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In the last 2 decades, Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) has gained significant prominence as a powerful imaging technique in the field of life sciences. This comprehensive review provides an in-depth overview of recent advancements in ToF-SIMS instrument technology and its applications in metabolomics, lipidomics, and single-cell analysis. We highlight the use of ToF-SIMS imaging for studying lipid distribution, composition, and interactions in cells and tissues, and discuss its application in metabolomics, including the analysis of metabolic pathways. Furthermore, we review recent progress in single-cell analysis using ToF-SIMS, focusing on sample preparation techniques, in situ investigation for subcellular distribution of drugs, and interactions between drug molecules and biological targets. The high spatial resolution and potential for multimodal analysis of ToF-SIMS make it a promising tool for unraveling the complex molecular landscape of biological systems. We also discuss future prospects and potential advancements of ToF-SIMS in the research of life sciences, with the expectation of a significant impact in the field.

Min oscillations are a fascinating mechanism used by Escherichia coli to find their middle. Beyond their biological role, they provide a convenient and relatively unexplored method to monitor the effect of sublethal environmental challenges on bacterial physiology in real-time and at the single-cell level. In this review, we discuss the original papers that put forward the idea of using Min oscillations as a reporting tool to monitor the effect of extracellular cationic compounds, including antibiotics. More recent work from our laboratory explores this tool to follow bacterial response to other challenges such as weak mechanical interactions with nanomaterials or photodynamic treatment. We discuss the physiological meaning of the changes in Min oscillation period, likely related to membrane potential dynamics, as well as the benefits and limitations of using oscillations as a reporter in fluorescence microscopy. Overall, Min oscillations are a useful addition to the fluorescence microscopy toolbox in order to visualize stress responses in E. coli, and have the potential to provide full mechanistic understanding of the events that lead to bacterial cell death in different contexts.

Abstract Mesenchymal stem/stromal cells (MSCs) are a promising resource for cell-based therapy because of their high immunomodulation ability, tropism towards inflamed and injured tissues, and their easy access and isolation. Currently, there are more than 1200 registered MSC clinical trials globally. However, a lack of standardized methods to characterize cell safety, efficacy, and biodistribution dramatically hinders the progress of MSC utility in clinical practice. In this review, we summarize the current state of MSC-based cell therapy, focusing on the systemic safety and biodistribution of MSCs. MSC-associated risks of tumor initiation and promotion and the underlying mechanisms of these risks are discussed. In addition, MSC biodistribution methodology and the pharmacokinetics and pharmacodynamics of cell therapies are addressed. Better understanding of the systemic safety and biodistribution of MSCs will facilitate future clinical applications of precision medicine using stem cells.

Central nervous system (CNS) infections are a major cause of human morbidity and mortality worldwide. Even patients that survive, CNS infections can have lasting neurological dysfunction resulting from immune and pathogen induced pathology. Developing approaches to noninvasively track pathology and immunity in the infected CNS is crucial for patient management and development of new therapeutics. Here, we develop novel MRI-based approaches to monitor virus-specific CD8+ T cells and their relationship to cerebrovascular pathology in the living brain. We studied a relevant murine model in which a neurotropic virus (vesicular stomatitis virus) was introduced intranasally and then entered the brain via olfactory sensory neurons – a route exploited by many pathogens in humans. Using T2*-weighted high-resolution MRI, we identified small cerebral microbleeds as an early form of pathology associated with viral entry into the brain. Mechanistically, these microbleeds occurred in the absence of peripheral immune cells and were associated with infection of vascular endothelial cells. We monitored the adaptive response to this infection by developing methods to iron label and track individual virus specific CD8+ T cells by MRI. Transferred antiviral T cells were detected in the brain within a day of infection and were able to reduce cerebral microbleeds. These data demonstrate the utility of MRI in detecting the earliest pathological events in the virally infected CNS as well as the therapeutic potential of antiviral T cells in mitigating this pathology.

Integrating single cell omics and single cell imaging allows for a more effective characterisation of the underlying mechanisms that drive a phenotype at the tissue level, creating a comprehensive profile at the cellular level. Although the use of imaging data is well established in biomedical research, its primary application has been to observe phenotypes at the tissue or organ level, often using medical imaging techniques such as MRI, CT, and PET. These imaging technologies complement omics-based data in biomedical research because they are helpful for identifying associations between genotype and phenotype, along with functional changes occurring at the tissue level. Single cell imaging can act as an intermediary between these levels. Meanwhile new technologies continue to arrive that can be used to interrogate the genome of single cells and its related omics datasets. As these two areas, single cell imaging and single cell omics, each advance independently with the development of novel techniques, the opportunity to integrate these data types becomes more and more attractive. This review outlines some of the technologies and methods currently available for generating, processing, and analysing single-cell omics- and imaging data, and how they could be integrated to further our understanding of complex biological phenomena like ageing. We include an emphasis on machine learning algorithms because of their ability to identify complex patterns in large multidimensional data.

Land plants evolved to quickly sense and adapt to temperature changes, such as hot days and cold nights. Given that calcium (Ca2+) signaling networks are implicated in most abiotic stress responses, heat-triggered changes in cytosolic Ca2+ were investigated in Arabidopsis leaves and pollen. Plants were engineered with a reporter called CGf, a ratiometric, genetically encoded Ca2+ reporter with an m C herry reference domain fused to an intensiometric Ca2+ reporter G CaMP6 f. Relative changes in [Ca2+]cyt were estimated based on CGf's apparent K D around 220 nM. The ratiometric output provided an opportunity to compare Ca2+ dynamics between different tissues, cell types, or subcellular locations. In leaves, CGf detected heat-triggered cytosolic Ca2+ signals, comprised of three different signatures showing similarly rapid rates of Ca2+ influx followed by differing rates of efflux (50% durations ranging from 5 to 19 min). These heat-triggered Ca2+ signals were approximately 1.5-fold greater in magnitude than blue light-triggered signals in the same leaves. In contrast, growing pollen tubes showed two different heat-triggered responses. Exposure to heat caused tip-focused steady growth [Ca2+]cyt oscillations to shift to a pattern characteristic of a growth arrest (22%), or an almost undetectable [Ca2+]cyt (78%). Together, these contrasting examples of heat-triggered Ca2+ responses in leaves and pollen highlight the diversity of Ca2+ signals in plants, inviting speculations about their differing kinetic features and biological functions. [ABSTRACT FROM AUTHOR]

Background: The extent of cellular heterogeneity in breast cancer could have potential impact on diagnosis and long-term outcome. However, pathology evaluation is limited to biomarker immunohistochemical staining and morphology of the bulk cancer. Inter-cellular heterogeneity of biomarkers is not usually assessed. As an initial evaluation of the extent of breast cancer cellular heterogeneity, we conducted quantitative and spatial imaging of Estrogen Receptor (ER), Progesterone Receptor (PR), Epidermal Growth Factor Receptor-2 (HER2), Ki67, TP53, CDKN1A (P21/WAF1), CDKN2A (P16INK4A), CD8 and CD20 of a tissue microarray (TMA) representing subtypes defined by St. Gallen surrogate classification.Methods: Quantitative, single cell-based imaging was conducted using an Immunofluorescence protein multiplexing platform (MxIF) to study protein co-expression signatures and their spatial localization patterns. The range of MxIF intensity values of each protein marker was compared to the respective IHC score for the TMA core. Extent of heterogeneity in spatial neighborhoods was analyzed using co-occurrence matrix and Diversity Index measures.Results: On the 101 cores from 59 cases studied, diverse expression levels and distributions were observed in MxIF measures of ER and PR among the hormonal receptor-positive tumor cores. As expected, Luminal A-like cancers exhibit higher proportions of cell groups that co-express ER and PR, while Luminal B-like (HER2-negative) cancers were composed of ER+, PR- groups. Proliferating cells defined by Ki67 positivity were mainly found in groups with PR-negative cells. Triple-Negative Breast Cancer (TNBC) exhibited the highest proliferative fraction and incidence of abnormal P53 and P16 expression. Among the tumors exhibiting P53 overexpression by immunohistochemistry, a group of TNBC was found with much higher MxIF-measured P53 signal intensity compared to HER2+, Luminal B-like and other TNBC cases. Densities of CD8 and CD20 cells were highest in HER2+ cancers. Spatial analysis demonstrated variability in heterogeneity in cellular neighborhoods in the cancer and the tumor microenvironment.Conclusions: Protein marker multiplexing and quantitative image analysis demonstrated marked heterogeneity in protein co-expression signatures and cellular arrangement within each breast cancer subtype. These refined descriptors of biomarker expressions and spatial patterns could be valuable in the development of more informative tools to guide diagnosis and treatment. [ABSTRACT FROM AUTHOR]

Land plants evolved to quickly sense and adapt to temperature changes, such as hot days and cold nights. Given that calcium (Ca2+) signaling networks are implicated in most abiotic stress responses, heat-triggered changes in cytosolic Ca2+ were investigated in Arabidopsis leaves and pollen. Plants were engineered with a reporter called CGf, a ratiometric, genetically encoded Ca2+ reporter with an mCherry reference domain fused to an intensiometric Ca2+ reporter GCaMP6f. Relative changes in [Ca2+]cyt were estimated based on CGf’s apparent KD around 220 nM. The ratiometric output provided an opportunity to compare Ca2+ dynamics between different tissues, cell types, or subcellular locations. In leaves, CGf detected heat-triggered cytosolic Ca2+ signals, comprised of three different signatures showing similarly rapid rates of Ca2+ influx followed by differing rates of efflux (50% durations ranging from 5 to 19 min). These heat-triggered Ca2+ signals were approximately 1.5-fold greater in magnitude than blue light-triggered signals in the same leaves. In contrast, growing pollen tubes showed two different heat-triggered responses. Exposure to heat caused tip-focused steady growth [Ca2+]cyt oscillations to shift to a pattern characteristic of a growth arrest (22%), or an almost undetectable [Ca2+]cyt (78%). Together, these contrasting examples of heat-triggered Ca2+ responses in leaves and pollen highlight the diversity of Ca2+ signals in plants, inviting speculations about their differing kinetic features and biological functions.

Abstract Fluorescence imaging of nucleic acids is a very important technique necessary to understand gene expression and the resulting changes in cell function. This mini-review focuses on sequence-specific fluorescence imaging of intracellular RNA and methylated DNA using fluorescent nucleic acid probes. A couple of functional fluorescent nucleic acid probes developed by our laboratory are introduced and the examples of their application to fluorescence imaging of intracellular nucleic acids are described.

Mesenchymal stem/stromal cells (MSCs) are a promising resource for cell-based therapy because of their high immunomodulation ability, tropism towards inflamed and injured tissues, and their easy access and isolation. Currently, there are more than 1200 registered MSC clinical trials globally. However, a lack of standardized methods to characterize cell safety, efficacy, and biodistribution dramatically hinders the progress of MSC utility in clinical practice. In this review, we summarize the current state of MSC-based cell therapy, focusing on the systemic safety and biodistribution of MSCs. MSC-associated risks of tumor initiation and promotion and the underlying mechanisms of these risks are discussed. In addition, MSC biodistribution methodology and the pharmacokinetics and pharmacodynamics of cell therapies are addressed. Better understanding of the systemic safety and biodistribution of MSCs will facilitate future clinical applications of precision medicine using stem cells. [ABSTRACT FROM AUTHOR]

Insights into the pharmacologic effect on cellular processes and the potential toxicological effects are vital to new drug development and evaluation, yet research on these subjects remains a great challenge due to the lack of information regarding the spatiotemporal distribution of drugs and metabolites within a single cell. Mass spectrometry imaging (MSI) has proven to be a label-free and high-throughput approach for visualizing drug distribution in spatial and temporal domains. However, single-cell drug imaging has been limited so far by detection sensitivity and microscale lateral resolution. Herein, we report near-field laser desorption/laser postionization mass spectrometry (NDPI-MS) for single-cell imaging of two structurally similar drugs, proflavine and ethacridine, and subcellular distributions of proflavine at different drug concentrations were investigated. The NDPI-MS imaging results indicate that proflavine was accumulated in lysosomes, which was verified by laser scanning confocal microscopy (LSCM). Additionally, a distinguished subcellular distribution pattern of ethacridine from proflavine could be visualized, highlighting the complexity of the interaction between the drugs and biological environment even though these two drugs possess similar structures. Taken together, the present results demonstrate the great potential of the integrated single-cell MSI platform for characterizing the drug distribution and its phenotype changes within individual cells, expediting the identification and evaluation of newly developed drugs. [ABSTRACT FROM AUTHOR]

Currently two techniques exist for 3D reconstruction of biological samples by time-of-flight secondary ion mass spectrometry (ToF-SIMS). The first, based on microtomy and combining of successive section images, is successfully applied for tissues, while the second, based on sputter depth profiling, is widely used for cells. In the present work, we report the first successful adaptation of sectioning technique for ToF-SIMS 3D imaging of a single cell—fully grown mouse germinal vesicle (GV) oocyte. In addition, microtomy was combined with sputter depth profiling of individual flat sections for three-dimensional reconstruction of intracellular organelles. GV oocyte sectioning allowed us to obtain molecule-specific 3D maps free from artifacts associated with surface topography and uneven etching depth. Sputter depth profiling of individual flat slices revealed fine structure of specific organelles inside the oocyte. Different oocyte organelles (cytoplasm, germinal vesicle, membranes, cumulus cells) were presented on the ion images. Atypical nucleoli referred to as "nucleolus-like body" (NLB) was detected inside the germinal vesicle in PO3− and CN− ions generated by nucleic acids and proteins respectively. Significant difference in PO3− intensity in the NLB central area and NLB border was found. This difference appears as a bright halo around the center area. The NLB size calculated for PO3− and CN− ion images is 12.9 ± 0.2 μm and 11.9 ± 0.2 μm respectively, which suggests that bright halo of PO3− ions is a chromatin compaction on the NLB surface. Areas of approximately 1.0–2.5 μm size inside nucleoplasm with increased PO3− and CN− signal were registered in germinal vesicle. Observed compartments have different sizes and shapes, and they are likely attributed to chromocenters or chromosomes. [ABSTRACT FROM AUTHOR]

Cancer stem cells (CSCs) are a heterogeneous and dynamic self‐renewing population that stands at the top of tumor cellular hierarchy and contribute to tumor recurrence and therapeutic resistance. As methods of CSC isolation and functional interrogation advance, there is a need for a reliable and accessible quantitative approach to assess heterogeneity and state transition dynamics in CSCs. We developed a high‐throughput automated single cell imaging analysis (HASCIA) approach for the quantitative assessment of protein expression with single‐cell resolution and applied the method to investigate spatiotemporal factors that influence CSC state transition using glioblastoma (GBM) CSCs (GSCs) as a model system. We were able to validate the quantitative nature of this approach through comparison of the protein expression levels determined by HASCIA to those determined by immunoblotting. A virtue of HASCIA was exemplified by detection of a subpopulation of SOX2‐low cells, which expanded in fraction size during state transition. HASCIA also revealed that GSCs were committed to loose stem cell state at an earlier time point than the average SOX2 level decreased. Functional assessment of stem cell frequency in combination with the quantification of SOX2 expression by HASCIA defined a stable cutoff of SOX2 expression level for stem cell state. We also developed an approach to assess local cell density and found that denser monolayer areas possess higher average levels of SOX2, higher cell diversity, and a presence of a sub‐population of slowly proliferating SOX2‐low GSCs. HASCIA is an open source software that facilitates understanding the dynamics of heterogeneous cell population such as that of GSCs and their progeny. It is a powerful and easy‐to‐use image analysis and statistical analysis tool available at https://hascia.lerner.ccf.org. © 2019 International Society for Advancement of Cytometry [ABSTRACT FROM AUTHOR]

Angela Ariza de Schellenberger,1 Harald Kratz,1 Tracy D Farr,2,3 Norbert Löwa,4 Ralf Hauptmann,1 Susanne Wagner,1 Matthias Taupitz,1 Jörg Schnorr,1 Eyk A Schellenberger1 1Department of Radiology, 2Department of Experimental Neurology, Center for Stroke Research Berlin, Charité – Universitätsmedizin Berlin, Berlin, Germany; 3School of Life Sciences, University of Nottingham, Medical School, Nottingham, UK; 4Department of Biomagnetic Signals, Physikalisch-Technische Bundesanstalt Berlin, Berlin, Germany Abstract: Sensitive cell detection by magnetic resonance imaging (MRI) is an important tool for the development of cell therapies. However, clinically approved contrast agents that allow single-cell detection are currently not available. Therefore, we compared very small iron oxide nanoparticles (VSOP) and new multicore carboxymethyl dextran-coated iron oxide nanoparticles (multicore particles, MCP) designed by our department for magnetic particle imaging (MPI) with discontinued Resovist® regarding their suitability for detection of single mesenchymal stem cells (MSC) by MRI. We achieved an average intracellular nanoparticle (NP) load of >10 pg Fe per cell without the use of transfection agents. NP loading did not lead to significantly different results in proliferation, colony formation, and multilineage in vitro differentiation assays in comparison to controls. MRI allowed single-cell detection using VSOP, MCP, and Resovist® in conjunction with high-resolution T2*-weighted imaging at 7 T with postprocessing of phase images in agarose cell phantoms and in vivo after delivery of 2,000 NP-labeled MSC into mouse brains via the left carotid artery. With optimized labeling conditions, a detection rate of ~45% was achieved; however, the experiments were limited by nonhomogeneous NP loading of the MSC population. Attempts should be made to achieve better cell separation for homogeneous NP loading and to thus improve NP-uptake-dependent biocompatibility studies and cell detection by MRI and future MPI. Additionally, using a 7 T MR imager equipped with a cryocoil resulted in approximately two times higher detection. In conclusion, we established labeling conditions for new high-relaxivity MCP, VSOP, and Resovist® for improved MRI of MSC with single-cell sensitivity. Keywords: magnetic field microdistortions, single-cell imaging, mesenchymal stem cells, VSOP, MCP, Resovist®

Autophagy is a highly conserved, intracellular recycling process by which cytoplasmic contents are degraded in the lysosome. This process occurs at a low level constitutively; however, it is induced robustly in response to stressors, in particular, starvation of critical nutrients such as amino acids and glucose. That said, the relative contribution of these inputs is ambiguous and many starvation medias are poorly defined or devoid of multiple nutrients. Here, we sought to generate a quantitative catalog of autophagy across multiple stages and in single, living cells under normal growth conditions as well as in media starved specifically of amino acids or glucose. We found that autophagy is induced by starvation of amino acids, but not glucose, in U2OS cells, and that MTORC1-mediated ULK1 regulation and autophagy are tightly linked to amino acid levels. While autophagy is engaged immediately during amino acid starvation, a heightened response occurs during a period marked by transcriptional upregulation of autophagy genes during sustained starvation. Finally, we demonstrated that cells immediately return to their initial, low-autophagy state when nutrients are restored, highlighting the dynamic relationship between autophagy and environmental conditions. In addition to sharing our findings here, we provide our data as a high-quality resource for others interested in mathematical modeling or otherwise exploring autophagy in individual cells across a population., Competing Interests: Competing Interests: J.P.M. has consulting agreements with Merck and scholarly activity with the Translational Genomics Research Institute (a non-profit organization). R.G.J. is a scientific advisor for Agios Pharmaceuticals and Servier Pharmaceuticals and is a member of the Scientific Advisory Board of Immunomet Therapeutics.

Short abstract Over the last decades a broad collection of sophisticated fluorescent protein-based probes was engineered with the aim to specifically monitor nitric oxide (NO), one of the most important signaling molecules in biology. Here we report and discuss the characteristics and fields of applications of currently available genetically encoded fluorescent sensors for the detection of NO and its metabolites in different cell types. Long abstract Because of its radical nature and short half-life, real-time imaging of NO on the level of single cells is challenging. Herein we review state-of-the-art genetically encoded fluorescent sensors for NO and its byproducts such as peroxynitrite, nitrite and nitrate. Such probes enable the real-time visualization of NO signals directly or indirectly on the level of single cells and cellular organelles and, hence, extend our understanding of the spatiotemporal dynamics of NO formation, diffusion and degradation. Here, we discuss the significance of NO detection in individual cells and on subcellular level with genetic biosensors. Currently available genetically encoded fluorescent probes for NO and nitrogen species are critically discussed in order to provide insights in the functionality and applicability of these promising tools. As an outlook we provide ideas for novel approaches for the design and application of improved NO probes and fluorescence imaging protocols. Graphical abstract fx1 Highlights • Genetically encoded fluorescent probes are indispensable tools in modern cell research. • Specific fluorescent biosensors allow detection of nitric oxide (NO) in individual cells. • Direct and indirect genetically encoded NO probes are available. • Genetically encoded NO probes will be improved in future. [ABSTRACT FROM AUTHOR]

In this study, we compared the depth discrimination and speed performance of multifoci Raman hyperspectral imaging with the reference standard of a single laser point confocal Raman mapping. A liquid crystal spatial light modulator was employed for the generation of multifoci laser beams, and a digital micromirror device was used as a software‐configurable reflective pinhole array. The patterns of the laser foci and pinhole array can be rapidly changed without requiring any hardware alterations. Confocal patterns with different distance‐to‐size ratios were tested and compared. After optimization of the laser‐foci pattern, we demonstrated the feasibility of multifoci Raman hyperspectral microscopy for recording depth‐resolved molecular maps of biological cells (Acanthamoeba castellanii trophozoites). Micrometric depth discrimination and short acquisition times (20 min for single plane confocal image) were achieved. A liquid crystal spatial light modulator was employed for the generation of multifoci laser beams, and a digital micromirror device was used as a software‐configurable reflective pinhole array. Confocal patterns with different distance‐to‐size ratios were tested to achieve depth‐resolved Raman molecular maps of biological cells. [ABSTRACT FROM AUTHOR]

Imaging techniques can be compromised by aberrations. Especially when imaging through biological specimens, sample-induced distortions can limit localization accuracy. In particular, this phenomenon affects localization microscopy, traction force measurements, and single-particle tracking, which offer high-resolution insights into biological tissue. Here we present a method for quantifying and correcting the optical distortions induced by single, adherent, living cells. The technique uses periodically patterned gold nanostructures as a reference framework to quantify optically induced displacements with micrometer-scale sampling density and an accuracy of a few nanometers. The 3D cell shape and a simplified geometrical optics approach are then utilized to remap the microscope image. Our experiments reveal displacements of up to several hundred nanometers, and in corrected images these distortions are reduced by a factor of 3. Conversely, the relationship between cell shape and distortion provides a novel method of 3D cell shape reconstruction from a single image, enabling label-free 3D cell analysis. [ABSTRACT FROM AUTHOR]

The transcription factor nuclear factor-κB (NF-κB) regulates neuronal differentiation, plasticity and survival. It is well established that excitatory neurotransmitters such as glutamate control NF-κB activity. Glutamate receptor overactivation is also involved in ischemic- and seizure-induced neuronal injury and neurodegeneration. However, little is known at the single cell-level how NF-κB signaling relates to neuronal survival during excitotoxic injury. We found that silencing of p65/NF-κB delayed N -methyl- d -aspartate (NMDA)-induced excitotoxic injury in hippocampal neurons, suggesting a functional role of p65 in excitotoxicity. Time-lapse imaging of p65 and its inhibitor IκBα using GFP and Cerulean fusion proteins revealed specific patterns of excitotoxic NF-κB activation. Nuclear translocation of p65 began on average 8 ± 3 min following 15 min of NMDA treatment and was observed in up to two thirds of hippocampal neurons. Nuclear translocation of IκBα preceded that of p65 suggesting independent translocation processes. In surviving neurons, the onset of p65 nuclear export correlated with mitochondrial membrane potential recovery. Dying neurons exhibited persistent nuclear accumulation of p65-eGFP until plasma membrane permeabilization. Our data demonstrate an important role for p65 activation kinetics in neuronal cell death decisions following excitotoxic injury. [ABSTRACT FROM AUTHOR]

Copper has a long historical role in the arena of materials with antimicrobial properties. Various forms of copper ranging from surfaces to impregnation in textiles and particles, have attracted considerable interest owing to their versatility, potency, chemical stability, and low cost. However, the effects and mechanisms of their antimicrobial action is still unclear. In this study, the effect of copper particles on Escherichia coli was studied at the nanoscale using atomic force microscopy (AFM). Time-lapse AFM images at the single cell level show the morphological changes on live E. coli during antimicrobial treatment, in which for the first time, this process was followed in situ on the same cell over time. AFM-based hydrophobicity mapping further showed that incubating cells with Cu decreased the surface hydrophobicity with an increase of incubation time. Specifically, we are able to visualize both morphology and physico-chemical nature of the bacterial cell surface change in response to copper treatment, leading to the membrane damage and cytoplasm leakage. Overall, the time-lapse AFM imaging combined with hydrophobicity mapping approach presented here provides spatio-temporal insight into the antimicrobial mechanisms of copper at the single cell level, and can be applied to design of better metallic antimicrobial materials as well as investigate different microorganisms. [ABSTRACT FROM AUTHOR]

We are developing a label-free nanoscale photoacoustic tomography (nPAT) for imaging a single living cell. nPAT uses a laser-induced acoustic pulse to generate a nanometer-scale image. The primary motivation behind this imaging technique is the imaging of biological cells in the context of diagnosis without fluorescent tagging. During this procedure, thermal damage due to the laser pulse is a potential risk that may damage the cells. A physical model is built to estimate the temperature rise and thermal relaxation during the imaging procedure. Through simulations using finite element methods, two lasers (532 nm at 5 ps pulse duration and 830 nm at 0.2 ps pulse duration) were simulated for imaging red blood cells (RBCs). We demonstrate that a single 5-ps pulse laser with a 400-Hz repetition rate will generate a steady state temperature rise of less than a Kelvin on the surface of the RBCs. All the simulation results show that there is no significant temperature rise in an RBC in either single pulse or multiple pulse illumination with a 532-nm laser with 219 W fluence. Therefore, our simulation results demonstrate the thermal safety of an nPAT system. The photoacoustic signal generated by this laser is on the order of 2.5 kPa, so it should still be large enough to generate high-resolution images with nPAT. Frequency analysis of this signal shows a peak at 1.47 GHz, with frequencies as high as 3.5 GHz still being present in the spectrum. We believe that nPAT will open an avenue for disease diagnosis and cell biology studies at the nanometer-level. [ABSTRACT FROM AUTHOR]

[Display omitted] • Lipid and protein bound water detected in cells with Raman spectroscopy. • Water is absent in lipid droplet/particle domains containing cholesterol. • Bound water distribution matches the corresponding biomolecule distributio. Lipids, proteins, and nucleic acids have closely associated water molecules (Bound water), which exhibit considerably different physical properties compared to bulk water. Here we investigate the possibility of resolving Raman spectra of the specific hydration shell of these biomolecules in intracellular regions using Raman imaging. Lipids and proteins + nucleic acids Raman spectral components resolved in the analysis showed associated water spectral features, which are uniquely different from that of bulk water. These spectral profiles agree with water spectral profile observed in the case of corresponding hydrated pure biomolecules. The results show the prospects of Raman imaging in examining intracellular hydration in biomolecules and its functional relation. [ABSTRACT FROM AUTHOR]

ABSTRACT The effect of Calotropis gigantea latex (CGLX) on human mammary carcinoma cells is not well established. We present the results of this drug activity at total population and single cell level. CGLX inhibited the growth of MCF7 cancer cells at lower IC50 concentration (17 µL/mL). Microscopy of IC50 drug treated cells at 24 hr confirming the appearance of morphological characteristics of apoptotic and necrotic cells, associated with 70% of DNA damage. FACS analysis confirmed that, 10 and 20% of the disruption of cellular mitochondrial nature by at 24 and 48 h, respectively. Microscopic image analysis of total population level proved that MMP changes were statistically significant with P values. The cell to cell variation was confirmed by functional heterogeneity analysis which proves that CGLX was able to induce the apoptosis without the contribution of mitochondria. We conclude that CGLX inhibits cell proliferation, survival, and heterogeneity of pathways in human mammary carcinoma cells. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 724-734, 2015. [ABSTRACT FROM AUTHOR]

Synchrotron X-ray fluorescence microscopy (SXRF) presents a valuable opportunity to study the metallome of single cells because it simultaneously provides high-resolution subcellular distribution and quantitative cellular content of multiple elements. Different sample preparation techniques have been used to preserve cells for observations with SXRF, with a goal to maintain fidelity of the cellular metallome. In this case study, mouse pancreatic beta-cells have been preserved with optimized chemical fixation. We show that cell-to-cell variability is normal in the metallome of beta-cells due to heterogeneity and should be considered when interpreting SXRF data. In addition, we determined the impact of several immunofluorescence (IF) protocols on metal distribution and quantification in chemically fixed beta-cells and found that the metallome of beta-cells was not well preserved for quantitative analysis. However, zinc and iron qualitative analysis could be performed after IF with certain limitations. To help minimize metal loss using samples that require IF, we describe a novel IF protocol that can be used with chemically fixed cells after the completion of SXRF., (© The Author(s) 2023. Published by Oxford University Press.)

Multiplexed, phenotypic, intravital cytometric imaging requires novel fluorophore conjugates that have an appropriate size for long circulation and diffusion and show virtually no nonspecific binding to cells/serum while binding to cells of interest with high specificity. In addition, these conjugates must be stable and maintain a high quantum yield in the in vivo environments. Here, we show that this can be achieved using compact (~15 nm in hydrodynamic diameter) and biocompatible quantum dot (QD) -Ab conjugates. We developed these conjugates by coupling whole mAbs to QDs coated with norbornene-displaying polyimidazole ligands using tetrazine-norbornene cycloaddition. Our QD immunoconstructs were used for in vivo single-cell labeling in bone marrow. The intravital imaging studies using a chronic calvarial bone window showed that our QD-Ab conjugates diffuse into the entire bone marrow and efficiently label single cells belonging to rare populations of hematopoietic stem and progenitor cells (Sca1+c-Kit+ cells). This in vivo cytometric technique may be useful in a wide range of structural and functional imaging to study the interactions between cells and between a cell and its environment in intact and diseased tissues. [ABSTRACT FROM AUTHOR]

Abstract: The Centre for Ion Beam Applications, National University of Singapore has recently expanded from three state-of-the-art beam lines to five. Two new beam lines have been constructed: A second generation proton beam writing line, and a high resolution single cell imaging facility. Both systems feature high demagnification lens systems based on compact Oxford Microbeams OM52 lenses, coupled with reduced lens/image distances. The single cell imaging facility is designed around OM52 compact lenses capable of operating in a variety of high demagnification configurations including the spaced Oxford triplet and the double crossover Russian quadruplet. The new facility has design specifications aimed at spatial resolutions below 50nm, with a variety of techniques including STIM, secondary electron and fluorescence imaging, and an in-built optical and fluorescence microscope for sample imaging, identification and positioning. Preliminary tests using the single space Oxford triplet configuration have indicated a beam spot size of 31×39nm in the horizontal and vertical directions respectively, at beam currents of ∼10,000 protons per second. However, a weakness in the specifications of the electrostatic scanning system has been identified, and a more stable scanning system needs to be implemented before we can fully realize the optimum performance. A single whole fibroblast cell has been scanned using 1.5MeV protons, and a median fit to the proton transmission energy loss data has shown that proton STIM gives excellent details of the cell structure despite the relatively poor contrast of proton STIM compared with alpha STIM. [Copyright &y& Elsevier]

We have designed and constructed fusion genes of C-terminal (Ct) or N-terminal (Nt) bmrA with EGFP vectors and successfully expressed them in ΔBmrA (BmrA deletion strain of Bacillus subtilis), generating two new strains of B. subtilis (Ct-BmrA-EGFP and Nt-BmrA-EGFP). The fusion genes were characterized using gel electrophoresis and DNA sequencing. Their expression in live cells was determined by measuring the fluorescence of EGFP in single live cells using fluorescence microscopy and spectroscopy. The efflux function of the new strains was studied by measuring their accumulation kinetics of intracellular Hoechst dye molecules (a pump substrate) using fluorescence spectroscopy, which were compared with wild-type (WT-BmrA) and ΔBmrA strains. Both new strains show lower accumulation rates than ΔBmrA, and their efflux kinetics are inhibited by a pump inhibitor (orthovanadate). The results suggest that both strains extrude the dye molecules and the fusion proteins retain the efflux function of BmrA (ATP-binding cassette, ABC, transporter). Notably, Nt-BmrA-EGFP strain shows lower accumulation rates (higher efflux rates) than Ct-BmrA-EGFP. Modeled structures of the fusion proteins illustrate a highly flexible linker region connecting EGFP with BmrA, suggesting a minimal obstruction of EGFP to the BmrA. A closer distance of two C termini (∼14 Å) than two N termini (47.9 Å) of the 'closed' BmrA dimer depicts the larger steric effect of C-terminal fusion. This study also shows that glucose affects the fluorescence study of efflux function of BmrA, suggesting that efflux kinetics of ABC membrane transporters in live cells must be characterized in the absence of glucose. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Abstract: Real time imaging of living cell activation is an increasing demand in disciplines of life science and medicine. We previously reported that surface plasmon resonance (SPR) sensors detect large changes of refractive index with living cells, such as mast cells, keratinocyte, human basophils and B-cells activated by biological stimuli. However, conventional SPR sensors detect only an average change of refractive index with thousands of cells at detectable area on a sensor chip. In this study, we developed an SPR imaging (SPRI) sensor with a CMOS camera and an objective lens in order to analyze refractive index of individual living cells and their changes upon stimuli. The SPRI sensor could detect reactions of individual rat basophilic leukemia (RBL-2H3) cells, mouse keratinocyte (PAM212) cells, and human epidermal carcinoma (A431) cells in response to either specific or non-specific stimuli, such as antigen, phorbol ester or epidermal growth factor, with or without their inhibitors, resembling signals obtained by a conventional SPR sensor. Moreover, we distinguished reactions of different type cells, co-cultured on a sensor chip, and revealed that the increase of refractive index around nuclei is rapid and potent as compared to that in peripheries in the reaction of RBL-2H3 cells against antigen. This system may be a useful tool to investigate the mechanism of refractive index-changes evoked in near-membrane fields of living cells, and to develop a system of high-throughput screening for clinical diagnosis. [ABSTRACT FROM AUTHOR]

Currently, molecular mechanisms of multidrug ABC (ATP-binding cassette) membrane transporters remain elusive. In this study, we synthesized and characterized purified spherically shaped silver nanoparticles (Ag NPs) (11.8 ± 2.6 nm in diameter), which were stable (non-aggregation) in PBS buffer and inside single living cells. We used the size-dependent localized surface plasmon resonance (LSPR) spectra of single Ag NPs to determine their sizes and to probe the size-dependent transport kinetics of the ABC (BmrA, BmrA-EGFP) transporters in single living cells (Bacillus subtilis) in real time at nanometer resolution using dark-field optical microscopy and spectroscopy (DFOMS). The results show that the smaller NPs stayed longer inside the cells than larger NPs, suggesting size-dependent efflux kinetics of the membrane transporter. Notably, accumulation and efflux kinetics of intracellular NPs for single living cells depended upon the cellular expression level of BmrA, NP concentrations, and a pump inhibitor (25 μM, orthovanadate), suggesting that NPs are substrates of BmrA transporters and that passive diffusion driven by concentration gradients is the primary mechanism by which the NPs enter the cells. The accumulation and efflux kinetics of intracellular NPs for given cells are similar to those observed using a substrate (Hoechst dye) of BmrA, demonstrating that NPs are suitable probes for study of multidrug membrane transporters of single living cells in real-time. Unlike fluorescent probes, single Ag NPs exibit size-dependent LSPR spectra and superior photostability, enabling them to probe the size-dependent efflux kinetics of membrane transporters of single living cells in real-time for better understanding of multidrug resistance. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

The toxicology of nanomaterials is a blooming field of study, yet it is difficult to keep pace with the innovations in new materials and material applications. Those applications are quickly being introduced in research, industrial, and consumer settings. Even though the cytotoxicity of many types of nanoparticles has been demonstrated, the behavior of those particles in a biological environment is not yet fully known. This work characterized the following over time: protein adsorption on silica particle surfaces, the internalization of particles in human lung carcinoma (A549) cells when coated with different specific proteins or no proteins at all, and the cellular loss of particles following the removal of extracellular particles. Proteins were shown to quickly saturate the particle surface, followed by a competitive process of particle agglomeration and protein adsorption. Uptake of particles peaked at 8–10 h, and it was determined that, in this system, the charge of the protein-coated particles changed the rate of uptake if the charge difference was great enough. Cells internalized particles lacking any adsorbed proteins with approximately 3 times the rate of protein-coated particles with the same charge. Although particles exited cells over time, the process was slower than uptake and did not near completion within 24 h. Finally, analysis at the single cell level afforded observations of particle agglomerates loosely associated with cell membranes when serum was present in the culture medium, but in the absence of serum, particles adhered to the dish floor and formed smaller agglomerates on cell surfaces. Although data trends were easily distinguished, all samples showed considerable variation from cell to cell. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Abstract: Scanning transmission ion microscopy (STIM) is a technique which utilizes the energy loss of high energy (MeV) ions passing through a sample to provide structural images. In this paper, we have successfully demonstrated STIM imaging of single cells at the nano-level using the high resolution capability of the proton beam writing facility at the Centre for Ion Beam Applications, National University of Singapore. MCF-7 breast cancer cells (American Type Culture Collection [ATCC]) were seeded on to silicon nitride windows, backed by a Hamamatsu pin diode acting as a particle detector. A reasonable contrast was obtained using 1MeV protons and excellent contrast obtained using 1MeV alpha particles. In a further experiment, nano-STIM was also demonstrated using cells seeded on to the pin diode directly, and high quality nano-STIM images showing the nucleus and multiple nucleoli were extracted before the detector was significantly damaged. [Copyright &y& Elsevier]

Cultured human and rat endothelial cells were used to study cellular toxicity and Ca2+ signalling upon exposure to reactive oxygen species. Superoxide and hydrogen peroxide (O2·–/H2O2) were produced by the hypoxanthine/xanthine oxidase system (HX/XO) and caused intracellular Ca2+ concentration ([Ca2+]i) to rise steadily when activities above 2 mU/ml were used. These Ca2+ increases were also measured when the glucose/glucose oxidase (G/GO) system above 5 mU/ml was used to produce hydrogen peroxide (H2O2). Gross morphological changes appeared to parallel elevated [Ca2+]i levels preceding cell death. However, when HX/XO or G/GO were used at non toxic doses rapid and transient changes in [Ca2+]i were measured. These treatments did not alter subsequent receptor mediated Ca2+ signalling induced by ATP (10 μM) or histamine (100 μM). Superoxide dismutase (50 U/ml), which dismutates O2·minus; into H2O2 al ient [Ca2+]i responses. H2O2 added directly was able to induce similar Ca2+ transients when concentrations of at least 500 μM were used. Buffering trace amounts of iron (o-phenanthroline; 200 μM) in order to inhibit úOH radical formation was not effective to alter Ca2+ changes. Experiments performed in Ca2+-free buffer showed a similar rise in [Ca2+]i and readdition of Ca2+ to the extracellular medium indicated the activation of store operated Ca2+ entry. Blocking Ca2+-ATPases of the endoplasmatic reticulum with thapsigargin (1 μM) inhibited ROS induced transient increases and cells preincubated with pertussis toxin (200 nM) showed unchanged Ca2+ transients after exposure to both enzyme systems. Phospholipase C inhibitor U73122 (2 μM) effectively reduced hydrogen peroxide induced emptying of intracellular stores. Taken together, we demonstrate that enzymatically produced non-toxic H2O2 rather than O· ndash; or · OH causes calcium signalling from thapsigargin sensitive stores, and activates store operated Ca2+ entry at least partially by activating phospholipase C. These changes clearly differ from pathological ‘oxidative stress’ associated with a progressive increase in [Ca2+]i. [ABSTRACT FROM AUTHOR]