immunolocalisation of various nuclear components, [...] Read more.
Nearly four decades have passed since fluorescence in situ hybridisation was first applied in plants to support molecular cytogenetic analyses across a wide range of species. Subsequent advances in DNA sequencing, bioinformatic analysis, and microscopy, together with the immunolocalisation of various nuclear components, have provided unprecedented insights into the cytomolecular organisation of the nuclear genome in both model and non-model plants, with crop species being perhaps the most significant. The ready availability of sequenced genomes is now facilitating the application of state-of-the-art cytomolecular techniques across diverse plant species. However, these same advances in genomics also pose a challenge to the future of plant molecular cytogenetics, as DNA sequence analysis is increasingly perceived as offering comparable insights into genome organisation. This perception persists despite the continued relevance of FISH-based approaches for the physical anchoring of genome assemblies to chromosomes. Furthermore, cytogenetic approaches cannot currently rival purely genomic methods in terms of throughput, standardisation, and automation. This review highlights the latest key topics in plant cytomolecular research, with particular emphasis on chromosome identification and karyotype evolution, chromatin and interphase nuclear organisation, chromosome structure, hybridisation and polyploidy, and cytogenetics-assisted crop improvement. In doing so, it underscores the distinctive contributions that cytogenetic techniques continue to offer in genomic research. Additionally, we critically assess future directions and emerging opportunities in the field, including those related to CRISPR/Cas-based live-cell imaging and chromosome engineering, as well as AI-assisted image analysis and karyotyping. Full article
(This article belongs to the Collection Feature Papers in Molecular Plant Sciences)
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26 pages, 38900 KiB  
Article
A Set of Fluorescent Protein-Based Markers for Major Vesicle Coat Proteins in Yeast
by Xue-Fei Cui, Zheng-Tan Zhang, Jing Zhu, Li Cui and Zhiping Xie
Membranes 2025, 15(7), 209; https://doi.org/10.3390/membranes15070209 - 13 Jul 2025
Viewed by 402
Abstract
In eukaryotic cells, vesicle-mediated transport interconnects the endomembrane system. These vesicles are formed by coat proteins via deformation of donor membranes. Here, we constructed a set of fluorescent protein-based markers for major coat protein complexes in the yeast model system, and examined their [...] Read more.
In eukaryotic cells, vesicle-mediated transport interconnects the endomembrane system. These vesicles are formed by coat proteins via deformation of donor membranes. Here, we constructed a set of fluorescent protein-based markers for major coat protein complexes in the yeast model system, and examined their subcellular localization patterns. Our markers covered COPII, COPI, AP-1, AP-2, AP-3, and retromer complexes. Our live cell imaging demonstrates that COPII puncta were primarily associated with the endoplasmic reticulum (ER), and occasionally with early Golgi. COPI was present on both early Golgi and late Golgi/early endosomes. AP-1 puncta were present on late Golgi/early endosomes. AP-2 was present on plasma membrane (PM)-associated puncta, and around the bud neck. AP-3 puncta were present on late Golgi/early endosomes and on the surface of vacuoles. Retromer was present on the surface of vacuoles, late endosomes, and other perivacuolar puncta. Notably, more than half of AP-1 puncta and AP-3 puncta were not associated with the donor compartments where they are thought to be generated, implying that these were coated transport vesicles. This work provides a convenient tool set for the investigation of vesicular transport in yeast and live cell imaging evidence for the presence of certain coated transport vesicles. Full article
(This article belongs to the Section Biological Membranes)
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22 pages, 1363 KiB  
Review
Live-Cell Imaging of Flaviviridae Family Virus Infections: Progress and Challenges
by Siena M. Centofanti and Nicholas S. Eyre
Viruses 2025, 17(6), 847; https://doi.org/10.3390/v17060847 - 13 Jun 2025
Viewed by 497
Abstract
The ability of a virus to be propagated within a host cell is dependent on a multitude of dynamic virus–host interactions. Live-cell imaging is an invaluable approach in the study of virus replication cycles and virus–host interactions as it can allow for the [...] Read more.
The ability of a virus to be propagated within a host cell is dependent on a multitude of dynamic virus–host interactions. Live-cell imaging is an invaluable approach in the study of virus replication cycles and virus–host interactions as it can allow for the direct visualisation of key events and interactions in real time. These details can provide unique insights into many aspects of viral infections including the cellular pathways that are exploited by viruses, the evasion of host immune defences, and viral pathogenesis. This review summarises the live-cell fluorescence imaging approaches that have been developed and applied to study Flaviviridae virus family members that are responsible for significant public health burdens and outbreaks which, in many instances, are increasing in frequency and severity. We discuss how these approaches have expanded our understanding of fundamental stages of viral replication cycles by enabling the direct visualisation of the localisation, trafficking, and interactions of virus particles, proteins, and genomes at distinct stages. The strategies that can be employed to enhance the biological relevance of live-cell fluorescence imaging acquisitions are discussed, along with how live-cell imaging approaches can be further developed to increase resolution, enable multi-colour imaging, and support the long-term visualisation of multiple stages of a viral replication cycle. Full article
(This article belongs to the Section Human Virology and Viral Diseases)
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13 pages, 3184 KiB  
Article
Furin-Triggered Peptide Self-Assembly Activates Coumarin Excimer Fluorescence for Precision Live-Cell Imaging
by Peiyao Chen, Liling Meng, Yuting Wang, Xiaoya Yan, Meiqin Li, Yun Deng and Yao Sun
Molecules 2025, 30(11), 2465; https://doi.org/10.3390/molecules30112465 - 4 Jun 2025
Viewed by 605
Abstract
Monomer-to-excimer transition has become a valuable technique in fluorescence imaging because of its ability to enhance imaging contrast. However, from a practical perspective, the accuracy of excimer formation at target sites warrants further exploration. Enzyme-triggered peptide self-assembly provides a promising solution to this [...] Read more.
Monomer-to-excimer transition has become a valuable technique in fluorescence imaging because of its ability to enhance imaging contrast. However, from a practical perspective, the accuracy of excimer formation at target sites warrants further exploration. Enzyme-triggered peptide self-assembly provides a promising solution to this limitation. As a proof-of-concept, in this study, we developed a furin-triggered peptide self-assembling fluorescent probe RF-Cou by coupling a coumarin dye 7-(diethylamino)-2-oxo-2H-chromene-3-carboxylic acid (Cou) with a furin-responsive peptide scaffold for precision live-cell imaging. Upon entering furin-overexpressing 4T1 tumor cells, RF-Cou underwent enzymatic cleavage, releasing an amphiphilic peptide motif and self-assembling into nanoparticles largely concentrated in the Golgi apparatus to confine the diffusion of Cou. During this process, the Cou excimers were formed and induced a red shift in the fluorescence emission, validating the feasibility of RF-Cou in efficient excimer imaging of furin-overexpressing tumor cells. We expect that our findings will highlight the potential of stimuli-responsive small molecular peptide probes to advance excimer-based imaging platforms, particularly for enzyme-specific cell imaging and therapeutic monitoring. Full article
(This article belongs to the Special Issue Metal-Based Molecular Photosensitizers: From Design to Applications)
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18 pages, 3767 KiB  
Article
Flow Cytometric Analysis and Sorting of Murine Enteric Nervous System Cells: An Optimized Protocol
by Faidra Karkala, Indy de Bosscher, Jonathan D. Windster, Savio Stroebel, Lars van Zanten, Maria M. Alves and Andrea Sacchetti
Int. J. Mol. Sci. 2025, 26(10), 4824; https://doi.org/10.3390/ijms26104824 - 18 May 2025
Viewed by 909
Abstract
Isolation of neurons and glia from the enteric nervous system (ENS) enables ex vivo studies, including the analysis of genomic and transcriptomic profiles. While we previously reported a fluorescence-activated cell sorting (FACS)-based isolation protocol for human ENS cells, no equivalent exists for mice. [...] Read more.
Isolation of neurons and glia from the enteric nervous system (ENS) enables ex vivo studies, including the analysis of genomic and transcriptomic profiles. While we previously reported a fluorescence-activated cell sorting (FACS)-based isolation protocol for human ENS cells, no equivalent exists for mice. As directly applying the human protocol to mouse tissue resulted in low recovery of live ENS cells, we optimized tissue dissociation using mouse colons. A 30 min Liberase-based digestion showed optimal recovery of viable ENS cells, with CD56 and CD24 emerging as the most reliable markers to select and subdivide these cells. ENS’ identity was further validated by FACS, using neuronal (TUBB3) and glial (SOX10) markers and reverse transcriptase quantitative PCR on sorted fractions. Overall, the mouse ENS expression profile significantly overlapped with the human one, showing that current dissociation protocols yield a mixed population of enteric neurons and glia. Nonetheless, using the imaging flow cytometer BD S8 FACS Discover and ELAVL4 as a neuronal soma-associated marker, we observed enrichment of neurons in a CD56/CD24TIP population. In conclusion, we present here a protocol for high-purity FACS-based isolation of viable mouse ENS cells, suitable for downstream applications. Full article
(This article belongs to the Special Issue Trends and Prospects of Flow Cytometry in Cell and Molecular Biology)
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17 pages, 2739 KiB  
Article
TP53 Mutation-Specific Dysregulation of Store-Operated Calcium Entry and Apoptotic Sensitivity in Triple-Negative Breast Cancer
by Kaneez E. Rabab, Paul J. Buchanan, Grace Colley, Anita White, Aisling Murphy, Chloe McCormack and Alex J. Eustace
Cancers 2025, 17(10), 1614; https://doi.org/10.3390/cancers17101614 - 10 May 2025
Cited by 1 | Viewed by 1055
Abstract
Background: Triple-negative breast cancer (TNBC) is an aggressive subtype lacking estrogen, progesterone, and HER2 receptors, and is associated with poor prognosis and limited targeted therapeutic options. TP53 mutations occur in the majority of TNBC cases, disrupting p53’s role in DNA repair and apoptosis. [...] Read more.
Background: Triple-negative breast cancer (TNBC) is an aggressive subtype lacking estrogen, progesterone, and HER2 receptors, and is associated with poor prognosis and limited targeted therapeutic options. TP53 mutations occur in the majority of TNBC cases, disrupting p53’s role in DNA repair and apoptosis. Beyond gene regulation, p53 also influences calcium signalling through store-operated calcium entry (SOCE), a critical pathway for cell survival and death. However, the impact of different TP53 mutation types on calcium signalling remains unclear. Methods: Calcium channel gene expression was analysed using publicly available TNBC datasets. Calcium channel expression and SOCE activity were assessed in TNBC cell lines with different TP53 mutations using quantitative PCR and calcium imaging (Fura-2AM). Cell proliferation was measured using acid phosphatase assays, while apoptosis was evaluated through caspase 3/7 activation using the Incucyte live-cell fluorescent imager. The p53 reactivator COTI-2 was tested for its ability to restore TP53 function and modulate calcium signalling. Results: Analysis revealed significant downregulation of CACNA1D in TP53-mutant TNBCs. TNBC cell lines harbouring frameshift and stop TP53 mutations exhibited reduced SOCE, lower CACNA1D expression, and resistance to thapsigargin-induced apoptosis compared to wild-type cells. In contrast, cells with the TP53 R273H missense mutation demonstrated similar calcium signalling and proliferation to TP53 wild-type cels. COTI-2 treatment restored CACNA1D expression and SOCE in frameshift and stop mutant cells, enhancing apoptotic sensitivity. Combined treatment with COTI-2 and thapsigargin resulted in a synergistic increase in apoptosis. Conclusions: This study identifies a novel link between TP53 mutation type and calcium signalling in TNBC. Reactivating mutant p53 with COTI-2 restores calcium-mediated apoptosis, supporting combination strategies targeting both TP53 dysfunction and calcium signalling. Full article
(This article belongs to the Special Issue Calcium Signaling in Cancer Cell Progression)
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17 pages, 3073 KiB  
Article
L-Lysine-Linked Modular Fluorescent Cholesteryl Mimics: Biophysical Properties, Molecular Interactions, and Cellular Applications
by Nicholas McInchak, Laura Stawikowska, Haylee Mesa, Jonathan Meade, Qi Zhang and Maciej J. Stawikowski
Sci 2025, 7(2), 56; https://doi.org/10.3390/sci7020056 - 7 May 2025
Viewed by 561
Abstract
Fluorescent cholesterol probes are indispensable tools for studying membrane structure, dynamics, and trafficking. To better understand the structure–function relationship of fluorescent cholesteryl probes, we developed a series of five new modular naphthalimide-containing cholesteryl probes (CND15–CND19). These probes incorporate an L-lysine linker between the [...] Read more.
Fluorescent cholesterol probes are indispensable tools for studying membrane structure, dynamics, and trafficking. To better understand the structure–function relationship of fluorescent cholesteryl probes, we developed a series of five new modular naphthalimide-containing cholesteryl probes (CND15–CND19). These probes incorporate an L-lysine linker between the cholesterol moiety and the fluorophore, along with a series of distinct head groups. We conducted extensive biophysical characterizations of these probes, including the determination of their solvatochromic properties and lipid partitioning behavior using giant unilamellar vesicles. Molecular dynamics simulations were employed to identify key molecular interactions of these probes within model lipid membranes. Furthermore, live-cell imaging in 3T3 fibroblasts demonstrated the potential applications of these analogs in live-cell imaging, measuring cellular membrane dynamics and studying cholesterol-related processes. The results of this study underscore the critical role of the linker and head group in designing fluorescent cholesterol-mimicking probes. These findings provide valuable insights into optimizing probe designs for future cholesterol and membrane biology research. Full article
(This article belongs to the Section Biology Research and Life Sciences)
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19 pages, 7264 KiB  
Article
Selective and Sensitive Dual Chromogenic Cyanide and Fluorescent Azide Probe
by Yousef M. Hijji, Rajeesha Rajan, Amjad M. Shraim, Bassam Attili, Sisay Uota and Fasil Abebe
Photochem 2025, 5(2), 12; https://doi.org/10.3390/photochem5020012 - 6 May 2025
Cited by 1 | Viewed by 664
Abstract
IR-780 is a heptamethine cyanine dye that exhibits strong absorbance in the near-infrared region. Herein, we report IR-780 dye as a dual sensor for chromogenic cyanide detection and azide’s fluorogenic sensing in acetonitrile. Cyanide and hydroxide cause instant, dramatic color changes in the [...] Read more.
IR-780 is a heptamethine cyanine dye that exhibits strong absorbance in the near-infrared region. Herein, we report IR-780 dye as a dual sensor for chromogenic cyanide detection and azide’s fluorogenic sensing in acetonitrile. Cyanide and hydroxide cause instant, dramatic color changes in the dye solution from green to yellow and dramatic spectral changes in the UV-Vis spectrum. The interaction of cyanide and hydroxide with the dye caused a dramatic decrease in the intensity of the strong absorption band at 780 nm and a concomitant band appearance at 435 nm. Other monovalent ions, including fluoride, chloride, bromide, iodide, dihydrogen phosphate, thiocyanate, acetate, and dihydrogen arsenate, caused no significant color or spectral changes. UV-Vis studies showed that the IR-780 dye is sensitive and selective to both ions. The detection limits for cyanide and azide are 0.39 µM and 0.50 µM, respectively. Interestingly, the IR-780 dye exhibited strong fluorescence at 535nm upon interaction with azide, while its initial emission at 809 nm was quenched. Both UV-Vis and fluorescence spectroscopy accomplished the detection of cyanide and azide using IR-780. Furthermore, the sensor’s effectiveness in fluorescence imaging of intracellular CN⁻ ions is demonstrated in live HeLa cells. Full article
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11 pages, 4512 KiB  
Article
Fluorescence Lifetime Multiplexing with Fluorogen-Activating FAST Protein Variants and Red-Shifted Arylidene–Imidazolone Derivative as Fluorogen
by Aidar R. Gilvanov, Ivan N. Myasnyanko, Sergey A. Goncharuk, Marina V. Goncharuk, Vadim S. Kublitski, Daria V. Bodunova, Svetlana V. Sidorenko, Eugene G. Maksimov, Mikhail S. Baranov and Yulia A. Bogdanova
Biosensors 2025, 15(5), 274; https://doi.org/10.3390/bios15050274 - 29 Apr 2025
Viewed by 537
Abstract
Fluorescence-lifetime imaging microscopy (FLIM) is a powerful technique for highly multiplexed imaging in live cells. In this work, we present a genetically encoded FLIM multiplexing platform based on a combination of fluorogen-activating protein FAST and red-shifted fluorogen N871b from the arylidene–imidazolone family. We [...] Read more.
Fluorescence-lifetime imaging microscopy (FLIM) is a powerful technique for highly multiplexed imaging in live cells. In this work, we present a genetically encoded FLIM multiplexing platform based on a combination of fluorogen-activating protein FAST and red-shifted fluorogen N871b from the arylidene–imidazolone family. We showed that a series of FAST protein mutants exhibit similar steady-state optical properties in complex with N871b fluorogen but have different fluorescence lifetimes. The similar brightness and binding strength of pairs of these FAST protein variants with N871b allows them to be successfully used for multiplexing up to three intracellular structures of living cells simultaneously. Full article
(This article belongs to the Section Optical and Photonic Biosensors)
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41 pages, 10319 KiB  
Review
BODIPY Dyes: A New Frontier in Cellular Imaging and Theragnostic Applications
by Panangattukara Prabhakaran Praveen Kumar, Shivanjali Saxena and Rakesh Joshi
Colorants 2025, 4(2), 13; https://doi.org/10.3390/colorants4020013 - 2 Apr 2025
Cited by 2 | Viewed by 4254
Abstract
BODIPY (Boron-Dipyrromethene) dyes have emerged as versatile fluorescent probes in cellular imaging and therapeutic applications owing to their unique chemical properties, including high fluorescence quantum yield, strong extinction coefficients, and remarkable photostability. This review synthesizes the recent advancements in BODIPY dyes, focusing on [...] Read more.
BODIPY (Boron-Dipyrromethene) dyes have emerged as versatile fluorescent probes in cellular imaging and therapeutic applications owing to their unique chemical properties, including high fluorescence quantum yield, strong extinction coefficients, and remarkable photostability. This review synthesizes the recent advancements in BODIPY dyes, focusing on their deployment in biological imaging and therapy. The exceptional ability of BODIPY dyes to selectively stain cellular structures enables precise visualization of lipids, proteins, and nucleic acids within live and tumor cells, thereby facilitating enhanced understanding of biochemical processes. Moreover, BODIPY derivatives are increasingly utilized in Photodynamic therapy (PDT) and Photothermal therapies (PTT) for targeting cancer cells, where their capability to generate cytotoxic reactive oxygen species upon light activation offers a promising approach to tumor treatment. Recently, BODIPY derivatives have been used for Boron Neutron Capture Therapy (BNCT) for various tumors, and it is a growing research field. Advancements in nanotechnology have allowed the fabrication of BODIPY dye-based nanomedicines, either alone or with the use of metallic nanoparticles as a matrix offering the development of a new class of bioimaging and theragnostic agents. This review also discusses innovative BODIPY-based formulations and strategies that amplify therapeutic efficacy while minimizing adverse effects, underscoring the potential of these dyes as integral components in next-generation diagnostic and therapeutic modalities. By summarizing current research and future perspectives, this review highlights the critical importance of BODIPY dyes in advancing the fields of cellular imaging and treatment methodologies. Full article
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19 pages, 3735 KiB  
Article
A Fluorescent Probe for Imaging and Treating S-Nitrosation Stress in OGD/R Cells
by Hui Ye, Chen Zhang, Lerong Li, Cunrui Li, Jiayue Yu, Duorui Ji, Zhuangzhuang Liang, Jianbing Wu and Zhangjian Huang
Antioxidants 2025, 14(3), 311; https://doi.org/10.3390/antiox14030311 - 4 Mar 2025
Cited by 1 | Viewed by 1099
Abstract
Protein S-nitrosation, a redox post-translational modification elicited by nitric oxide (NO), is essential for modulating diverse protein functions and signaling pathways. Dysregulation of S-nitrosation is implicated in various pathological processes, including oxygen-glucose deprivation/reperfusion (OGD/R) injury, a widely used model for ischemia-reperfusion diseases. The [...] Read more.
Protein S-nitrosation, a redox post-translational modification elicited by nitric oxide (NO), is essential for modulating diverse protein functions and signaling pathways. Dysregulation of S-nitrosation is implicated in various pathological processes, including oxygen-glucose deprivation/reperfusion (OGD/R) injury, a widely used model for ischemia-reperfusion diseases. The dynamic changes in S-nitrosothiols (SNOs) during ischemia-reperfusion highlight the need for theranostic strategies to monitor and modulate SNO levels based on pathological progression. However, to date, no theranostic strategies have been reported for addressing dysregulated SNO in disease models, particularly in OGD/R conditions. Here, we report the development of a selective probe P-EHC, which could specifically react with SNOs to release EHC, not only exhibiting turn-on fluorescence with high quantum yield and good water solubility but also demonstrating macrophage migration inhibitory factor (MIF) inhibitory activity. In an OGD/R model of SH-SY5Y cells, we observed elevated SNO levels by using live-cell confocal imaging. Treatment of P-EHC significantly reduced intracellular reactive oxygen species (ROS), lowered total NOx species, and improved cell viability in the OGD/R model. In summary, the simplicity and versatility of P-EHC suggest its broad applicability for monitoring SNO in various biological models and therapeutic contexts, particularly in ischemia-reperfusion diseases. Full article
(This article belongs to the Special Issue Nitric Oxide and Redox Mechanisms)
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15 pages, 15102 KiB  
Article
The Curse of the Red Pearl: A Fibroblast-Specific Pearl-Necklace Mitochondrial Phenotype Caused by Phototoxicity
by Irene M. G. M. Hemel, Kèvin Knoops, Carmen López-Iglesias and Mike Gerards
Biomolecules 2025, 15(2), 304; https://doi.org/10.3390/biom15020304 - 19 Feb 2025
Viewed by 716
Abstract
The dynamic nature of mitochondria makes live cell imaging an important tool in mitochondrial research. Although imaging using fluorescent probes is the golden standard in studying mitochondrial morphology, these probes might introduce aspecific features. In this study, live cell fluorescent imaging was applied [...] Read more.
The dynamic nature of mitochondria makes live cell imaging an important tool in mitochondrial research. Although imaging using fluorescent probes is the golden standard in studying mitochondrial morphology, these probes might introduce aspecific features. In this study, live cell fluorescent imaging was applied to investigate a pearl-necklace-shaped mitochondrial phenotype that arises when mitochondrial fission is restricted. In this fibroblast-specific pearl-necklace phenotype, constricted and expanded mitochondrial regions alternate. Imaging studies revealed that the formation time of this pearl-necklace phenotype differs between laser scanning confocal, widefield and spinning disk confocal microscopy. We found that the phenotype formation correlates with the excitation of the fluorescent probe and is the result of phototoxicity. Interestingly, the phenotype only arises in cells stained with red mitochondrial dyes. Serial section electron tomography of the pearl-necklace mitochondria revealed that the mitochondrial membranes remained intact, while the cristae structure was altered. Furthermore, filaments and ER were present at the constricted sites. This study illustrates the importance of considering experimental conditions for live cell imaging to prevent imaging artifacts that can have a major impact on the obtained results. Full article
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12 pages, 4717 KiB  
Article
A Ratiometric Fluorescence Nano pH Biosensor for Live-Cell Imaging Using Cerasome
by Zhongqiao Zhang, Xiaoshan Luo, Xuanbo Wang, Meng Liu, Xiuli Yue and Zhaozhu Zheng
Biosensors 2025, 15(2), 114; https://doi.org/10.3390/bios15020114 - 16 Feb 2025
Viewed by 996
Abstract
The development of a robust and biocompatible pH-sensing platform is critical for monitoring intracellular processes and diagnosing diseases. Here, we present a smart ultrastable ratiometric fluorescence nano pH sensor based on silica-coated liposome nanoparticles (cerasome, 138.4 nm). The sensor integrates pH-sensitive dye, pyranine, [...] Read more.
The development of a robust and biocompatible pH-sensing platform is critical for monitoring intracellular processes and diagnosing diseases. Here, we present a smart ultrastable ratiometric fluorescence nano pH sensor based on silica-coated liposome nanoparticles (cerasome, 138.4 nm). The sensor integrates pH-sensitive dye, pyranine, within cerasome, achieving enhanced photostability, sensitivity, and biocompatibility. Its unique ratiometric design enables precise pH monitoring with minimal photobleaching and quenching, covering a linear detection range of pH 6.25–8.5. The hybrid nanoparticles exhibit high morphological stability, making them suitable for real-time intracellular pH measurement. This novel platform shows great promise for applications in cellular biology, disease diagnosis, and therapeutic monitoring, offering a versatile tool for biomedical research. Full article
(This article belongs to the Special Issue Nanotechnology-Based Optical Sensors for Biomedical Applications)
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