Fluorescence Imaging Of Cells Research Articles

Integrated Point-of-Care Immune Cell Analyzer with Rapid Blood Sample Reaction and Wide Field-of-View Detection.

The development of affordable, reliable, and rapid diagnostic devices is crucial for monitoring immunological responses using a drop of blood. However, conventional automated diagnostic devices typically involve expensive and impractical robotic fluid-handling approaches. Herein, we developed an integrated cell analyzer comprising a cylindrical sample cartridge connected to a direct current motor and a compact fluorescence imaging module. Sample mixing and loading are performed automatically by a programmable sequence of single motor rotation controlled by an Android application. Two distinct stained immune cell samples can be identified by using two types of fluorescence imaging modes. The effectiveness of mixing performance in antigen-antibody (Ag-Ab) reactions was assessed through a compound objective lens that collects weak fluorescence emitted by the cell membrane. Active mixing with bidirectional rotation of the cartridge in a confined space shortened the Ag-Ab reaction time by a factor of 3.3 and achieved cell counting with higher accuracy while reducing reagent consumption by 4 times compared to the conventional incubation method. High-intensity fluorescence images of cells labeled with a nucleic acid stain were acquired through a single-lens-based fluorescence imaging module with a large field of view (FOV) in an unconventional detection chamber with a curved substrate. Compared with a flat chamber, the curved detection chamber reduces the effects of field curvature and provides aberration-free wide-FOV images, even with a simple lens. Our integrated cell analyzer thus offers a practical and cost-effective solution for monitoring patient immune responses in point-of-care settings.

Biomimetic Synthesis and Chemical Proteomics Reveal the Mechanism of Action and Functional Targets of Phloroglucinol Meroterpenoids.

Natural products perennially serve as prolific sources of drug leads and chemical probes, fueling the development of numerous therapeutics. Despite their scarcity, natural products that modulate protein function through covalent interactions with lysine residues hold immense potential to unlock new therapeutic interventions and advance our understanding of the biological processes governed by these modifications. Phloroglucinol meroterpenoids constitute one of the most expansive classes of natural products, displaying a plethora of biological activities. However, their mechanism of action and cellular targets have, until now, remained elusive. In this study, we detail the concise biomimetic synthesis, computational mechanistic insights, physicochemical attributes, kinetic parameters, molecular mechanism of action, and functional cellular targets of several phloroglucinol meroterpenoids. We harness synthetic clickable analogues of natural products to probe their disparate proteome-wide reactivity and subcellular localization through in-gel fluorescence scanning and cell imaging. By implementing sample multiplexing and a redesigned lysine-targeting probe, we streamline a quantitative activity-based protein profiling, enabling the direct mapping of global reactivity and ligandability of proteinaceous lysines in human cells. Leveraging this framework, we identify numerous lysine-meroterpenoid interactions in breast cancer cells at tractable protein sites across diverse structural and functional classes, including those historically deemed undruggable. We validate that phloroglucinol meroterpenoids perturb biochemical functions through stereoselective and site-specific modification of lysines in proteins vital for breast cancer metabolism, including lipid signaling, mitochondrial respiration, and glycolysis. These findings underscore the broad potential of phloroglucinol meroterpenoids for targeting functional lysines in the human proteome.

Water-soluble near-infrared CuInS2/ZnS fluorescent quantum dots for Cu2+ detection and cellular imaging

The detection and imaging of intracellular copper ions (Cu2+) play a crucial role in understanding their physiological implications and their involvement in various cellular processes. This study focuses on the synthesis and characterization of water-soluble near-infrared fluorescent CIS/ZnS core–shell quantum dots (QDs) with excellent biocompatibility and stability. The QDs exhibit exceptional selectivity and sensitivity for Cu2+ detection, allowing for precise quantification of Cu2+ content within biological systems. Furthermore, Co-incubation of the QDs with HeLa cells demonstrates their impressive cellular imaging capabilities, facilitated by fluorescence bursts in response to changing Cu2+ content. Overall, our proposed QDs are of great significance in advancing the research and application of near-infrared semiconductor quantum dots in biomedicine, and their near-infrared fluorescence properties and high fluorescence quantum yield (34.48 %) make them very advantageous for the precise detection of intracellular Cu2+ and cell fluorescence imaging. This research contributes to the understanding of copper ion dynamics within cells and opens avenues for further exploration of its role in cellular physiology.

Fluorescence and magnetic resonance imaging of ONL-93 cells in a rat model of ischemic

ObjectivesThe current methods for detecting myelin changes in ischemic stroke are indirect and cannot accurately reflect their status. This study aimed to develop a novel fluorescent-magnetic resonance dual-modal molecular imaging probe for direct imaging of myelin. MethodsCompounds 7a and 7b were synthesized by linking the MeDAS group and Gadolinium (III) 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate. Compound 7a was selected for characterization and further study. Cell uptake, cytotoxicity, and magnetic resonance imaging scans were performed on cells. In vitro experiments on frozen brain sections from 7-day-old, 8-week-old, and ischemic stroke rats were compared with commercially available Luxol Fast Blue staining. After HPLC and MR scanning, brain tissue was soaked in 7a and scanned using T1WI and T1maps sequences. ResultsSpectrophotometer results showed that compounds 7a and 7b had fluorescent properties. MR scans indicated that the compounds had contrast agent properties. Cells could uptake 7a and exhibited high signals in imaging scans. Compound 7a brain tissue staining showed more fluorescence in myelin-rich regions and identified injury sites in ischemic stroke rats. MR scanning of brain sections provided clear myelin contrast. ConclusionA novel fluorescent-magnetic resonance dual-modal molecular imaging probe for direct imaging of myelin was successfully developed and tested in rats with ischemic stroke. These findings provide new insights for the clinical diagnosis of demyelinating diseases.

DNA-Based Fluorescent Nanoprobe for Cancer Cell Membrane Imaging.

As an important barrier between the cytoplasm and the microenvironment of the cell, the cell membrane is essential for the maintenance of normal cellular physiological activities. An abnormal cell membrane is a crucial symbol of body dysfunction and the occurrence of variant diseases; therefore, the visualization and monitoring of biomolecules associated with cell membranes and disease markers are of utmost importance in revealing the biological functions of cell membranes. Due to their biocompatibility, programmability, and modifiability, DNA nanomaterials have become increasingly popular in cell fluorescence imaging in recent years. In addition, DNA nanomaterials can be combined with the cell membrane in a specific manner to enable the real-time imaging of signal molecules on the cell membrane, allowing for the real-time monitoring of disease occurrence and progression. This article examines the recent application of DNA nanomaterials for fluorescence imaging on cell membranes. First, we present the conditions for imaging DNA nanomaterials in the cell membrane microenvironment, such as the ATP, pH, etc. Second, we summarize the imaging applications of cell membrane receptors and other molecules. Finally, some difficulties and challenges associated with DNA nanomaterials in the imaging of cell membranes are presented.

Red fluorescent BODIPY-based nanoparticles for targeted cancer imaging-guided photodynamic therapy.

Imaging-guided diagnosis and treatment of cancer hold potential to significantly improve therapeutic accuracies and efficacies. Central to this theragnostic approach has been the use of multicomponent-based multimodal nanoparticles (NPs). Apart from this conventional approach, here we propose a design strategy for the simple and straightforward formulation of NPs based on boron dipyrromethene (BODIPY) derivatives, LaB-X (X = H, Et, and Br). Specifically, the conjugation of lactose to the inherently hydrophobic BODIPY promoted the formation of LaB-X NPs in water. Furthermore, the BODIPY backbone was subjected to distyrylation, dibromination, and diethylation to tailor the optical window and the balance between fluorescence and singlet oxygen generation capabilities. We demonstrate that while the photoinduced anticancer activities of LaB-H and LaB-Et NPs were trivial, LaB-Br NPs effectively induced the apoptotic death of hepatocellular carcinoma cells under red light irradiation while allowing fluorescence cell imaging in the phototherapeutic window. This dual fluorescence photosensitizing activity of LaB-Br NPs could be switched off and on, so that both fluorescence and singlet oxygen generation were paused during NP formation in an aqueous solution, while both processes resumed after cellular uptake, likely due to NP disassembly.

Synthesis and Application of a Near-Infrared Light-Emitting Fluorescent Probe for Specific Imaging of Cancer Cells with High Sensitivity and Selectivity.

The preclinical diagnosis of tumors is of great significance to cancer treatment. Near-infrared fluorescence imaging technology is promising for the in-situ detection of tumors with high sensitivity. Here, a fluorescent probe was synthesized on the basis of Au nanoclusters with near-infrared light emission and applied to fluorescent cancer cell labeling. Near-infrared methionine-N-Hydroxy succinimide Au nanoclusters (Met-NHs-AuNCs) were prepared successfully by one-pot synthesis using Au nanoclusters, methionine, and N-Hydroxy succinimide as frameworks, reductants, and stabilizers, respectively. The specific fluorescence imaging of tumor cells or tissues by fluorescent probe was studied on the basis of SYBYL Surflex-DOCK simulation model of LAT1 active site of overexpressed receptor on cancer cell surface. The results showed that Met-NHs-AuNCs interacted with the surface of LAT1, and C_Score scored the conformation of the probe and LAT1 as five. Characterization and in vitro experiments were conducted to explore the Met-NHs-AuNCs targeted uptake of cancer cells. The prepared near-infrared fluorescent probe (Met-NHs-AuNCs) can specifically recognize the overexpression of L-type amino acid transporter 1 (LAT1) in cancer cells so that it can show red fluorescence in cancer cells. Meanwhile, normal cells (H9c2) have no fluorescence. The fluorescent probe demonstrates the power of targeting and imaging cancer cells.

Infectious Virus Tracking by Fluorescent Live Cell Imaging in Primary Cells.

To successfully infect a cell, HIV-1 has to overcome several host barriers while exploiting cellular cofactors. HIV-1 infection is highly inefficient with the great majority of viral particles not being able to successfully integrate into the target cell genome. Nonproductive HIV-1 particles are degraded or accumulated in cellular compartments. Thus, it becomes hard to distinguish between viral behaviors that lead to effectively infecting the cell from the ones that do not by using traditional methods. Here, we describe the infectious virus tracking method that detects and quantifies individual fluorescent viral particles over time and links viral particle behavior to its infectivity. This method employs live-cell imaging at ultra-low MOIs to detect the outcome of infection for every HIV-1 particle.

Designing functionalized nanodiamonds with hyaluronic acid-phospholipid conjugates for enhanced cancer cell targeting and fluorescence imaging capabilities.

Nanomedicine aims to develop smart approaches for treating cancer and other diseases to improve patient survival and quality of life. Novel nanoparticles as nanodiamonds (NDs) represent promising candidates to overcome current limitations. In this study, NDs were functionalized with a 200 kDa hyaluronic acid-phospholipid conjugate (HA/DMPE), enhancing the stability of the nanoparticles in water-based solutions and selectivity for cancer cells overexpressing specific HA cluster determinant 44 (CD44) receptors. These nanoparticles were characterized by diffuse reflectance Fourier-transform infrared spectroscopy, Raman spectroscopy, and photoluminescence spectroscopy, confirming the efficacy of the functionalization process. Scanning electron microscopy was employed to evaluate the size distribution of the dry particles, while dynamic light scattering and zeta potential measurements were utilized to evaluate ND behavior in a water-based medium. Furthermore, the ND biocompatibility and uptake mediated by CD44 receptors in three different models of human adenocarcinoma cells were assessed by performing cytofluorimetric assay and confocal microscopy. HA-functionalized nanodiamonds demonstrated the advantage of active targeting in the presence of cancer cells expressing CD44 on the surface, suggesting higher drug delivery to tumors over non-tumor tissues. Even CD44-poorly expressing cancers could be targeted by the NDs, thanks to their good passive diffusion within cancer cells.

Monoamine oxidase A probe synthesis and fluorescence imaging in cells and zebrafish based on AIE mechanism

Monoamine oxidase A (MAO-A) is a crucial mammalian flavoenzyme that plays a significant role in various neurodegenerative diseases such as depression, Parkinson’s disease, schizophrenia, and epilepsy, as well as cancer, cardiac disorders, and liver disease. Thus, a real-time and targeted MAO-A detection approach is desperately needed to study the onset and management of disorders linked to MAO-A. In this study, we have successfully synthesized a red-emitting fluorescent probe AIE-M, which is based on the specificity of MAOs for the structural catalysis of propylamine group substrates. This probe utilizes quinoline-malononitrile (QM) with AIE properties as the fluorescent master. The probe containing QM exhibits excellent aggregation-induced luminescence (AIE), biocompatibility, and high photostability, enabling specific recognition of MAO-A. Moreover, it can be employed for the detection and imaging of endogenous MAO-A in neuroblastoma cells and zebrafish, thereby offering a novel research instrument for diagnosing associated ailments.

Real-time imaging of individual electropores proves their longevity in cells

With over 50 years of electroporation research, the nature of cell membrane permeabilization remains elusive. The lifetime of electropores in molecular models is limited to nano- or microseconds, whereas the permeabilization of electroporated cells can last minutes. This study aimed at resolving a longstanding debate on whether the prolonged permeabilization is due to the formation of long-lived pores in cells. We developed a method for dynamic monitoring and conductance measurements of individual electropores. This was accomplished by time-lapse total internal reflection fluorescence (TIRF) imaging in HEK cells loaded with CAL-520 dye and placed on an indium tin oxide (ITO) surface. Applying a 1-ms, 0 to −400 mV pulse between the patch pipette and ITO evoked focal Ca2+ transients that identified individual electropores. Some transients disappeared in milliseconds but others persisted for over a minute. Persistent transients (“Ca2+ plumes”) faded over time to a stable or a randomly fluctuating level that could include periods of full quiescence. Single pore conductance, measured by 0 to −50 mV, 50 ms steps at 30 and 60 s after the electroporation, ranged from 80 to 200 pS. These experiments proved electropore longevity in cells, in stark contrast to molecular simulations and many findings in lipid bilayers.

A novel “turn-on” fluorescent probe based on thiocarbamoyl-DHP for Hg2+ detection in water samples and living cells

In this study, two fluorescent sensing probes, dihydropyridine (DHP) derivatives (DHP-CT1 and DHP-CT2) bearing phenoxy thiocarbonyl group, have been developed for Hg2+ detection. The tandem trimerization-cyclization of methylpropiolate with ammonium acetate gave 1.4-DHP and 1,2-DHP derivatives, which were reacted with O-phenylcarbonochloridothioate to produce DHP-CT1 and DHP-CT2, respectively. DHP-CT1 exhibits superior sensitivity and selectivity of fluorescence enhancement towards Hg2+ in aqueous media. The fluorescence intensity shows a good linear relationship with the concentration of Hg2+ in the range of 0–10 µM providing the extremely low LOD of 346 nM (69.4 ppb). The fluorescence enhancement is caused by the Hg2+ promoted hydrolysis of the thioamide bond releasing the fluorescent 1,4-DHP that was confirmed by NMR and HRMS. The quantitative analysis of Hg2+ in water samples using DHP-CT1 probe was demonstrated in aqueous solution and paper-based sensing strips. Furthermore, DHP-CT1 was also applied for monitoring intracellular Hg2+ in living RAW264.7 macrophages through fluorescence cell imaging.

Dynamic altruistic cooperation within breast tumors

BackgroundSocial behaviors such as altruism, where one self-sacrifices for collective benefits, critically influence an organism’s survival and responses to the environment. Such behaviors are widely exemplified in nature but have been underexplored in cancer cells which are conventionally seen as selfish competitive players. This multidisciplinary study explores altruism and its mechanism in breast cancer cells and its contribution to chemoresistance.MethodsMicroRNA profiling was performed on circulating tumor cells collected from the blood of treated breast cancer patients. Cancer cell lines ectopically expressing candidate miRNA were used in co-culture experiments and treated with docetaxel. Ecological parameters like relative survival and relative fitness were assessed using flow cytometry. Functional studies and characterization performed in vitro and in vivo include proliferation, iTRAQ-mass spectrometry, RNA sequencing, inhibition by small molecules and antibodies, siRNA knockdown, CRISPR/dCas9 inhibition and fluorescence imaging of promoter reporter-expressing cells. Mathematical modeling based on evolutionary game theory was performed to simulate spatial organization of cancer cells.ResultsOpposing cancer processes underlie altruism: an oncogenic process involving secretion of IGFBP2 and CCL28 by the altruists to induce survival benefits in neighboring cells under taxane exposure, and a self-sacrificial tumor suppressive process impeding proliferation of altruists via cell cycle arrest. Both processes are regulated concurrently in the altruists by miR-125b, via differential NF-κB signaling specifically through IKKβ. Altruistic cells persist in the tumor despite their self-sacrifice, as they can regenerate epigenetically from non-altruists via a KLF2/PCAF-mediated mechanism. The altruists maintain a sparse spatial organization by inhibiting surrounding cells from adopting the altruistic fate via a lateral inhibition mechanism involving a GAB1-PI3K-AKT-miR-125b signaling circuit.ConclusionsOur data reveal molecular mechanisms underlying manifestation, persistence and spatial spread of cancer cell altruism. A minor population behave altruistically at a cost to itself producing a collective benefit for the tumor, suggesting tumors to be dynamic social systems governed by the same rules of cooperation in social organisms. Understanding cancer cell altruism may lead to more holistic models of tumor evolution and drug response, as well as therapeutic paradigms that account for social interactions. Cancer cells constitute tractable experimental models for fields beyond oncology, like evolutionary ecology and game theory.

A structural optimized fluorescent probe for monitoring hydrogen sulfide in cells and zebrafish

In this work, we reported a fluorescent probe Fur-SH, a derivative of benzofuranone, which was used to detect H2S in living cells and zebrafish. Based on the three structural characteristics of the probe, the effects of different structural modifications on the optical properties of the fluorophore were compared. Then, the fluorophore Fur-OH was synthesized by modifying diethylamino group with benzofuranone as the main skeleton. With 2,4-dinitrofluorobenzene as the recognition group and diethylamino as the electron donor, the push–pull electron effect occurred with nitro group, which led to fluorescence quenching, and an openable fluorescent probe Fur-SH was formed. The probe Fur-SH (λex = 510 nm; λem = 570 nm) had the advantages of smaller full width at half maxima, rapid response (5 min) and wide pH window. The quantitative properties of the probe were excellent, reaching saturation at 50 equivalents of substrate. The probe Fur-SH showed high sensitivity to H2S, with LOD of 48.9 nM and LOQ of 50 nM. At present, the probe Fur-SH had been applied to fluorescence imaging of MCF-7 cells and zebrafish. By comparing the effects of different structures on the optical properties of fluorophores, this work was expected to be helpful to the development of fluorescent probes in the future.

The Na/K-ATPase α1/Src Signaling Axis Regulates Mitochondrial Metabolic Function and Redox Signaling in Human iPSC-Derived Cardiomyocytes.

Na/K-ATPase (NKA)-mediated regulation of Src kinase, which involves defined amino acid sequences of the NKA α1 polypeptide, has emerged as a novel regulatory mechanism of mitochondrial function in metazoans. Mitochondrial metabolism ensures adequate myocardial performance and adaptation to physiological demand. It is also a critical cellular determinant of cardiac repair and remodeling. To assess the impact of the proposed NKA/Src regulatory axis on cardiac mitochondrial metabolic function, we used a gene targeting approach in human cardiac myocytes. Human induced pluripotent stem cells (hiPSC) expressing an Src-signaling null mutant (A420P) form of the NKA α1 polypeptide were generated using CRISPR/Cas9-mediated genome editing. Total cellular Na/K-ATPase activity remained unchanged in A420P compared to the wild type (WT) hiPSC, but baseline phosphorylation levels of Src and ERK1/2 were drastically reduced. Both WT and A420P mutant hiPSC readily differentiated into cardiac myocytes (iCM), as evidenced by marker gene expression, spontaneous cell contraction, and subcellular striations. Total NKA α1-3 protein expression was comparable in WT and A420P iCM. However, live cell metabolism assessed functionally by Seahorse extracellular flux analysis revealed significant reductions in both basal and maximal rates of mitochondrial respiration, spare respiratory capacity, ATP production, and coupling efficiency. A significant reduction in ROS production was detected by fluorescence imaging in live cells, and confirmed by decreased cellular protein carbonylation levels in A420P iCM. Taken together, these data provide genetic evidence for a role of NKA α1/Src in the tonic stimulation of basal mitochondrial metabolism and ROS production in human cardiac myocytes. This signaling axis in cardiac myocytes may provide a new approach to counteract mitochondrial dysfunction in cardiometabolic diseases.

Multifunctional phthalocyanine NIR sensor for fluorescent and colorimetric dual-imaging and removal of intracellular and environmental Cu2+

Cu2+ sensing and removal are of great significance for environment protection and human health. Herein, multifunctional Cu2+ sensor system for both detection and removal of intracellular and environmental Cu2+ based on trans/cis interconversion of silicon phthalocyanine (SiPc) was developed. This phthalocyanine based Cu2+ sensor shows remarkably dual-mode NIR fluorescence and colorimetric responses for recognition of Cu2+, resulting in insoluble chelate for Cu2+ removal as well as the extremely high anti-interference in the presence of various anions and cations. The binding mechanism and stoichiometry ratio were systematically revealed. Intracellular fluorescence imaging in MCF-7 cells demonstrated that Cu2+ could be efficiently detected in living cells and form insoluble chelate for clearance. As the result, this novel phthalocyanine based multifunctional sensor can be used for dual-mode NIR fluorescence/colorimetric detection and removal of Cu2+ in either complicated environment or biological systems.

Synthesis of a super-low detection limit fluorescent probe for Al3+ and its application in fluorescence imaging of zebrafish and cells

A novel fluorescent probe N'-(2-hydroxybenzylidene)-indole-3-formylhydrazine (JHK) was designed and synthesized based on the condensation reaction of indole-3-formylhydrazine and salicylaldehyde. The probe JHK solution could highly selectively recognize Al3+ by the obvious fluorescence enhancement (288-fold) after adding Al3+. And the probe solution with Al3+ had a very high fluorescence quantum yield (89.29 %). The detection limit was calculated to be 1.135 nM, which was significantly lower than many reported detection limits, indicating that the probe JHK had pretty good sensitivity. The ratio of JHK to Al3+ (1:1) and the sensing mechanism were determined by Job's plot, 1H NMR spectra, FTIR spectra, ESI-MS and Gaussian calculation. The probe solution and medium-speed filter paper were successfully used to make test papers for more convenient detection of Al3+. Furthermore, the probe JHK had been successfully applied to the detection of Al3+ in real water, zebrafish and living cells.

Relay recognition of Cu2+ and S2− using naphthalimide-based fluorescent probe and its applications in molecular logic gate and bioimaging

A fluorescent chemosensor based on naphthalimide derivative (HL) was prepared and characterized. HL exhibited strong fluorescence emission at 528 nm, which was attributed to naphthalimide moiety, while the addition of Cu2+ almost completely quenched the fluorescence emission. However, with the addition of S2−, the fluorescence at 528 nm reoccurred remarkably. The detection limits for Cu2+ and S2− were measured to be 1.72 × 10−7 M and 2.66 × 10−7 M, respectively. As determined, the fluorescence behavior of HL in the presence of Cu2+ and S2− could be manufactured to IMPLICATION type logic gate. Moreover, confocal fluorescence imaging in HepG2 cells indicated that the HL could act as a useful fluorescent probe for intracellular Cu2+ and S2− detection with negligible cytotoxicity.

A triple-targeting fluorescent probe reveals the glutathione and viscosity crosstalk in mitochondria, endoplasmic reticulum, and nucleoli in cells during ferroptosis

Ferroptosis, a recently termed cell death form, is featured by iron accumulation and lipid peroxidation, which plays various roles in physiological and pathological processes such as cancer evolution and therapy. Redox homeostasis profoundly varies in cells during ferroptosis, but many events remain to be explored, especially at the sub-cellular level. Glutathione (GSH) is the most abundant bio-reductant and mainly distributes in mitochondria (Mito), endoplasmic reticulum (ER), and nucleoli (Nuc) except the cytoplasm. The GSH level is intrinsically related to the redox equilibriums in the three organelles. Viscosity is an index of the intracellular micro-environments related to redox homeostasis. The dynamics of the GSH and viscosity levels in organelles are vital to understanding cell ferroptosis but are in a veil due to the lack of the appropriate tools. Here we report a fluorescent probe (FPY) with dual-response to GSH and viscosity, characteristic of the triple-targeting to Mito, ER, and Nuc. We apply FPY in fluorescent imaging of cells during ferroptosis and disclose the GSH-viscosity crosstalk at the sub-cellular level. We discover an abnormal GSH recruitment in Nuc from the cytoplasm in the late stage of ferroptosis.

Lysosome-targeted dual-emissive carbon dots for ratiometric optical dual-readout and smartphone-assisted visual determination of Hg2+ and SO32−

Smartphone-assisted visual assay not only expands quantitative analysis but also enhance real-time on-site sensing capabilities. Herein, lysosome-targeted dual-emissive carbon dots (L-CDs) can not only recognize Hg2+ and SO32− by ratiometric fluorescence and ratiometric absorption, but also visually quantify Hg2+ and SO32− by smartphone-assisted method. With monitoring of intrinsic ratiometric fluorescent variation (I580/I468), L-CDs are developed as an effective sensing platform for ratiometric fluorescent successive identification of Hg2+ and SO32− accompanying with continuous fluorescence variation of blue, purplish pink, pink, and light yellow. With detecting of inherent ratiometric absorption change (A538/A206), L-CDs are also constructed as an efficacious sensing terrace for ratiometric absorption consecutive discrimination of Hg2+ and SO32−. More crucially, integrating continuous fluorescence color change and color recognizer APP in the smartphone, visual quantification of Hg2+ and SO32− can be accomplished on the basis of the ratio of red value and blue value (R/B) with linear ranges of 0–130 and 0–850 µM, respectively, as well as LOD of 8.4 and 12.9 nM, respectively. More interestingly, confocal fluorescent imaging of HeLa cells further verifies that L-CDs can be regarded as favorable biosensors for on-site, real-time differentiation of Hg2+ and SO32− in vitro and in vivo with ratiometric manners.