In Vivo MR Imaging of Glioma Recruitment of Adoptive T‐Cells Labeled with NaGdF4‐TAT Nanoprobes

Adoptive T lymphocyte immunotherapy is one of the most promising methods to treat residual lesions after glioma surgery. However, the fate of the adoptively transferred T‐cells in vivo is unclear, hampering the understanding of this emerging therapy. Thus, it is highly desirable to develop noninvasive and quantitative in vivo tracking of these T‐cells to glioma for better identification of the migratory fate and to provide objective evaluation of outcomes of adoptive T‐cell immunotherapy targeting glioma. In this work, ultrasmall T₁ MR‐based nanoprobes, NaGdF₄‐TAT, as molecular probes with high longitudinal relaxivity (8.93 mm ^?1 s^?1) are designed. By means of HIV‐1 transactivator (TAT) peptides, nearly 95% of the adoptive T‐cells are labeled with the NaGdF₄‐TAT nanoprobes without any measurable side effects on the labeled T‐cells, which is remarkably superior to that of the control fluorescein isothiocyanate‐NaGdF₄ concerning labeling efficacy. Labeled adoptive T‐cell clusters can be sensitively tracked in an orthotopic GL261‐glioma model 24 h after intravenous infusion of 10⁷ labeled T‐cells by T₁‐weighted MR imaging. Both in vitro and in vivo experiments show that the NaGdF₄‐TAT nanoprobes labeling of T‐cells may be a promising method to track adoptive T‐cells to improve our understanding of the pathophysiology in adoptive immunotherapy for gliomas.     

Lysosome‐Targeted Bioprobes for Sequential Cell Tracking from Macroscopic to Microscopic Scales

Longitudinal tracking of living cells is crucial to understanding the mechanism of action and toxicity of cell‐based therapeutics. To quantify the presence of administered cells in the host tissue without sacrifice of animals, labeling of the target cells with a nontoxic and stable contrast agent is a prerequisite. However, such long‐term live cell tracking is currently limited by the lack of fluorophores with steady optical and physicochemical properties in the near‐infrared (NIR) window. Herein, for the first time, the design of fixable cell‐tracking NIR fluorophores (CTNFs) with high optical properties, excellent cell permeation and retention, and high stability against chemical treatments is reported. Efficient cellular labeling and tracking of CTNFs using intraoperative optical fluorescence imaging by following the fate of NIR‐labeled cells from the time of injection into animals to ex vivo cellular analysis after resection of the target tissue is demonstrated. Due to the lipophilic cationicity and primary amine docking group, CTNF126 outperforms the other tested fluorophores with rapid diffusion into the cytoplasmic membrane and sequestration inside the lysosomes, which prevents cellular efflux and improves cellular retention. Thus, CTNF126 will be useful to track cells in living organisms for the mechanism of action at the single cell level.     

Retrosynthesis of Tunable Fluorescent Carbon Dots for Precise Long‐Term Mitochondrial Tracking

Mitochondria play a significant role in many cellular processes. Precise long‐term tracking of mitochondrial status and behavior is very important for regulating cell fate and treating mitochondrial diseases. However, developing probes with photostability, long‐term tracking capability, and tunable long‐wavelength fluorescence has been a challenge in mitochondrial targeting. Carbon dots (CDs) as new fluorescent nanomaterials with low toxicity and high stability show increasing advantages in bioimaging. Herein, the mitochondria tracking CDs (MitoTCD) with intrinsic mitochondrial imaging capability and tunable long‐wavelength fluorescence from green to red are synthesized where the lipophilic cation of rhodamine is served as the luminescent center of CDs. Due to the excellent photostability, superior fluorescence properties and favorable biocompatibility, these MitoTCD are successfully used for mitochondrial targeting imaging of HeLa cells in vitro and can be tracked as long as six passages, which is suitable for long‐term cell imaging. Moreover, these MitoTCD can also be used for zebrafish imaging in vivo.     

A Bioresponsive Near‐Infrared Fluorescent Probe for Facile and Persistent Live‐Cell Tracking

Molecular imaging significantly transforms the field of biomedical science and facilitates the visualization, characterization, and quantification of biologic processes. However, it is still challenging to monitor cell localization in vivo, which is essential to the study of tumor metastasis and in the development of cell‐based therapies. While most conventional small‐molecule fluorescent probes cannot afford durable cell labeling, transfection of cells with fluorescent proteins is limited by their fixed fluorescence, poor tissue penetration, and interference of autofluorescence background. Here, a bioresponsive near‐infrared fluorescent probe is reported as facile and reliable tool for real‐time cell tracking in vivo. The design of this probe relies on a new phenomenon observed upon fluorobenzene‐conjugated fluorescent dyes, which can form complexes with cytosolic glutathione and actively translocates to lysosomes, exhibiting enhanced and stable cell labeling. Fluorobenzene‐coupled hemicyanine, a near‐infrared fluorophore manifests to efficiently staining tumor cells without affecting their invasive property and enables persistent monitoring of cell migration in metastatic tumor murine models at high resolution for one week. The method of fluorobenzene functionalization also provides a simple and universal “add‐on” strategy to render ordinary fluorescent probes suitable for long‐term live‐cell tracking, for which currently there is a deficit of suitable molecular tools.     

Amino‐polyvinyl Alcohol Coated Superparamagnetic Iron Oxide Nanoparticles are Suitable for Monitoring of Human Mesenchymal Stromal Cells In Vivo

Mesenchymal stromal cells (MSCs) are promising candidates in regenerative cell‐therapies. However, optimizing their number and route of delivery remains a critical issue, which can be addressed by monitoring the MSCs’ bio‐distribution in vivo using super‐paramagnetic iron‐oxide nanoparticles (SPIONs). In this study, amino‐polyvinyl alcohol coated (A‐PVA) SPIONs are introduced for cell‐labeling and visualization by magnetic resonance imaging (MRI) of human MSCs. Size and surface charge of A‐PVA‐SPIONs differ depending on their solvent. Under MSC‐labeling conditions, A‐PVA‐SPIONs have a hydrodynamic diameter of 42 ± 2 nm and a negative Zeta potential of 25 ± 5 mV, which enable efficient internalization by MSCs without the need to use transfection agents. Transmission X‐ray microscopy localizes A‐PVA‐SPIONs in intracellular vesicles and as cytosolic single particles. After identifying non‐interfering cell‐assays and determining the delivered and cellular dose, in addition to the administered dose, A‐PVA‐SPIONs are found to be non‐toxic to MSCs and non‐destructive towards their multi‐lineage differentiation potential. Surprisingly, MSC migration is increased. In MRI, A‐PVA‐SPION‐labeled MSCs are successfully visualized in vitro and in vivo. In conclusion, A‐PVA‐SPIONs have no unfavorable influences on MSCs, although it becomes evident how sensitive their functional behavior is towards SPION‐labeling. And A‐PVA‐SPIONs allow MSC‐monitoring in vivo.     

Nanohybrids with Magnetic and Persistent Luminescence Properties for Cell Labeling,Tracking, In Vivo Real‐Time Imaging,and Magnetic Vectorization

Once injected into a living organism, cells diffuse or migrate around the initial injection point and become impossible to be visualized and tracked in vivo. The present work concerns the development of a new technique for therapeutic cell labeling and subsequent in vivo visualization and magnetic retention. It is hypothesized and subsequently demonstrated that nanohybrids made of persistent luminescence nanoparticles and ultrasmall superparamagnetic iron oxide nanoparticles incorporated into a silica matrix can be used as an effective nanoplatform to label therapeutic cells in a nontoxic way in order to dynamically track them in real‐time in vitro and in living mice. As a proof‐of‐concept, it is shown that once injected, these labeled cells can be visualized and attracted in vivo using a magnet. This first step suggests that these nanohybrids represent efficient multifunctional nanoprobes for further imaging guided cell therapies development.     

Water‐Dispersible,pH‐Stable and Highly‐Luminescent Organic Dye Nanoparticles with Amplified Emissions for In Vitro and In Vivo Bioimaging

A new strategy is presented for using doped small‐molecule organic nanoparticles (NPs) to achieve high‐performance fluorescent probes with strong brightness, large Stokes shifts and tunable emissions for in vitro and in vivo imaging. The host organic NPs are used not only as carriers to encapsulate different doped dyes, but also as fluorescence resonance energy transfer donors to couple with the doped dyes (as acceptors) to achieve multicolor luminescence with amplified emissions (AE). The resulting optimum green emitting NPs show high brightness with quantum yield (QY) of up to 45% and AE of 12 times; and the red emitting NPs show QY of 14% and AE of 10 times. These highly‐luminescent doped NPs can be further surface modified with poly(maleic anhydride‐alt‐1‐octadecene)‐polyethylene glycol (C18PMH‐PEG), endowing them with excellent water dispersibility and robust stability in various bio‐environments covering wide pH values from 2 to 10. In this study, cytotoxicity studies and folic acid targeted cellular imaging of these multicolor probes are carried out to demonstrate their potential for in vitro imaging. On this basis, applications of the NP probes in in vivo and ex vivo imaging are also investigated. Intense fluorescent signals of the doped NPs are distinctly, selectively and spatially resolved in tumor sites with high sensitivity, due to the preferential accumulation of the NPs in tumor sites through the passive enhanced permeability and retention effect. The results clearly indicate that these doped NPs are promising fluorescent probes for biomedical applications.     

Multicolor Photo‐Crosslinkable AIEgens toward Compact Nanodots for Subcellular Imaging and STED Nanoscopy

Aggregation induced emission (AIE) has attracted considerable interest for the development of fluorescence probes. However, controlling the bioconjugation and cellular labeling of AIE dots is a challenging problem. Here, this study reports a general approach for preparing small and bioconjugated AIE dots for specific labeling of cellular targets. The strategy is based on the synthesis of oxetane‐substituted AIEgens to generate compact and ultrastable AIE dots via photo‐crosslinking. A small amount of polymer enriched with oxetane groups is cocondensed with most of the AIEgens to functionalize the nanodot surface for subsequent streptavidin bioconjugation. Due to their small sizes, good stability, and surface functionalization, the cell‐surface markers and subcellular structures are specifically labeled by the AIE dot bioconjugates. Remarkably, stimulated emission depletion imaging with AIE dots is achieved for the first time, and the spatial resolution is significantly enhanced to ≈95 nm. This study provides a general approach for small functional molecules for preparing small sized and ultrastable nanodots.     

Receptor‐Mediated Cellular Uptake of Folate‐Conjugated Fluorescent Nanodiamonds: A Combined Ensemble and Single‐Particle Study

Fluorescent nanodiamonds (FNDs) are nontoxic and photostable nanomaterials, ideal for long‐term in vivo imaging applications. This paper reports that FNDs with a size of ≈140 nm can be covalently conjugated with folic acid (FA) for receptor‐mediated targeting of cancer cells at the single‐particle level. The conjugation is made by using biocompatible polymers, such as polyethylene glycol, as crosslinked buffer layers. Ensemble‐averaged measurements with flow cytometry indicate that more than 50% of the FA‐conjugated FND particles can be internalized by the cells (such as HeLa cells) through receptor‐mediated endocytosis, as confirmed by competitive inhibition assays. Confocal fluorescence microscopy reveals that these FND particles accumulate in the perinuclear region. The absolute number of FNDs internalized by HeLa cells after 3 h of incubation at a particle concentration of 10 µg mL^?1 is in the range of 100 particles per cell. The receptor‐mediated uptake process is further elucidated by single‐particle tracking of 35‐nm FNDs in three dimensions and real time during the endocytosis.     

Molecular Cancer Imaging in the Second Near‐Infrared Window Using a Renal‐Excreted NIR‐II Fluorophore‐Peptide Probe

In vivo molecular imaging of tumors targeting a specific cancer cell marker is a promising strategy for cancer diagnosis and imaging guided surgery and therapy. While targeted imaging often relies on antibody‐modified probes, peptides can afford targeting probes with small sizes, high penetrating ability, and rapid excretion. Recently, in vivo fluorescence imaging in the second near‐infrared window (NIR‐II, 1000–1700 nm) shows promise in reaching sub‐centimeter depth with microscale resolution. Here, a novel peptide (named CP) conjugated NIR‐II fluorescent probe is reported for molecular tumor imaging targeting a tumor stem cell biomarker CD133. The click chemistry derived peptide‐dye (CP‐IRT dye) probe afforded efficient in vivo tumor targeting in mice with a high tumor‐to‐normal tissue signal ratio (T/NT > 8). Importantly, the CP‐IRT probes are rapidly renal excreted (≈87% excretion within 6 h), in stark contrast to accumulation in the liver for typical antibody‐dye probes. Further, with NIR‐II emitting CP‐IRT probes, urethra of mice can be imaged fluorescently for the first time noninvasively through intact tissue. The NIR‐II fluorescent, CD133 targeting imaging probes are potentially useful for human use in the clinic for cancer diagnosis and therapy.     

A Self‐Assembled DNA Origami‐Gold Nanorod Complex for Cancer Theranostics

A self‐assembled DNA origami (DO)‐gold nanorod (GNR) complex, which is a dual‐functional nanotheranostics constructed by decorating GNRs onto the surface of DNA origami, is demonstrated. After 24 h incubation of two structured DO‐GNR complexes with human MCF7 breast cancer cells, significant enhancement of cell uptake is achieved compared to bare GNRs by two‐photon luminescence imaging. Particularly, the triangle shaped DO‐GNR complex exhibits optimal cellular accumulation. Compared to GNRs, improved photothermolysis against tumor cells is accomplished for the triangle DO‐GNR complex by two‐photon laser or NIR laser irradiation. Moreover, the DO‐GNR complex exhibits enhanced antitumor efficacy compared with bare GNRs in nude mice bearing breast tumor xenografts. The results demonstrate that the DO‐GNR complex can achieve optimal two‐photon cell imaging and photothermal effect, suggesting a promising candidate for cancer diagnosis and therapy both in vitro and in vivo.     

Albumin Nanoshell Encapsulation of Near‐Infrared‐Excitable Rare‐Earth Nanoparticles Enhances Biocompatibility and Enables Targeted Cell Imaging

The use of traditional fluorophores for in vivo imaging applications is limited by poor quantum yield, poor tissue penetration of the excitation light, and excessive tissue autofluorescence, while the use of inorganic fluorescent particles that offer a high quantum yield is frequently limited due to particle toxicity. Rare‐earth‐doped nanoparticles that utilize near‐infrared upconversion overcome the optical limitations of traditional fluorophores, but are not typically suitable for biological application due to their insolubility in aqueous solution, lack of functional surface groups for conjugation of biomolecules, and potential cytotoxicity. A new approach to establish highly biocompatible and biologically targetable nanoshell complexes of luminescent rare‐earth‐doped NaYF₄ nanoparticles (REs) excitable with 920–980 nm near‐infrared light for biomedical imaging applications is reported. The approach involves the encapsulation of NaYF₄ nanoparticles doped with Yb and Er within human serum albumin nanoshells to create water‐dispersible, biologically functionalizable composite particles. These particles exhibit narrow size distributions around 200 nm and are stable in aqueous solution for over 4 weeks. The albumin shell confers cytoprotection and significantly enhances the biocompatibility of REs even at concentrations above 200 µg REs mL^?1. Composite particles conjugated with cyclic arginine‐glycine‐aspartic acid (cRGD) specifically target both human glioblastoma cell lines and melanoma cells expressing α_vβ₃ integrin receptors. These findings highlight the promise of albumin‐encapsulated rare‐earth nanoparticles for imaging cancer cells in vitro and the potential for targeted imaging of disease sites in vivo.     

Fluorescent Polymer Nanoparticles for Cell Barcoding In Vitro and In Vivo

Fluorescent polymer nanoparticles for long‐term labeling and tracking of living cells with any desired color code are developed. They are built from biodegradable poly(lactic‐co‐glycolic acid) polymer loaded with cyanine dyes (DiO, DiI, and DiD) with the help of bulky fluorinated counterions, which minimize aggregation‐caused quenching. At the single particle level, these particles are ≈20‐fold brighter than quantum dots of similar color. Due to their identical 40 nm size and surface properties, these nanoparticles are endocytosed equally well by living cells. Mixing nanoparticles of three colors in different proportions generates a homogeneous RGB (red, green, and blue) barcode in cells, which is transmitted through many cell generations. Cell barcoding is validated on 7 cell lines (HeLa, KB, embryonic kidney (293T), Chinese hamster ovary, rat basophilic leucemia, U97, and D2A1), 13 color codes, and it enables simultaneous tracking of co‐cultured barcoded cell populations for >2 weeks. It is also applied to studying competition among drug‐treated cell populations. This technology enabled six‐color imaging in vivo for (1) tracking xenografted cancer cells and (2) monitoring morphogenesis after microinjection in zebrafish embryos. In addition to a robust method of multicolor cell labeling and tracking, this work suggests that multiple functions can be co‐localized inside cells by combining structurally close nanoparticles carrying different functions.     

Three‐Dimensional Exploration and Mechano‐Biophysical Analysis of the Inner Structure of Living Cells

A novel mechanobiological method is presented to explore qualitatively and quantitatively the inside of living biological cells in three dimensions, paving the way to sense intracellular changes during dynamic cellular processes. For this purpose, holographic optical tweezers, which allow the versatile manipulation of nanoscopic and microscopic particles by means of tailored light fields, are combined with self‐interference digital holographic microscopy. This biophotonic holographic workstation enables non‐contact, minimally invasive, flexible, high‐precision optical manipulation and accurate 3D tracking of probe particles that are incorporated by phagocytosis in cells, while simultaneously quantitatively phase imaging the cell morphology. In a first model experiment, internalized polystyrene microspheres with 1 μm diameter are three‐dimensionally moved and tracked in order to quantify distances within the intracellular volume with submicrometer accuracy. Results from investigations on cell swelling provoked by osmotic stimulation demonstrate the homogeneous stretching of the cytoskeleton network, and thus that the proposed method provides a new way for the quantitative 3D analysis of the dynamic intracellular morphology.     

Quantum dots with phenylboronic acid tags for specific labeling of sialic acids on living cells

Sialic acids with a nine-carbon backbone are commonly found at the terminal position of the glycans structures on cell membranes. The unique distribution and ubiquitous existence of sialic acid on the cell membrane make them important mediators in various biological and pathological processes. We report a new class of imaging probes based on semiconductor quantum dots with small molecular phenylboronic acid tags for highly specific and efficient labeling of sialic acid on living cells. Our results have shown that the use of these probes enables one-step labeling and continuous tracking of the cell surface sialic acid moieties without any pretreatment of living cells. The one-step procedure with fast binding kinetics and the biocompatibility of these probes make it an ideal noninvasive technology for living cell imaging. We also find that the labeled sialic acids undergo quick internalization shortly after surface binding via endocytosis and eventually distribute in the perinuclear region. This distribution pattern is consistent with the notion that sialylated glycoproteins are populated on cell membranes and recycled through the vesicular exocytotic and endocytic pathways. The superior photostability and brightness of quantum dots enable quantitative analysis of the diffusion dynamics of sialic acids, which has been a significant challenge for glycan imaging.     

New Templated Ostwald Ripening Process of Mesostructured FeOOH for Third‐Harmonic Generation Bioimaging

Emerging advances in iron oxide nanoparticles exploit their high magnetization for various applications, such as bioseparation, hyperthermia, and magnetic resonance imaging. In contrast to their excellent magnetic performance, the harmonic generation and luminescence properties of iron oxide nanoparticles have not been thoroughly explored, thus limiting their development as a tool in photomedicine. In this work, a seed/growth‐inspired synthesis is developed combined with primary mineralization and a ligand‐assisted secondary growth strategy to prepare mesostructured α‐FeOOH nanorods (NRs). The sub‐wavelength heterogeneity of the refractive index leads to enhanced third‐harmonic generation (THG) signals under near‐infrared excited wavelengths at 1230 nm. The as‐prepared NRs exhibit an 11‐fold stronger THG intensity compared to bare α‐FeOOH NRs. Using these unique nonlinear optical properties, it is demonstrated that mesostructured α‐FeOOH NRs can serve as biocompatible and nonbleaching contrast agents in THG microscopy for long‐term labeling of cells as well as in angiography in vivo by modifying lectin to enhance the binding efficiency to the glycocalyx layers on the wall of blood vessels. These results provide a new insight into Fe‐based nanoplatforms capable of emitting coherent light as molecular probes in optical microscopy, thus establishing a complementary microscopic imaging method for macroscopic magnetic imaging systems.     

Label‐Free Optofluidic Nanobiosensor Enables Real‐Time Analysis of Single‐Cell Cytokine Secretion

Single‐cell analysis of cytokine secretion is essential to understand the heterogeneity of cellular functionalities and develop novel therapies for multiple diseases. Unraveling the dynamic secretion process at single‐cell resolution reveals the real‐time functional status of individual cells. Fluorescent and colorimetric‐based methodologies require tedious molecular labeling that brings inevitable interferences with cell integrity and compromises the temporal resolution. An innovative label‐free optofluidic nanoplasmonic biosensor is introduced for single‐cell analysis in real time. The nanobiosensor incorporates a novel design of a multifunctional microfluidic system with small volume microchamber and regulation channels for reliable monitoring of cytokine secretion from individual cells for hours. Different interleukin‐2 secretion profiles are detected and distinguished from single lymphoma cells. The sensor configuration combined with optical spectroscopic imaging further allows us to determine the spatial single‐cell secretion fingerprints in real time. This new biosensor system is anticipated to be a powerful tool to characterize single‐cell signaling for basic and clinical research.     

Tumor Microenvironment‐Triggered Aggregation of Antiphagocytosis 99mTc‐Labeled Fe3O4 Nanoprobes for Enhanced Tumor Imaging In Vivo

A tumor microenvironment responsive nanoprobe is developed for enhanced tumor imaging through in situ crosslinking of the Fe₃O₄ nanoparticles modified with a responsive peptide sequence in which a tumor‐specific Arg‐Gly‐Asp peptide for tumor targeting and a self‐peptide as a “mark of self” are linked through a disulfide bond. Positioning the self‐peptide at the outmost layer is aimed at delaying the clearance of the nanoparticles from the bloodstream. After the self‐peptide is cleaved by glutathione within tumor microenvironment, the exposed thiol groups react with the remaining maleimide moieties from adjacent particles to crosslink the particles in situ. Both in vitro and in vivo experiments demonstrate that the aggregation substantially improves the magnetic resonance imaging (MRI) contrast enhancement performance of Fe₃O₄ particles. By labeling the responsive particle probe with ^99mTc, single‐photon emission computed tomography is enabled not only for verifying the enhanced imaging capacity of the crosslinked Fe₃O₄ particles, but also for achieving sensitive dual modality imaging of tumors in vivo. The novelty of the current probe lies in the combination of tumor microenvironment‐triggered aggregation of Fe₃O₄ nanoparticles for boosting the T₂ MRI effect, with antiphagocytosis surface coating, active targeting, and dual‐modality imaging, which is never reported before.     

Unraveling In Vivo Brain Transport of Protein‐Coated Fluorescent Nanodiamonds

Nanotheranostics, combining diagnostics and therapy, has the potential to revolutionize treatment of neurological disorders. But one of the major obstacles for treating central nervous system diseases is the blood–brain barrier (BBB) preventing systemic delivery of drugs and optical probes into the brain. To overcome these limitations, nanodiamonds (NDs) are investigated in this study as they are a powerful sensing and imaging platform for various biological applications and possess outstanding stable far‐red fluorescence, do not photobleach, and are highly biocompatible. Herein, fluorescent NDs encapsulated by a customized human serum albumin–based biopolymer (polyethylene glycol) coating (dcHSA‐PEG) are taken up by target brain cells. In vitro BBB models reveal transcytosis and an additional direct cell–cell transport via tunneling nanotubes. Systemic application of dcHSA‐NDs confirms their ability to cross the BBB in a mouse model. Tracking of dcHSA‐NDs is possible at the single cell level and reveals their uptake into neurons and astrocytes in vivo. This study shows for the first time systemic NDs brain delivery and suggests transport mechanisms across the BBB and direct cell–cell transport. Fluorescent NDs are envisioned as traceable transporters for in vivo brain imaging, sensing, and drug delivery.     

STED Microscopy to Monitor Agglomeration of Silica Particles Inside A549 Cells

The widespread use of engineered nanomaterials increases the exposure of the materials to humans. Therefore, it is necessary to know how these materials interact with cells. One approach is to trace particles by fluorescent labeling. The aim of the present work was to study the behavior of silica particles in A549 cells. For the first time, we applied stimulated emission depletion (STED) microscopy for this approach. Therefore, SiO₂ particles conjugated with Atto647N were prepared by L ‐arginine‐catalyzed hydrolysis of tetraethoxysilane. The Atto647N labeled SiO₂ particles exhibit a mean size of 128 ± 7 nm and a zeta‐potential of ?11 mV in cell culture medium. STED microscopy enables subdiffraction resolution imaging of single Atto647N labeled SiO₂ particles not only in pure solution but also in a cellular environment. To visualize Atto647N labeled SiO₂ particles inside A549 cells, the membrane was labeled and image stacks, that give three‐dimensional information, were taken after 5, 24, and 48 h exposure of particles to cells. During this incubation period, an increase in particle uptake was observed and STED micrographs allowed us to evaluate the agglomeration of Atto647N labeled SiO₂ particles inside A549 cells. Our results show that STED microscopy is a powerful technique to study particles in a cellular environment.