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Abnormalities in cellular metabolism are seen early in Alzheimer's disease (AD). Astrocyte support for neuronal function has a high metabolic demand, and astrocyte glucose metabolism plays a key role in encoding memory. This indicates that astrocyte metabolic dysfunction might be an early event in the development of AD. In this paper we interrogate glycolytic and mitochondrial functional changes and mitochondrial structural alterations in patients' astrocytes derived with a highly efficient direct conversion protocol. In astrocytes derived from patients with sporadic (sAD) and familial AD (fAD) we identified reductions in extracellular lactate, total cellular ATP and an increase in mitochondrial reactive oxygen species. sAD and fAD astrocytes displayed significant reductions in mitochondrial spare respiratory capacity, have altered mitochondrial membrane potential and a stressed mitochondrial network. A reduction in glycolytic reserve and glycolytic capacity is seen. Interestingly, glycolytic reserve, mitochondrial spare respiratory capacity and extracellular lactate levels correlated positively with neuropsychological tests of episodic memory affected early in AD. We identified a deficit in the glycolytic enzyme hexokinase 1 (HK1), and correcting this deficit improved the metabolic phenotype in sAD not fAD astrocytes. Importantly, the amount of HK1 at the mitochondria was shown to be reduced in sAD astrocytes, and not in fAD astrocytes. Overexpression of HK1 in sAD astrocytes increases mitochondrial HK1 levels. In fAD astrocytes HK1 levels were unaltered at the mitochondria after overexpression. This study highlights a clear metabolic deficit in AD patient-derived astrocytes and indicates how HK1, with its roles in both oxidative phosphorylation and glycolysis, contributes to this.

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The dysregulation of pH has been linked to the onset of chronic conditions, such as cancer and neurological diseases. Consequently, the development of a highly sensitive tool for intracellular pH sensing is imperative to investigate the interplay between pH and the biochemical changes accompanying disease pathogenesis. Here, we present the development of a ratiometric fluorescent nanoprobe, NpRhoDot, designed for precisely measuring pH levels. We demonstrate its efficacy in sensitively reporting intracellular pH in monolayer A549 lung cancer cells, primary fibroblast cells, and 3D tumor spheroids derived from the DLD-1 colorectal adenocarcinoma cell line. NpRhoDot leverages a novel design, where stable carbon dots are functionalized with a pH-responsive ratiometric fluorescent probe comprising a naphthalimide-rhodamine moiety, NpRho1. This design confers NpRhoDot with the high pH sensitivity characteristics of organic fluorescent probes, along with excellent photostability up to 1 h and biocompatibility of carbon dots. Through one-photon and two-photon fluorescence microscopy, we validate the reliability of NpRhoDot for biosensing intracellular pH in monolayer and three-dimensional tumor models from pH 4 to 7.

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Imaging with multiple modalities can maximise the information gained from the analysis of a single sample. probes for optical fluorescence and X-ray fluorescence microscopy based on brominated 4-amino-1,8-naphthalimide and BODIPY scaffolds have been successfully designed and synthesised. Herein we show that these prototype probes, based on each of these scaffolds, can be imaged in two different cancer cell lines, and that the respective optical fluorescence and X-ray fluorescence signals are well correlated in these images.

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To fully understand and predict the impact of nanotechnologies, a truly multidisciplinary approach is required. However, the practicalities relating to how innovation, commercialisation, risk assessment, informatics, and governance in nanotechnology should intersect remain somewhat of a black box. To begin to shed light on this intersection, we identify a need to place 'purpose' at the heart of the nanotechnology innovation ecosystem. There is a growing appetite for responsible, sustainable, and purposeful innovation from business, financiers, regulators, consumers, and other stakeholders - an appetite that we foresee will permeate all spheres of commercialisation, including that of nanotechnology. Ultimately, nanotechnologies will only have the ability to sustainably address the global challenges of the 21st century if they are developed and implemented with purpose, and in full consideration of their social and environmental impacts. We (re)define purpose as it relates to sustainable nanotechnology innovation, in an effort to create a more-broadly shared language that can bridge the diverse stakeholder needs and perspectives that are required to address these challenges. To enable innovation, standardisation, promote interdisciplinarity, increase transparency, and enhance regulatory and corporate accountability, we propose a four stage, principles-based framework for purposeful nanotechnology development. This framework offers a practical way forward for nanotechnology innovation, shedding light on how nano-impact can be approached by multidisciplinary teams and describing how interrelated systems and stakeholders can interact successfully to achieve shared goals.

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Copper participates in a range of critical functions in the nervous system and human brain. Disturbances in brain copper content is strongly associated with neurological diseases. For example, changes in the level and distribution of copper are reported in neuroblastoma, Alzheimer's disease, and Lewy body disorders, such as Parkinson disease and dementia with Lewy bodies (DLB). There is a need for more sensitive techniques to measure intracellular copper levels to have a better understanding of the role of copper homeostasis in neuronal disorders. Here, we report a reaction-based near-infrared (NIR) ratiometric fluorescent probe CyCu1 for imaging Cu2+ in biological samples. High stability and selectivity of CyCu1 enabled the probe to be deployed as a sensor in a range of systems, including SH-SY5Y cells and neuroblastoma tumors. Furthermore, it can be used in plant cells, reporting on copper added to Arabidopsis roots. We also used CyCu1 to explore Cu2+ levels and distribution in post-mortem brain tissues from patients with DLB. We found significant decreases in Cu2+ content in the cytoplasm, neurons, and extraneuronal space in the degenerating substantia nigra in DLB compared with healthy age-matched control tissues. These findings enhance our understanding of Cu2+ dysregulation in Lewy body disorders. Our probe also shows promise as a photoacoustic imaging agent, with potential for applications in bimodal imaging.

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Although transition metals constitute less than 0.1% of the total mass within a human body, they have a substantial impact on fundamental biological processes across all kingdoms of life. Indeed, these nutrients play crucial roles in the physiological functions of enzymes, with the redox properties of many of these metals being essential to their activity. At the same time, imbalances in transition metal pools can be detrimental to health. Modern analytical techniques are helping to illuminate the workings of metal homeostasis at a molecular and atomic level, their spatial localization in real time, and the implications of metal dysregulation in disease pathogenesis. Fluorescence microscopy has proven to be one of the most promising non-invasive methods for studying metal pools in biological samples. The accuracy and sensitivity of bioimaging experiments are predominantly determined by the fluorescent metal-responsive sensor, highlighting the importance of rational probe design for such measurements. This review covers activity- and binding-based fluorescent metal sensors that have been applied to cellular studies. We focus on the essential redox-active metals: iron, copper, manganese, cobalt, chromium, and nickel. We aim to encourage further targeted efforts in developing innovative approaches to understanding the biological chemistry of redox-active metals.

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Self-assembly of misfolded proteins can lead to the formation of amyloids, which are implicated in the onset of many pathologies including Alzheimer's disease and Parkinson's disease. The facile detection and discrimination of different amyloids are crucial for early diagnosis of amyloid-related pathologies. Here, we report the development of a fluorescent coumarin-based two-sensor array that is able to correctly discriminate between four different amyloids implicated in amyloid-related pathologies with 100% classification. The array was also applied to mouse models of Alzheimer's disease and was able to discriminate between samples from mice corresponding to early (6 months) and advanced (12 months) stages of Alzheimer's disease. Finally, the flexibility of the array was assessed by expanding the analytes to include functional amyloids. The same two-sensor array was able to correctly discriminate between eight different disease-associated and functional amyloids with 100% classification.

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Cell-penetrating peptides (CPPs) play a significant role in the delivery of cargos into human cells. We report the first CPPs based on peptide-bismuth bicycles, which can be readily obtained from commercially available peptide precursors, making them accessible for a wide range of applications. These CPPs enter human cells as demonstrated by live-cell confocal microscopy using fluorescently labelled peptides. We report efficient sequences that demonstrate increased cellular uptake compared to conventional CPPs like the TAT peptide (derived from the transactivating transcriptional activator of human immunodeficiency virus 1) or octaarginine (R8 ), despite requiring only three positive charges. Bicyclization triggered by the presence of bismuth(III) increases cellular uptake by more than one order of magnitude. Through the analysis of cell lysates using inductive coupled plasma mass spectrometry (ICP-MS), we have introduced an alternative approach to examine the cellular uptake of CPPs. This has allowed us to confirm the presence of bismuth in cells after exposure to our CPPs. Mechanistic studies indicated an energy-dependent endocytic cellular uptake sensitive to inhibition by rottlerin, most likely involving macropinocytosis.

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Imaging techniques permit the study of the molecular interactions that underlie health and disease. Each imaging technique collects unique chemical information about the cellular environment. Multimodal imaging, using a single probe that can be detected by multiple imaging modalities, can maximise the information extracted from a single cellular sample by combining the results of different imaging techniques. Of particular interest in biological imaging is the combination of the specificity and sensitivity of optical fluorescence microscopy (OFM) with the quantitative and element-specific nature of X-ray fluorescence microscopy (XFM). Together, these techniques give a greater understanding of how native elements or therapeutics affect the cellular environment. This review focuses on recent studies where both techniques were used in conjunction to study cellular systems, demonstrating the breadth of biological models to which this combination of techniques can be applied and the potential for these techniques to unlock untapped knowledge of disease states.

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Small-molecule fluorophores are powerful tools for biological research. They have enabled researchers to study cellular architecture and decipher biological processes. Responsive fluorescent sensors have enabled the study of a wide range of analytes and their effects on biological phenomena in situ. The application of fluorescent sensors to studies in living organisms is complicated by challenges such as biocompatibility, chemostability, photostability and sufficient penetration of light through living tissues. Translation to in vivo imaging is therefore not straightforward and requires innovative approaches. Recent advances in the design of fluorophores with improved photophysical properties and the development of long-wavelength-emitting fluorophore scaffolds that can be modularly functionalised with targeting and sensing groups have allowed the application of fluorogenic, ratiometric and reversible sensors in vivo.

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The COVID-19 pandemic has had a devastating impact on global health, highlighting the need to understand how the SARS-CoV-2 virus damages the lungs in order to develop effective treatments. Recent research has shown that patients with COVID-19 experience severe oxidative damage to various biomolecules. We propose that the overproduction of reactive oxygen species (ROS) in SARS-CoV-2 infection involves an interaction between copper ions and the virus's spike protein. We tested two peptide fragments, Ac-ELDKYFKNH-NH2 (L1) and Ac-WSHPQFEK-NH2 (L2), derived from the spike protein of the Wuhan strain and the ß variant, respectively, and found that they bind Cu(II) ions and form a three-nitrogen complexes at lung pH. Our research demonstrates that these complexes trigger the overproduction of ROS, which can break both DNA strands and transform DNA into its linear form. Using A549 cells, we demonstrated that ROS overproduction occurs in the mitochondria, not in the cytoplasm. Our findings highlight the importance of the interaction between copper ions and the virus's spike protein in the development of lung damage and may aid in the development of therapeutic procedures.

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While metal ions play an important role in the proper functioning of all life, many questions remain unanswered about exactly how different metals contribute to health and disease. The development of fluorescent probes, which respond to metals, has allowed greater understanding of the cellular location, concentration and speciation of metals in living systems, giving a new appreciation of their function. While the focus of studies using these fluorescent tools has largely been on mammalian organisms, there has been relatively little application of these powerful tools to other organisms. In this review, we highlight recent examples of molecular fluorophores, which have been applied to sensing metals in non-mammalian organisms.

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Decoding cellular processes requires visualization of the spatial distribution and dynamic interactions of biomolecules. It is therefore not surprising that innovations in imaging technologies have facilitated advances in biomedical research. The advent of super-resolution imaging technologies has empowered biomedical researchers with the ability to answer long-standing questions about cellular processes at an entirely new level. Fluorescent probes greatly enhance the specificity and resolution of super-resolution imaging experiments. Here, we introduce key super-resolution imaging technologies, with a brief discussion on single-molecule localization microscopy (SMLM). We evaluate the chemistry and photochemical mechanisms of fluorescent probes employed in SMLM. This Review provides guidance on the identification and adoption of fluorescent probes in single molecule localization microscopy to inspire the design of next-generation fluorescent probes amenable to single-molecule imaging.

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Imbalances in metal homeostasis have been implicated in the progression and drug response of cancer cells. Understanding these changes will enable identification of new treatment regimes and precision medicine approaches to cancer treatment. In particular, there has been considerable interest in the interplay between copper homeostasis and response to platinum-based chemotherapeutic agents. Here, we have studied differences in the Cu uptake and distributions in the ovarian cancer cell line, A2780, and its cisplatin resistant form, A2780.CisR, by measuring total Cu content and the bioavailable Cu pool. Atomic absorption spectroscopy (AAS) revealed a lower total Cu uptake in A2780.CisR compared to A2780 cells. Conversely, live-cell confocal microscopy studies with the ratiometric Cu(I)-sensitive fluorescent dye, InCCu1, revealed higher relative cellular content of labile Cu in A2780.CisR cells compared with A2780 cells. These results demonstrate that Cu trafficking, homeostasis and speciation are different in the Pt-sensitive and resistant cells and may be associated with the predominance of different phenotypes for A2780 (epithelial) and A2780.CisR (mesenchymal) cells.

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Mitochondrial health is crucial to sperm quality and male fertility, but the precise role of mitochondria in sperm function remains unclear. SDHA is a component of the succinate dehydrogenase (SDH) complex and plays a critical role in mitochondria. In humans, SDH activity is positively correlated with sperm quality, and mutations in SDHA are associated with Leigh Syndrome. Here we report that the C. elegans SDHA orthologue SDHA-2 is essential for male fertility: sdha-2 mutants produce dramatically fewer offspring due to defective sperm activation and motility, have hyperfused sperm mitochondria, and disrupted redox balance. Similar sperm motility defects in sdha-1 and icl-1 mutant animals suggest an imbalance in metabolites may underlie the fertility defect. Our results demonstrate a role for SDHA-2 in sperm motility and male reproductive health and establish an animal model of SDH deficiency-associated infertility.

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Human activities, such as mining and manufacturing, expose society and the natural environment to harmful levels of metal ions. Recently, optical sensor arrays for metal ion detection have become popular owing to their favourable features, such as facile sample preparation and the requirement of less expensive instrumentation compared to traditional, spectrometry-based analysis techniques. Sensor arrays usually consist of numerous optical probes that are used in combination to generate unique analyte responses. In contrast, here we present an array that comprises a single fluorescent sensor, Coum4-DPA, that produces unique responses to metal ions in different pH environments. With this simple sensing platform, we were able to classify 10 metal ions in different water sources and quantify Pb2+ in tap water using just one fluorescent sensor, a few pH buffers and two sets of spectral data. This novel approach significantly decreases time and costs associated with probe synthesis and data collection, making it highly transferrable to real-world metal sensing applications.

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The lipid content of mammalian cells varies greatly between cell type. Current methods for analysing lipid components of cells are technically challenging and destructive. Here, we report a facile, inexpensive method to identify lipid content - intracellular flow cytometric lipid analysis (IFCLA). Distinct lipid classes can be distinguished by Nile Blue fluorescence, Nile Red fluorescence or violet autofluorescence. Nile Blue is fluorescent in the presence of unsaturated fatty acids with a carbon chain length greater than 16. Cis-configured fatty acids induce greater Nile Blue fluorescence than their trans-configured counterparts. In contrast, Nile Red exhibits greatest fluorescence in the presence of cholesterol, cholesteryl esters, some triglycerides and phospholipids. Multiparametric spanning-tree progression analysis for density-normalized events (SPADE) analysis of hepatic cellular lipid distribution, including vitamin A autofluorescence, is presented. This flow cytometric system allows for the rapid, inexpensive and non-destructive identification of lipid content, and highlights the differences in lipid biology between cell types by imaging and flow cytometry.

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Many soluble proteins can self-assemble into macromolecular structures called amyloids, a subset of which are implicated in a range of neurodegenerative disorders. The nanoscale size and structural heterogeneity of prefibrillar and early aggregates, as well as mature amyloid fibrils, pose significant challenges for the quantification of amyloid morphologies. We report a fluorescent amyloid sensor AmyBlink-1 and its application in super-resolution imaging of amyloid structures. AmyBlink-1 exhibits a 5-fold increase in ratio of the green (thioflavin T) to red (Alexa Fluor 647) emission intensities upon interaction with amyloid fibrils. Using AmyBlink-1, we performed nanoscale imaging of four different types of amyloid fibrils, achieving a resolution of ≈30 nm. AmyBlink-1 enables nanoscale visualization and subsequent quantification of morphological features, such as the length and skew of individual amyloid aggregates formed at different times along the amyloid assembly pathway.

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Fluorescent probes for biological imaging have revealed much about the functions of biomolecules in health and disease. Fluorogenic probes, which are fluorescent only upon a bioorthogonal reaction with a specific partner, are particularly advantageous as they ensure that fluorescent signals observed in biological imaging arise solely from the intended target. In this work, we report the first series of naphthalimide tetrazines for bioorthogonal fluorogenic labelling. We establish that all of these compounds can be used for imaging through photophysical, analytical and biological studies. The best candidate was Np6mTz, where the tetrazine ring is appended to the naphthalimide at its 6-position via a phenyl linker in a meta configuration. Taking our synthetic scaffold, we generated two targeted variants, LysoNpTz and MitoNpTz, which successfully localized within the lysosomes and mitochondria respectively, without the requirement of genetic modification. In addition, the naphthalimide tetrazine system was used for the no-wash imaging of insulin amyloid fibrils in vitro, providing a new method that can monitor their growth kinetics and morphology. Since our synthetic approach is simple and modular, these new naphthalimide tetrazines provide a novel scaffold for a range of bioorthogonal tetrazine-based imaging agents for selective staining and sensing of biomolecules.

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Fluorescent tools have emerged as an important tool for studying the distinct chemical microenvironments of organelles, due to their high specificity and ability to be used in non-destructive, live cellular studies. These tools fall largely in two categories: exogenous fluorescent dyes, or endogenous labels such as genetically encoded fluorescent proteins. In both cases, the probe must be targeted to the organelle of interest. To date, many organelle-targeted fluorescent tools have been reported and used to uncover new information about processes that underpin health and disease. However, the majority of these tools only apply a handful of targeting groups, and less-studied organelles have few robust targeting strategies. While the development of new, robust strategies is difficult, it is essential to develop such strategies to allow for the development of new tools and broadening the effective study of organelles. This review aims to provide a comprehensive overview of the major targeting strategies for both endogenous and exogenous fluorescent cargo, outlining the specific challenges for targeting each organelle type and as well as new developments in the field.

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