Subcellular Structures Research Articles

Single-Molecule Spectroscopy and Super-Resolution Mapping of Physicochemical Parameters in Living Cells.

By superlocalizing the positions of millions of single molecules over many camera frames, a class of super-resolution fluorescence microscopy methods known as single-molecule localization microscopy (SMLM) has revolutionized how we understand subcellular structures over the past decade. In this review, we highlight emerging studies that transcend the outstanding structural (shape) information offered by SMLM to extract and map physicochemical parameters in living mammalian cells at single-molecule and super-resolution levels. By encoding/decoding high-dimensional information-such as emission and excitation spectra, motion, polarization, fluorescence lifetime, and beyond-for every molecule, and mass accumulating these measurements for millions of molecules, such multidimensional and multifunctional super-resolution approaches open new windows into intracellular architectures and dynamics, as well as their underlying biophysical rules, far beyond the diffraction limit.

Cellular Alterations Due to Direct and Indirect Interaction of Nanomaterials with Nucleic Acids.

Deoxyribonucleic acid (DNA) represents the main reservoir of genetic information in the cells, which is why it is protected in the nucleus. Entry into the nucleus is, in general, difficult, as the nuclear membrane is a selective barrier to molecules longer than 40 kDa. However, in some cases, the size of certain nanoparticles (NPs) allows their internalization into the nucleus, thus causing a direct effect on the DNA structure. NPs can also induce indirect effects on DNA through reactive oxygen species (ROS) generation. In this context, nanomaterials are emerging as a disruptive tool for the development of novel therapies in a broad range of biomedical fields; although their effect on cell viability is commonly studied, further interactions with DNA or indirect alterations triggered by the internalization of these materials are not always clarified, since the small size of these materials makes them perfectly suitable for interaction with subcellular structures, such as the nucleus. In this context, and using as a reference the predicted interactions presented in a computational model, we describe and discuss the observed direct and indirect effects of the implicated nanomaterials on DNA.

Functional Relevance of Extracellular Vesicle-Derived Long Non-Coding and Circular RNAs in Cancer Angiogenesis.

Extracellular vesicles (EVs) are defined as subcellular structures limited by a bilayer lipid membrane that function as important intercellular communication by transporting active biomolecules, such as proteins, amino acids, metabolites, and nucleic acids, including long non-coding RNAs (lncRNAs). These cargos can effectively be delivered to target cells and induce a highly variable response. LncRNAs are functional RNAs composed of at least 200 nucleotides that do not code for proteins. Nowadays, lncRNAs and circRNAs are known to play crucial roles in many biological processes, including a plethora of diseases including cancer. Growing evidence shows an active presence of lnc- and circRNAs in EVs, generating downstream responses that ultimately affect cancer progression by many mechanisms, including angiogenesis. Moreover, many studies have revealed that some tumor cells promote angiogenesis by secreting EVs, which endothelial cells can take up to induce new vessel formation. In this review, we aim to summarize the bioactive roles of EVs with lnc- and circRNAs as cargo and their effect on cancer angiogenesis. Also, we discuss future clinical strategies for cancer treatment based on current knowledge of circ- and lncRNA-EVs.

Asymptotic analysis of conversion-limited phase separation

Liquid–liquid phase separation plays a major role in the formation and maintenance of various membrane-less subcellular structures in the cytoplasm and nucleus of cells. Biological condensates contain enhanced concentrations of proteins and RNA, many of which can be continually exchanged with the surrounding medium. Coarsening is an important step in the kinetics of phase separation, whereby an emulsion of polydisperse condensates transitions to a single condensate in thermodynamic equilibrium with a surrounding dilute phase. A key feature of biological phase separation is the co-existence of multiple condensates over significant time scales, which is consistent with experimental observations showing a slowing of coarsening rates. It has recently been proposed that one rate limiting step could be the slow interfacial conversion of a molecular constituent between the dilute and dense phases. In this paper, we analyse conversion-limited phase separation within the framework of diffusion in singularly perturbed domains, which exploits the fact that biological condensates tend to be much smaller than the size of a cell. Using matched asymptotic analysis, we solve the quasi-static diffusion equation for the concentration in the dilute phase, and then derive kinetic equations for the slow growth/shrinkage of the condensates. This provides a systematic way of obtaining corrections to mean-field theory that take into account the geometry of the cell and the locations of all the condensates.

Asymmetric response emerges between creation and disintegration of force-bearing subcellular structures as revealed by percolation analysis.

Cells dynamically remodel their internal structures by modulating the arrangement of actin filaments (AFs). In this process, individual AFs exhibit stochastic behavior without knowing the macroscopic higher-order structures they are meant to create or disintegrate, but the mechanism allowing for such stochastic process-driven remodeling of subcellular structures remains incompletely understood. Here we employ percolation theory to explore how AFs interacting only with neighboring ones without recognizing the overall configuration can nonetheless create a substantial structure referred to as stress fibers (SFs) at particular locations. We determined the interaction probabilities of AFs undergoing cellular tensional homeostasis, a fundamental property maintaining intracellular tension. We showed that the duration required for the creation of SFs is shortened by the increased amount of preexisting actin meshwork, while the disintegration occurs independently of the presence of actin meshwork, suggesting that the coexistence of tension-bearing and non-bearing elements allows cells to promptly transition to new states in accordance with transient environmental changes. The origin of this asymmetry between creation and disintegration, consistently observed in actual cells, is elucidated through a minimal model analysis by examining the intrinsic nature of mechano-signal transmission. Specifically, unlike the symmetric case involving biochemical communication, physical communication to sense environmental changes is facilitated via AFs under tension, while other free AFs dissociated from tension-bearing structures exhibit stochastic behavior. Thus, both the numerical and minimal models demonstrate the essence of intracellular percolation, in which macroscopic asymmetry observed at the cellular level emerges not from microscopic asymmetry in the interaction probabilities of individual molecules, but rather only as a consequence of the manner of the mechano-signal transmission. These results provide novel insights into the role of the mutual interplay between distinct subcellular structures with and without tension-bearing capability. Insight: Cells continuously remodel their internal elements or structural proteins in response to environmental changes. Despite the stochastic behavior of individual structural proteins, which lack awareness of the larger subcellular structures they are meant to create or disintegrate, this self-assembly process somehow occurs to enable adaptation to the environment. Here we demonstrated through percolation simulations and minimal model analyses that there is an asymmetry in the response between the creation and disintegration of subcellular structures, which can aid environmental adaptation. This asymmetry inherently arises from the nature of mechano-signal transmission through structural proteins, namely tension-mediated information exchange within cells, despite the stochastic behavior of individual proteins lacking asymmetric characters in themselves.

Investigation of the functional impact of CHED- and FECD4-associated SLC4A11 mutations in human corneal endothelial cells.

Mutations in the solute linked carrier family 4 member 11 (SLC4A11) gene are associated with congenital hereditary endothelial dystrophy (CHED) and Fuchs corneal endothelial dystrophy type 4 (FECD4), both characterized by corneal endothelial cell (CEnC) dysfunction and/or cell loss leading to corneal edema and visual impairment. In this study, we characterize the impact of CHED-/FECD4-associated SLC4A11 mutations on CEnC function and SLC4A11 protein localization by generating and comparing human CEnC (hCEnC) lines expressing wild type SLC4A11 (SLC4A11WT) or mutant SLC4A11 harboring CHED-/FECD4-associated SLC4A11 mutations (SLC4A11MU). SLC4A11WT and SLC4A11MU hCEnC lines were generated to express either SLC4A11 variant 2 (V2WT and V2MU) or variant 3 (V3WT and V3MU), the two major variants expressed in ex vivo hCEnC. Functional assays were performed to assess cell barrier, proliferation, viability, migration, and NH3-induced membrane conductance. We demonstrate SLC4A11-/- and SLC4A11MU hCEnC lines exhibited increased migration rates, altered proliferation and decreased cell viability compared to SLC4A11WT hCEnC. Additionally, SLC4A11-/- hCEnC demonstrated decreased cell-substrate adhesion and membrane capacitances compared to SLC4A11WT hCEnC. Induction with 10mM NH4Cl led SLC4A11WT hCEnC to depolarize; conversely, SLC4A11-/- hCEnC hyperpolarized and the majority of SLC4A11MU hCEnC either hyperpolarized or had minimal membrane potential changes following NH4Cl induction. Immunostaining of primary hCEnC and SLC4A11WT hCEnC lines for SLC4A11 demonstrated predominately plasma membrane staining with poor or partial colocalization with mitochondrial marker COX4 within a subset of punctate subcellular structures. Overall, our findings suggest CHED-associated SLC4A11 mutations likely lead to hCEnC dysfunction, and ultimately CHED, by interfering with cell migration, proliferation, viability, membrane conductance, barrier function, and/or cell surface localization of the SLC4A11 protein in hCEnC. Additionally, based on their similar subcellular localization and exhibiting similar cell functional profiles, protein isoforms encoded by SLC4A11 variant 2 and variant 3 likely have highly overlapping functional roles in hCEnC.

Encapsulation of Lactobacillus rhamnosus GG in double emulsions: Role of prebiotics in improving probiotics survival during spray drying and storage

The protective effect of solid fat on probiotics to reduce heat damage during spray drying was revealed in previous study. However, the direct dispersion of fresh cells in the thermo-protectants did not encapsulate the probiotics in the oil phase, but dispersed in the water phase. Therefore, this study aims to improve the protective effect of solid fat to probiotics during spray drying by encapsulating Lactobacillus rhamnosus GG (LGG) in double emulsion (W/O/W) containing solid fat. In addition, various prebiotics were added in W/O/W as wall materials, and their ability of improving the vitality of probiotics was evaluated by combining spray drying and re-culture. The results of fluorescence microscope and scanning electron microscope demonstrated the successful encapsulation of LGG in double emulsions during spray drying, which increased the survival rate from 43.23% to 65.16% while improved the integrity of the subcellular structure. In terms of prebiotic effects, the microcapsules of W/O/W + inulin showed the lowest water activity and higher glass transition temperature, leading to the best survival of LGG during storage in all tested formulations, while W/O/W + FOS had the best proliferation-promoting in re-culture due to the fact that LGG consumed more FOS than other prebiotics during re-culture. Therefore, the current study has provided valuable information for the selection of carriers and wall materials in improving the viability of probiotics during spray drying.

Soft X-ray Microscopy in Cell Biology: Current Status, Contributions and Prospects.

The recent advances achieved in microscopy technology have led to a significant breakthrough in biological research. Super-resolution fluorescent microscopy now allows us to visualize subcellular structures down to the pin-pointing of the single molecules in them, while modern electron microscopy has opened new possibilities in the study of protein complexes in their native, intracellular environment at near-atomic resolution. Nonetheless, both fluorescent and electron microscopy have remained beset by their principal shortcomings: the reliance on labeling procedures and severe sample volume limitations, respectively. Soft X-ray microscopy is a candidate method that can compensate for the shortcomings of both technologies by making possible observation of the entirety of the cellular interior without chemical fixation and labeling with an isotropic resolution of 40-70 nm. This will thus bridge the resolution gap between light and electron microscopy (although this gap is being narrowed, it still exists) and resolve the issue of compatibility with the former, and possibly in the near future, the latter methods. This review aims to assess the current state of soft X-ray microscopy and its impact on our understanding of the subcellular organization. It also attempts to look into the future of X-ray microscopy, particularly as relates to its seamless integration into the cell biology toolkit.

Foliar spraying of Zn/Si affects Cd accumulation in paddy grains by regulating the remobilization and transport of Cd in vegetative organs

The reduction of cadmium (Cd) accumulation in rice grains through biofortification of essential nutrients like zinc (Zn) and silicon (Si) is an area of study that has gained significant attention. However, there is limited understanding of the mechanism of Zn/Si interaction on Cd accumulation and remobilization in rice plants. This work used a pot experiment to examine the effects of Zn and Si applied singly or in combination on the physiological metabolism of Cd in different rice organs under Cd stress. The results revealed that: Zn/Si application led to a significant decrease in root Cd concentration and reduce the value of Tf Soil-Root in filling stage. The content of phytochelatin (PCs, particularly PC2) and glutathione (GSH) in roots, top and basal nodes were increased with Zn/Si treatment application. Furthermore, Zn/Si treatment promoted the distribution of Cd in cell wall during Cd stress. These findings suggest that Zn/Si application facilitates the compartmentalization of Cd within subcellular structures and enhances PCs production in vegetative organs, thereby reducing Cd remobilization. Zn/Si treatment upregulated the metabolism of amino acid components involved in osmotic regulation, secondary metabolite synthesis, and plant chelating peptide synthesis in vegetative organs. Additionally, it significantly decreased the accumulation of Cd in globulin, albumin, and glutelin, resulting in an average reduction of 50.87% in Cd concentration in milled rice. These results indicate that Zn/Si nutrition plays a crucial role in mitigating heavy metal stress and improving the nutritional quality of rice by regulating protein composition and coordinating amino acid metabolism balance.

Development and immunological evaluation of an mRNA-based vaccine targeting Naegleria fowleri for the treatment of primary amoebic meningoencephalitis

More than 95% of patients fall victim to primary amoebic meningoencephalitis (PAM), a fatal disease attacking the central nervous system. Naegleria fowleri, a brain-eating microorganism, is PAM's most well-known pathogenic ameboflagellate. Despite the use of antibiotics, the fatality rate continues to rise as no clinical trials have been conducted against this disease. To address this, we mined the UniProt database for pathogenic proteins and selected assumed epitopes to create an mRNA-based vaccine. We identified thirty B-cell and T-cell epitopes for the vaccine candidate. These epitopes, secretion boosters, subcellular trafficking structures, and linkers were used to construct the vaccine candidate. Through predictive modeling and confirmation via the Ramachandran plot (with a quality factor of 92.22), we assessed secondary and 3D structures. The adjuvant RpfE was incorporated to enhance the vaccine construct's immunogenicity (GRAVY index: 0.394, instability index: 38.99, antigenicity: 0.8). The theoretical model of immunological simulations indicated favorable responses from both innate and adaptive immune cells, with memory cells expected to remain active for up to 350 days post-vaccination, while the antigen was eliminated from the body within 24 h. Notably, strong interactions were observed between the vaccine construct and TLR-4 (− 11.9 kcal/mol) and TLR-3 (− 18.2 kcal/mol).

TRIB3, as a robust prognostic biomarker for HNSC, is associated with poor immune infiltration and cancer cell immune evasion.

As a pseudokinase, Tribbles Pseudokinase 3 (TRIB3) is implicated in a wide array of biological processes, including cell signal transduction, metabolic regulation, stress responses, and immune regulation. While its significant role in the immune regulation of certain cancers is well-established, the specific functions and impact of TRIB3 in head and neck squamous cell carcinoma (HNSC) remain unclear. The data of RNA-sequence was acquired from the TCGA database to analyze the expression patterns of TRIB3 and elucidate its prognostic value in HNSC patients. Furthermore, the correlation between TRIB3 and tumor mutation burden, clinical data, immune checkpoint genes, and immune cell infiltration was explored. Moreover, the TRIB3 location in tumor tissues and subcellular structures was identified via Tisch in the HPA database, and the potential protein interaction molecules for TRIB3 were elucidated in the STRING database. The potential TRIB3 gene function was assessed using gene set enrichment analysis (GSEA), whereas the TRIB3 expression levels in clinical HNSC samples were verified by RT-qPCR and immunohistochemistry. the role of TRIB3 in enhancing the malignant behavior of HNSC cells was validated in vitro through a series of methods including RT-qPCR, CCK8 assay, wound healing assay, and transwell assay. It was revealed that TRIB3 was significantly overexpressed in the nucleus and cytoplasm of HNSC. Furthermore, this overexpression markedly enhanced the migration ability of tumor cells. As an independent prognostic factor, TRIB3 was associated with advanced tumor T stage and was significantly involved with tumor mutation burden and immune cell infiltration in HNSC. Moreover, it was observed that TRIB3 was not a predicted factor for PD1/PDL1 and ATL4 inhibitor treatment; however, it was substantially correlated with various immune evasion-related genes in HNSC. TRIB3 could serve as a potential prognostic marker for HNSC and might be a key gene mediating HNSC immune evasion.

Deep-Learning-Based Nanomechanical Vibration for Rapid and Label-Free Assay of Epithelial Mesenchymal Transition.

Cancer is a profound danger to our life and health. The classification and related studies of epithelial and mesenchymal phenotypes of cancer cells are key scientific questions in cancer research. Here, we investigated cancer cell colonies from a mechanical perspective and developed an assay for classifying epithelial/mesenchymal cancer cell colonies using the biomechanical fingerprint in the form of "nanovibration" in combination with deep learning. The classification method requires only 1 s of vibration data and has a classification accuracy of nearly 92.5%. The method has also been validated for the screening of anticancer drugs. Compared with traditional methods, the method has the advantages of being nondestructive, label-free, and highly sensitive. Furthermore, we proposed a perspective that subcellular structure influences the amplitude and spectrum of nanovibrations and demonstrated it using experiments and numerical simulation. These findings allow internal changes in the cell colony to be manifested by nanovibrations. This work provides a perspective and an ancillary method for cancer cell phenotype diagnosis and promotes the study of biomechanical mechanisms of cancer progression.

AI-driven projection tomography with multicore fibre-optic cell rotation

Optical tomography has emerged as a non-invasive imaging method, providing three-dimensional insights into subcellular structures and thereby enabling a deeper understanding of cellular functions, interactions, and processes. Conventional optical tomography methods are constrained by a limited illumination scanning range, leading to anisotropic resolution and incomplete imaging of cellular structures. To overcome this problem, we employ a compact multi-core fibre-optic cell rotator system that facilitates precise optical manipulation of cells within a microfluidic chip, achieving full-angle projection tomography with isotropic resolution. Moreover, we demonstrate an AI-driven tomographic reconstruction workflow, which can be a paradigm shift from conventional computational methods, often demanding manual processing, to a fully autonomous process. The performance of the proposed cell rotation tomography approach is validated through the three-dimensional reconstruction of cell phantoms and HL60 human cancer cells. The versatility of this learning-based tomographic reconstruction workflow paves the way for its broad application across diverse tomographic imaging modalities, including but not limited to flow cytometry tomography and acoustic rotation tomography. Therefore, this AI-driven approach can propel advancements in cell biology, aiding in the inception of pioneering therapeutics, and augmenting early-stage cancer diagnostics.

Subcellular biochemistry and biology of filamentous entomopathogenic fungi.

Filamentous entomopathogenic fungi (EPF) function as important biotic factors regulating the arthropod population in natural ecosystems and have great potential as biocontrol agents in modern agriculture. In the infection cycle, EPF undergo a plethora of physiological processes, including metabolism (e.g., cuticle hydrolysis and nutrient utilization), development (e.g., dimorphism and conidiation), stress response (e.g., oxidative and osmotic stresses), and immune evasion from the host. In-depth explorations of the mechanisms involved in the lifecycle of EPF offer excellent opportunities to increase their virulence and stability, which increases the efficacy of EPF in biocontrol programs. This review discusses the current state of knowledge relating to the biological roles and regulatory mechanisms of organelles and subcellular structures in the physiology of EPF, as well as some suggestions for future investigation.

A correlated light and electron microscopy approach to study the subcellular localization of phosphorylated vimentin in human lung tissue.

Correlative microscopy is an important approach for bridging the resolution gap between fluorescence light and electron microscopy. Here, we describe a fast and simple method for correlative immunofluorescence and immunogold labeling on the same section to elucidate the localization of phosphorylated vimentin (P-Vim), a robust feature of pulmonary vascular remodeling in cells of human lung small arteries. The lung is a complex, soft and difficult tissue to prepare for transmission electron microscopy (TEM). Detailing the molecular composition of small pulmonary arteries (<500μm) would be of great significance for research and diagnostics. Using the classical methods of immunochemistry (either hydrophilic resin or thin cryosections), is difficult to locate small arteries for analysis by TEM. To address this problem and to observe the same structures by both light and electron microscopy, correlative microscopy is a reliable approach. Immunofluorescence enables us to know the distribution of P-Vim in cells but does not provide ultrastructural detail on its localization. Labeled structures selected by fluorescence microscope can be identified and further analyzed by TEM at high resolution. With our method, the morphology of the arteries is well preserved, enabling the localization of P-Vim inside pulmonary endothelial cells. By applying this approach, fluorescent signals can be directly correlated to the corresponding subcellular structures in areas of interest.

Investigating Liquid-Liquid Phase Separation in Virus-Generated Inclusion Bodies Using Fluorescence Recovery After Photobleaching of Fluorescently Labeled Host Proteins.

Many negative-sense single-stranded RNA viruses within the order Mononegavirales harm humans. A common feature shared among cells infected by these viruses is the formation of subcellular membraneless structures called biomolecular condensates, also known as inclusion bodies (IBs), that form through a process called liquid-liquid phase separation (LLPS). Like many other membraneless organelles, viral IBs enrich a specific subset of viral and host proteins involved in the formation of viral particles. Elucidation of the properties and regulation of these IBs as they mature throughout the viral replication process are important for our understanding of viral replication, which may also lead to the development of alternative antiviral treatments. The protocol outlined in this chapter aims to characterize the intrinsic properties of LLPS within themeasles virus (MeV, a member of Mononegavirales) IBs by using an imaging approach that fluorescently tags an IB-associated host protein. This method uses common laboratory techniques and is generalizable to any host factors as well as other viral systems.

Neisseria gonorrhoeae carbonic anhydrase inhibition.

Carbonic anhydrases (CAs) are ubiquitous enzymes that are found in all kingdoms of life. Though different classes of CAs vary in their roles and structures, their primary function is to catalyze the reaction between carbon dioxide and water to produce bicarbonate and a proton. Neisseria gonorrhoeae encodes for three distinct CAs (NgCAs) from three different families: an α-, a β-, and a γ-isoform. This chapter details the differences between the three NgCAs, summarizing their subcellular locations, roles, essentiality, structures, and enzyme kinetics. These bacterial enzymes have the potential to be drug targets; thus, previous studies have investigated the inhibition of NgCAs-primarily the α-isoform. Therefore, the classes of inhibitors that have been shown to bind to the NgCAs will be discussed as well. These classes include traditional CA inhibitors, such as sulfonamides, phenols, and coumarins, as well as non-traditional inhibitors including anions and thiocarbamates.

Advancements in nanoscale coherent emitters: The development of substrate-free surface plasmon nanolasers

The surface plasmon effect can be used to confine electromagnetic fields to a small footprint measuring tens of nanometers. The resultant resonant cavities function as optimal coherent light sources with subwavelength scale configurations. The plasmonic laser sources based on nanoshell structures, in particular, have demonstrated the potential for use in the detection of subcellular mesoscopic molecular structures. However, this structure has a high plasmon dephasing rate, which can increase the threshold of the device, making it difficult to achieve electrically excited structures, thereby rendering them unsuitable as an active component for integration into optoelectronic circuits. A different approach to confining electromagnetic fields involves using a propagating surface plasmon laser structured on a planar layered semiconductor–insulator–metal. This design enables the surface plasmon to propagate along the direction of the nanowire and offers the potential to achieve electrically driven structures by injecting current into the semiconductor nanowire. Consequently, this structure is more effective in guiding energy into integrated optoelectronic circuits compared to the isotropic radiation of nanoshell structures. However, this design also necessitates a supporting substrate, resulting in the actual device volume exceeding the nanoscale and, in some cases, even larger than the size of a cell. This limitation hinders the application of integrated optoelectronic circuits at the micro/nanoscale for bio-applications. To address these challenges, we developed a substrate-free surface plasmon polariton laser. We demonstrated that allowing direct contact between the film and the air significantly reduced the laser threshold. Furthermore, the device maintained its operational capability across different surfaces.

Preparation of Bunyavirus-Infected Cells for Electron Cryo-Tomography.

Cellular electron cryo-tomography (cryoET) produces high-resolution three-dimensional images of subcellular structures in a near-native frozen-hydrated state. These three-dimensional images are obtained by recording a series of two-dimensional tilt images on a transmission electron cryo-microscope that are subsequently back-projected to form a tomogram. Key to a successful experiment is however a high-quality sample. This chapter outlines a basic workflow for the preparation of cellular cryoET samples. It covers the preparation of infected cells on electron cryo-microscopy grids and the vitrification by plunge-freezing and clipping of grids into AutoGrid rims. It also provides a general overview of the workflow for thinning the vitrified cells by focused ion beam (FIB) milling. Although this book is dedicated to Rift Valley fever virus research, the present protocol may also be applied to any other research subject where high-resolution structural insight into intracellular processes is desired.

Comprehensive review on fluorescent carbon dots and their applications in nucleic acid detection, nucleolus targeted imaging and gene delivery.

Carbon dots (CDs), including carbon quantum dots, graphene quantum dots, carbon nanodots, and polymer dots, have gained significant attention due to their unique structural and fluorescence characteristics. This review provides a comprehensive overview of the classification, structural characteristics, and fluorescence properties of CDs, followed by an exploration of various fluorescence sensing mechanisms and their applications in gene detection, nucleolus imaging, and gene delivery. Furthermore, the functionalization of CDs with diverse surface ligand molecules, including dye molecules, nucleic acid probes, and metal derivatives, for sensitive nucleic acid detection is systematically examined. Fluorescence imaging of the cell nucleolus plays a vital role in examining intracellular processes and the dynamics of subcellular structures. By analyzing the mechanism of fluorescence and structure-function relationships inherent in CDs, the nucleolus targeting abilities of CDs in various cell lines have been discussed. Additionally, challenges such as the insufficient organelle specificity of CDs and the inconsistent mechanisms underlying nucleolus targeting have also been highlighted. The unique physical and chemical properties of CDs, particularly their strong affinity toward deoxyribonucleic acid (DNA), have spurred interest in gene delivery applications. The use of nuclear-targeting peptides, polymers, and ligands in conjunction with CDs for improved gene delivery applications have been systematically reviewed. Through a comprehensive analysis, the review aims to contribute to a deeper understanding of the potential and challenges associated with CDs in biomedical applications.