Nucleoside supplements as treatments for mitochondrial DNA depletion syndrome.

Introduction: In mitochondrial DNA (mtDNA) depletion syndrome (MDS), patients cannot maintain sufficient mtDNA for their energy needs. MDS presentations range from infantile encephalopathy with hepatopathy (Alpers syndrome) to adult chronic progressive external ophthalmoplegia. Most are caused by nucleotide imbalance or by defects in the mtDNA replisome. There is currently no curative treatment available. Nucleoside therapy is a promising experimental treatment for TK2 deficiency, where patients are supplemented with exogenous deoxypyrimidines. We aimed to explore the benefits of nucleoside supplementation in POLG and TWNK deficient fibroblasts. Methods: We used high-content fluorescence microscopy with software-based image analysis to assay mtDNA content and membrane potential quantitatively, using vital dyes PicoGreen and MitoTracker Red CMXRos respectively. We tested the effect of 15 combinations (A, T, G, C, AT, AC, AG, CT, CG, GT, ATC, ATG, AGC, TGC, ATGC) of deoxynucleoside supplements on mtDNA content of fibroblasts derived from four patients with MDS (POLG1, POLG2, DGUOK, TWNK) in both a replicating (10% dialysed FCS) and quiescent (0.1% dialysed FCS) state. We used qPCR to measure mtDNA content of supplemented and non-supplemented fibroblasts following mtDNA depletion using 20 µM ddC and after 14- and 21-day recovery in a quiescent state. Results: Nucleoside treatments at 200 µM that significantly increased mtDNA content also significantly reduced the number of cells remaining in culture after 7 days of treatment, as well as mitochondrial membrane potential. These toxic effects were abolished by reducing the concentration of nucleosides to 50 µM. In POLG1 and TWNK cells the combination of ATGC treatment increased mtDNA content the most after 7 days in non-replicating cells. ATGC nucleoside combination significantly increased the rate of mtDNA recovery in quiescent POLG1 cells following mtDNA depletion by ddC. Conclusion: High-content imaging enabled us to link mtDNA copy number with key read-outs linked to patient wellbeing. Elevated G increased mtDNA copy number but severely impaired fibroblast growth, potentially by inhibiting purine synthesis and/or causing replication stress. Combinations of nucleosides ATGC, T, or TC, benefited growth of cells harbouring POLG mutations. These combinations, one of which reflects a commercially available preparation, could be explored further for treatment of POLG patients.

Longitudinal characterization of TK6 cells sequentially adapted to animal product-free, chemically defined culture medium: considerations for genotoxicity studies.

Introduction: In vitro approaches are an essential tool in screening for toxicity of new chemicals, products and therapeutics. To increase the reproducibility and human relevance of these in vitro assessments, it is advocated to remove animal-derived products such as foetal bovine serum (FBS) from the cell culture system. Currently, FBS is routinely used as a supplement in cell culture medium, but batch-to-batch variability may introduce inconsistency in inter- and intra-lab assessments. Several chemically defined serum replacements (CDSR) have been developed to provide an alternative to FBS, but not every cell line adapts easily and successfully to CDSR-supplemented medium, and the long-term effect on cell characteristics remains uncertain. Aim: The aim of this study was to adapt the TK6 cell line to animal-product free CDSR-supplemented medium and evaluate the long-term effects on cell health, growth, morphology, phenotype, and function. This included a provisional assessment to determine the suitability of the transitioned cell line for standardised genotoxicity testing using the "in vitro mammalian cell micronucleus test" (OECD TG 487). Materials and methods: Gradual adaptation and direct adaptation methodologies were compared by assessing the cell proliferation, size and viability every passage until the cells were fully adapted to animal-free CDSR. The metabolic activity and membrane integrity was assessed every 4-8 passages by PrestoBlue and CytoTox-ONE™ Homogeneous Membrane Integrity Assay respectively. A detailed morphology study by high content imaging was performed and the expression of cell surface markers (CD19 and CD20) was conducted via flow cytometry to assess the potential for phenotypic drift during longer term culture of TK6 in animal-free conditions. Finally, functionality of cells in the OECD TG 487 assay was evaluated. Results: The baseline characteristics of TK6 cells cultured in FBS-supplemented medium were established and variability among passages was used to set up acceptance criteria for CDSR adapted cells. TK6 were adapted to CDSR supplemented medium either via direct or gradual transition reducing from 10% v/v FBS to 0% v/v FBS. The cell growth rate was compromised in the direct adaptation and therefore the gradual adaptation was preferred to investigate the long-term effects of animal-free CDSR on TK6 cells. The new animal cells showed comparable (p > 0.05) viability and cell size as the parent FBS-supplemented cells, with the exception of growth rate. The new animal free cells showed a lag phase double the length of the original cells. Cell morphology (cellular and nuclear area, sphericity) and phenotype (CD19 and CD20 surface markers) were in line (p > 0.05) with the original cells. The new cells cultured in CDSR-supplemented medium performed satisfactory in a pilot OECD TG 487 assay with compounds not requiring metabolic activation. Conclusion: TK6 cells were successfully transitioned to FBS- and animal product-free medium. The new cell cultures were viable and mimicked the characteristics of FBS-cultured cells. The gradual transition methodology utilised in this study can also be applied to other cell lines of interest. Maintaining cells in CDSR-supplemented medium eliminates variability from FBS, which in turn is likely to increase the reproducibility of in vitro experiments. Furthermore, removal of animal derived products from cell culture techniques is likely to increase the human relevance of in vitro methodologies.

Small-Molecule Polθ Inhibitors Provide Safe and Effective Tumor Radiosensitization in Preclinical Models.

PURPOSE: DNA polymerase theta (Polθ, encoded by the POLQ gene) is a DNA repair enzyme critical for microhomology mediated end joining (MMEJ). Polθ has limited expression in normal tissues but is frequently overexpressed in cancer cells and, therefore, represents an ideal target for tumor-specific radiosensitization. In this study we evaluate whether targeting Polθ with novel small-molecule inhibitors is a feasible strategy to improve the efficacy of radiotherapy. EXPERIMENTAL DESIGN: We characterized the response to Polθ inhibition in combination with ionizing radiation in different cancer cell models in vitro and in vivo. RESULTS: Here, we show that ART558 and ART899, two novel and specific allosteric inhibitors of the Polθ DNA polymerase domain, potently radiosensitize tumor cells, particularly when combined with fractionated radiation. Importantly, noncancerous cells were not radiosensitized by Polθ inhibition. Mechanistically, we show that the radiosensitization caused by Polθ inhibition is most effective in replicating cells and is due to impaired DNA damage repair. We also show that radiosensitization is still effective under hypoxia, suggesting that these inhibitors may help overcome hypoxia-induced radioresistance. In addition, we describe for the first time ART899 and characterize it as a potent and specific Polθ inhibitor with improved metabolic stability. In vivo, the combination of Polθ inhibition using ART899 with fractionated radiation is well tolerated and results in a significant reduction in tumor growth compared with radiation alone. CONCLUSIONS: These results pave the way for future clinical trials of Polθ inhibitors in combination with radiotherapy.

Automated 96-well format high throughput colony formation assay for siRNA library screen.

The colony formation assay is the gold-standard technique to assess cell viability after treatment with cytotoxic reagents, ionizing radiation, and cytotoxic combinatorial treatments. This protocol describes a high-throughput automated and high-content imaging approach to screen siRNA molecular libraries in HeLa cervical cancer cells in 96-well format. We detail reverse transfection of cells with siRNAs, followed by ionizing radiation, fixing, and staining of the plates for automated colony counting. This protocol can be used across a broad range of cell types. For complete details on the use and execution of this protocol, please refer to Tiwana et al. (2015).

A phenotypic high-content, high-throughput screen identifies inhibitors of NLRP3 inflammasome activation.

Inhibition of the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome has recently emerged as a promising therapeutic target for several inflammatory diseases. After priming and activation by inflammation triggers, NLRP3 forms a complex with apoptosis-associated speck-like protein containing a CARD domain (ASC) followed by formation of the active inflammasome. Identification of inhibitors of NLRP3 activation requires a well-validated primary high-throughput assay followed by the deployment of a screening cascade of assays enabling studies of structure-activity relationship, compound selectivity and efficacy in disease models. We optimized a NLRP3-dependent fluorescent tagged ASC speck formation assay in murine immortalized bone marrow-derived macrophages and utilized it to screen a compound library of 81,000 small molecules. Our high-content screening assay yielded robust assay metrics and identified a number of inhibitors of NLRP3-dependent ASC speck formation, including compounds targeting HSP90, JAK and IKK-ß. Additional assays to investigate inflammasome priming or activation, NLRP3 downstream effectors such as caspase-1, IL-1ß and pyroptosis form the basis of a screening cascade to identify NLRP3 inflammasome inhibitors in drug discovery programs.

Germline and Somatic Genetic Variants in the p53 Pathway Interact to Affect Cancer Risk, Progression, and Drug Response.

Insights into oncogenesis derived from cancer susceptibility loci (SNP) hold the potential to facilitate better cancer management and treatment through precision oncology. However, therapeutic insights have thus far been limited by our current lack of understanding regarding both interactions of these loci with somatic cancer driver mutations and their influence on tumorigenesis. For example, although both germline and somatic genetic variation to the p53 tumor suppressor pathway are known to promote tumorigenesis, little is known about the extent to which such variants cooperate to alter pathway activity. Here we hypothesize that cancer risk-associated germline variants interact with somatic TP53 mutational status to modify cancer risk, progression, and response to therapy. Focusing on a cancer risk SNP (rs78378222) with a well-documented ability to directly influence p53 activity as well as integration of germline datasets relating to cancer susceptibility with tumor data capturing somatically-acquired genetic variation provided supportive evidence for this hypothesis. Integration of germline and somatic genetic data enabled identification of a novel entry point for therapeutic manipulation of p53 activities. A cluster of cancer risk SNPs resulted in increased expression of prosurvival p53 target gene KITLG and attenuation of p53-mediated responses to genotoxic therapies, which were reversed by pharmacologic inhibition of the prosurvival c-KIT signal. Together, our results offer evidence of how cancer susceptibility SNPs can interact with cancer driver genes to affect cancer progression and identify novel combinatorial therapies. SIGNIFICANCE: These results offer evidence of how cancer susceptibility SNPs can interact with cancer driver genes to affect cancer progression and present novel therapeutic targets.

Modifying nutritional substrates induces macrovesicular lipid droplet accumulation and metabolic alterations in a cellular model of hepatic steatosis.

BACKGROUND AND AIMS: Nonalcoholic fatty liver disease (NAFLD) begins with steatosis, where a mixed macrovesicular pattern of large and small lipid droplets (LDs) develops. Since in vitro models recapitulating this are limited, the aims of this study were to develop mixed macrovesicular steatosis in immortalized hepatocytes and investigate effects on intracellular metabolism by altering nutritional substrates. METHODS: Huh7 cells were cultured in 11 mM glucose and 2% human serum (HS) for 7 days before additional sugars and fatty acids (FAs), either with 200 µM FAs (low fat low sugar; LFLS), 5.5 mM fructose + 200 µM FAs (low fat high sugar; LFHS), or 5.5 mM fructose + 800 µM FAs (high fat high sugar; HFHS), were added for 7 days. FA metabolism, lipid droplet characteristics, and transcriptomic signatures were investigated. RESULTS: Between the LFLS and LFHS conditions, there were few notable differences. In the HFHS condition, intracellular triacylglycerol (TAG) was increased and the LD pattern and distribution was similar to that found in primary steatotic hepatocytes. HFHS-treated cells had lower levels of de novo-derived FAs and secreted larger, TAG-rich lipoprotein particles. RNA sequencing and gene set enrichment analysis showed changes in several pathways including those involved in metabolism and cell cycle. CONCLUSIONS: Repeated doses of HFHS treatment resulted in a cellular model of NAFLD with a mixed macrovesicular LD pattern and metabolic dysfunction. Since these nutrients have been implicated in the development of NAFLD in humans, the model provides a good physiological basis for studying NAFLD development or regression in vitro.

HighVia-A Flexible Live-Cell High-Content Screening Pipeline to Assess Cellular Toxicity.

High-content screening to monitor disease-modifying phenotypes upon small-molecule addition has become an essential component of many drug and target discovery platforms. One of the most common phenotypic approaches, especially in the field of oncology research, is the assessment of cell viability. However, frequently used viability readouts employing metabolic proxy assays based on homogeneous colorimetric/fluorescent reagents are one-dimensional, provide limited information, and can in many cases yield conflicting or difficult-to-interpret results, leading to misinterpretation of data and wasted resources.The resurgence of high-content, phenotypic screening has significantly improved the quality and breadth of cell viability data, which can be obtained at the very earliest stages of drug and target discovery. Here, we describe a relatively inexpensive, high-throughput, high-content, multiparametric, fluorescent imaging protocol using a live-cell method of three fluorescent probes (Hoechst, Yo-Pro-3, and annexin V), that is amenable to the addition of further fluorophores. The protocol enables the accurate description and profiling of multiple cell death mechanisms, including apoptosis and necrosis, as well as accurate determination of compound IC50, and has been validated on a range of high-content imagers and image analysis software. To validate the protocol, we have used a small library of approximately 200 narrow-spectrum kinase inhibitors and clinically approved drugs. This fully developed, easy-to-use pipeline has subsequently been implemented in several academic screening facilities, yielding fast, flexible, and rich cell viability data for a range of early-stage high-throughput drug and target discovery programs.

TCR and Inflammatory Signals Tune Human MAIT Cells to Exert Specific Tissue Repair and Effector Functions.

MAIT cells are an unconventional T cell population that can be activated through both TCR-dependent and TCR-independent mechanisms. Here, we examined the impact of combinations of TCR-dependent and TCR-independent signals in human CD8+ MAIT cells. TCR-independent activation of these MAIT cells from blood and gut was maximized by extending the panel of cytokines to include TNF-superfamily member TL1A. RNA-seq experiments revealed that TCR-dependent and TCR-independent signals drive MAIT cells to exert overlapping and specific effector functions, affecting both host defense and tissue homeostasis. Although TCR triggering alone is insufficient to drive sustained activation, TCR-triggered MAIT cells showed specific enrichment of tissue-repair functions at the gene and protein levels and in in vitro assays. Altogether, these data indicate the blend of TCR-dependent and TCR-independent signaling to CD8+ MAIT cells may play a role in controlling the balance between healthy and pathological processes of tissue inflammation and repair.

Defining the Functional Role of NaV1.7 in Human Nociception.

Loss-of-function mutations in NaV1.7 cause congenital insensitivity to pain (CIP); this voltage-gated sodium channel is therefore a key target for analgesic drug development. Utilizing a multi-modal approach, we investigated how NaV1.7 mutations lead to human pain insensitivity. Skin biopsy and microneurography revealed an absence of C-fiber nociceptors in CIP patients, reflected in a reduced cortical response to capsaicin on fMRI. Epitope tagging of endogenous NaV1.7 revealed the channel to be localized at the soma membrane, axon, axon terminals, and the nodes of Ranvier of induced pluripotent stem cell (iPSC) nociceptors. CIP patient-derived iPSC nociceptors exhibited an inability to properly respond to depolarizing stimuli, demonstrating that NaV1.7 is a key regulator of excitability. Using this iPSC nociceptor platform, we found that some NaV1.7 blockers undergoing clinical trials lack specificity. CIP, therefore, arises due to a profound loss of functional nociceptors, which is more pronounced than that reported in rodent models, or likely achievable following acute pharmacological blockade. VIDEO ABSTRACT.

Combined flow cytometry and high-throughput image analysis for the study of essential genes in Caenorhabditis elegans.

BACKGROUND: Advances in automated image-based microscopy platforms coupled with high-throughput liquid workflows have facilitated the design of large-scale screens utilising multicellular model organisms such as Caenorhabditis elegans to identify genetic interactions, therapeutic drugs or disease modifiers. However, the analysis of essential genes has lagged behind because lethal or sterile mutations pose a bottleneck for high-throughput approaches, and a systematic way to analyse genetic interactions of essential genes in multicellular organisms has been lacking. RESULTS: In C. elegans, non-conditional lethal mutations can be maintained in heterozygosity using chromosome balancers, commonly expressing green fluorescent protein (GFP) in the pharynx. However, gene expression or function is typically monitored by the use of fluorescent reporters marked with the same fluorophore, presenting a challenge to sort worm populations of interest, particularly at early larval stages. Here, we develop a sorting strategy capable of selecting homozygous mutants carrying a GFP stress reporter from GFP-balanced animals at the second larval stage. Because sorting is not completely error-free, we develop an automated high-throughput image analysis protocol that identifies and discards animals carrying the chromosome balancer. We demonstrate the experimental usefulness of combining sorting of homozygous lethal mutants and automated image analysis in a functional genomic RNA interference (RNAi) screen for genes that genetically interact with mitochondrial prohibitin (PHB). Lack of PHB results in embryonic lethality, while homozygous PHB deletion mutants develop into sterile adults due to maternal contribution and strongly induce the mitochondrial unfolded protein response (UPRmt). In a chromosome-wide RNAi screen for C. elegans genes having human orthologues, we uncover both known and new PHB genetic interactors affecting the UPRmt and growth. CONCLUSIONS: The method presented here allows the study of balanced lethal mutations in a high-throughput manner. It can be easily adapted depending on the user's requirements and should serve as a useful resource for the C. elegans community for probing new biological aspects of essential nematode genes as well as the generation of more comprehensive genetic networks.

Imaging-Based Screen Identifies Laminin 411 as a Physiologically Relevant Niche Factor with Importance for i-Hep Applications.

Use of hepatocytes derived from induced pluripotent stem cells (i-Heps) is limited by their functional differences in comparison with primary cells. Extracellular niche factors likely play a critical role in bridging this gap. Using image-based characterization (high content analysis; HCA) of freshly isolated hepatocytes from 17 human donors, we devised and validated an algorithm (Hepatocyte Likeness Index; HLI) for comparing the hepatic properties of cells against a physiological gold standard. The HLI was then applied in a targeted screen of extracellular niche factors to identify substrates driving i-Heps closer to the standard. Laminin 411, the top hit, was validated in two additional induced pluripotent stem cell (iPSC) lines, primary tissue, and an in vitro model of α1-antitrypsin deficiency. Cumulatively, these data provide a reference method to control and screen for i-Hep differentiation, identify Laminin 411 as a key niche protein, and underscore the importance of combining substrates, soluble factors, and HCA when developing iPSC applications.

Collar occupancy: A new quantitative imaging tool for morphometric analysis of oligodendrocytes.

BACKGROUND: Oligodendrocytes (OL) are the myelinating cells of the central nervous system. OL differentiation from oligodendrocyte progenitor cells (OPC) is accompanied by characteristic stereotypical morphological changes. Quantitative imaging of those morphological alterations during OPC differentiation is commonly used for characterization of new molecules in cell differentiation and myelination and screening of new pro-myelinating drugs. Current available imaging analysis methods imply a non-automated morphology assessment, which is time-consuming and prone to user subjective evaluation. NEW METHOD: Here, we describe an automated high-throughput quantitative image analysis method entitled collar occupancy that allows morphometric ranking of different stages of in vitro OL differentiation in a high-content analysis format. Collar occupancy is based on the determination of the percentage of area occupied by OPC/OL cytoplasmic protrusions within a defined region that contains the protrusion network, the collar. RESULTS: We observed that more differentiated cells have higher collar occupancy and, therefore, this parameter correlates with the degree of OL differentiation. COMPARISON WITH EXISTING METHODS: In comparison with the method of manual categorization, we found the collar occupancy to be more robust and unbiased. Moreover, when coupled with myelin basic protein (MBP) staining to quantify the percentage of myelinating cells, we were able to evaluate the role of new molecules in OL differentiation and myelination, such as Dusp19 and Kank2. CONCLUSIONS: Altogether, we have successfully developed an automated and quantitative method to morphologically characterize OL differentiation in vitro that can be used in multiple studies of OL biology.

Morphometric Characterization of Rat and Human Alveolar Macrophage Cell Models and their Response to Amiodarone using High Content Image Analysis.

PURPOSE: Progress to the clinic may be delayed or prevented when vacuolated or "foamy" alveolar macrophages are observed during non-clinical inhalation toxicology assessment. The first step in developing methods to study this response in vitro is to characterize macrophage cell lines and their response to drug exposures. METHODS: Human (U937) and rat (NR8383) cell lines and primary rat alveolar macrophages obtained by bronchoalveolar lavage were characterized using high content fluorescence imaging analysis quantification of cell viability, morphometry, and phospholipid and neutral lipid accumulation. RESULTS: Cell health, morphology and lipid content were comparable (p < 0.05) for both cell lines and the primary macrophages in terms of vacuole number, size and lipid content. Responses to amiodarone, a known inducer of phospholipidosis, required analysis of shifts in cell population profiles (the proportion of cells with elevated vacuolation or lipid content) rather than average population data which was insensitive to the changes observed. CONCLUSIONS: A high content image analysis assay was developed and used to provide detailed morphological characterization of rat and human alveolar-like macrophages and their response to a phospholipidosis-inducing agent. This provides a basis for development of assays to predict or understand macrophage vacuolation following inhaled drug exposure.

Bridging Functional and Structural Cardiotoxicity Assays Using Human Embryonic Stem Cell-Derived Cardiomyocytes for a More Comprehensive Risk Assessment.

More relevant and reliable preclinical cardiotoxicity tests are required to improve drug safety and reduce the cost of drug development. Current in vitro testing strategies predominantly take the form of functional assays to predict the potential for drug-induced ECG abnormalities in vivo. Cardiotoxicity can also be structural in nature, so a full and efficient assessment of cardiac liabilities for new chemical entities should account for both these phenomena. As well as providing a more appropriate nonclinical model for in vitro cardiotoxicity testing, human stem cell-derived cardiomyocytes offer an integrated system to study drug impact on cardiomyocyte structure as well as function. Employing human embryonic stem cell-derived cardiacmyocytes (hESC-CMs) on 3 assay platforms with complementary insights into cardiac biology (multielectrode array assay, electrophysiology; impedance assay, cell movement/beating; and high content analysis assay, subcellular structure) we profiled a panel of 13 drugs with well characterized cardiac liabilities (Amiodarone, Aspirin, Astemizole, Axitinib, AZT, Bepridil, Doxorubicin, E-4031, Mexiletine, Rosiglitazone, Sunitinib, Sibutramine, and Verapamil). Our data show good correlations with previous studies and reported clinical observations. Using multiparameter phenotypic profiling techniques we demonstrate the dynamic relationship that exists between functional and structural toxicity, and the benefits of this more holistic approach to risk assessment. We conclude by showing for the first time how the advent of transparent MEA plate technology enables functional and structural cardiotoxic responses to be recorded from the same cell population. This approach more directly links changes in morphology of the hESC-CMs with recorded electrophysiology signatures, offering even greater insight into the wide range of potential drug impacts on cardiac physiology, with a throughput that is more amenable to early drug discovery.

In Vitro Multiparameter Assay Development Strategy toward Differentiating Macrophage Responses to Inhaled Medicines.

Although foamy macrophages (FMΦ) are commonly observed during nonclinical development of medicines for inhalation, there are no accepted criteria to differentiate adaptive from adverse FMΦ responses in drug safety studies. The purpose of this study was to develop a multiparameter in vitro assay strategy to differentiate and characterize different mechanisms of drug-induced FMΦ. Amiodarone, staurosporine, and poly(vinyl acetate) nanoparticles were used to induce distinct FMΦ phenotypes in J774A.1 cells, which were then compared with negative controls. Treated macrophages were evaluated for morphometry, lipid accumulation, gene expression, apoptosis, cell activation, and phagocytosis. Analysis of vacuolization (number/area vacuoles per cell) and phospholipid content revealed inducer-dependent distinctive patterns, which were confirmed by electron microscopy. In contrast to the other inducers, amiodarone increased vacuole size rather than number and resulted in phospholipid accumulation. No pronounced dysregulation of transcriptional activity or apoptosis was observed in response to sublethal concentrations of all inducers. Functionally, FMΦ induction did not affect macrophage activation by lipopolysaccharide, but it reduced phagocytic capacity, with different patterns of induction, severity, and resolution observed with the different inducers. An in vitro multiparameter assay strategy is reported that successfully differentiates and characterizes mechanisms leading to FMΦ induction by different types of agents.

Nanoparticle vesicle encoding for imaging and tracking cell populations.

For phenotypic behavior to be understood in the context of cell lineage and local environment, properties of individual cells must be measured relative to population-wide traits. However, the inability to accurately identify, track and measure thousands of single cells via high-throughput microscopy has impeded dynamic studies of cell populations. We demonstrate unique labeling of cells, driven by the heterogeneous random uptake of fluorescent nanoparticles of different emission colors. By sequentially exposing a cell population to different particles, we generated a large number of unique digital codes, which corresponded to the cell-specific number of nanoparticle-loaded vesicles and were visible within a given fluorescence channel. When three colors are used, the assay can self-generate over 17,000 individual codes identifiable using a typical fluorescence microscope. The color-codes provided immediate visualization of cell identity and allowed us to track human cells with a success rate of 78% across image frames separated by 8 h.

Stabilisation of a heptamethine cyanine dye by rotaxane encapsulation.

The crystal structure of a cyanine dye rotaxane shows that the cyclodextrin is tightly threaded round the polymethine bridge of the dye; encapsulation dramatically increases the kinetic chemical stability of the radicals formed on oxidation and reduction of the dye, making it possible to observe the rotaxane radical dication by ESR and UV-vis-NIR spectroscopy.