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Natural hydrogen (H 2) emanations in intracratonic areas offer potentially exploitable carbon-free energy. To date, H 2 seepages have been detected in more than sixty sites with exploration ongoing in many locations. One mechanism of natural hydrogen generation is the oxidation of Fe2+ in Fe-rich lithologies, and estimating the potential for hydrogen generation by this pathway is an important aspect of characterizing H 2 -generating rocks. However, accurate estimation of Fe2+ can be challenging due to large-scale heterogeneities and small sample sizes used in conventional analysis. Here, we propose a correlative imaging technique to assess H 2 generation potential in Fe2+-rich source rocks by integrating 2D chemical information with 3D volumes of the rock imaged using X-ray computed tomography (micro-CT). The advantage of this method lies in its ability to analyze a whole drill core of the source rock to obtain the most representative values while preserving sample integrity. Our method, validated on fractured monzo-diorite from a natural H 2 -emitting well in Kansas, USA, yields an estimate of 707.93 ± 49.18 mol (H 2)/ton (source rock), as the upper limit. The proposed method could be useful in characterizing source rocks and estimating their natural H 2 generation potential in the early stages of natural H 2 exploration. • We propose a method to quantify Fe2+ in a H 2 source rock by an imaging technique. • Upper limit of H 2 generation in mols (H 2)/ton (rock) was obtained assuming that all Fe2+ reacted to generate H 2. • Method allows imaging large representative samples such as drill cores (1 m). • Ensures minimal sample destruction and high accuracy. [ABSTRACT FROM AUTHOR]

Ultrafast electron microscopy (UEM) has a broad scope of application across material systems and scientific disciplines. In UEM, we investigate multiscale dynamics in the spatial domain ranging from micrometres to ångströms, in reciprocal space, and on timescales from microseconds to attoseconds, with an energy resolution of a few electronvolts or less. Notably, UEM has played a pivotal role in visualisation of ultrafast structural dynamics with high local selectivity, enabling the exploration of the dynamic nature of chemical bonding in non-equilibrium states and investigation of electron – photon interactions to manipulate free-electron wavefunctions. This breakthrough has created new opportunities in condensed matter physics, chemical dynamics, and quantum electrodynamics. In addition, efforts are underway to achieve high-resolution UEM in correlative real-space microscopy to diffractography and spectroscopy in a single instrument for comprehensive investigations of light – matter interactions and structure – dynamics – function relations. In this review, we provide an overview of the current state of UEM imaging capabilities and scientific interests, outlining the technological challenges faced by UEM in related fields and exploring potential approaches to overcome these challenges. Furthermore, we highlight the emerging fields of interest and present future perspectives that can further extend the imaging capabilities of UEM.

Despite extensive research efforts, cancer continues to stand as one of the leading causes of death on a global scale. To gain profound insights into the intricate mechanisms underlying cancer onset and progression, it is imperative to possess methodologies that allow the study of cancer cells at the single-cell level, focusing on critical parameters such as cell morphology, metabolism, and molecular characteristics. These insights are essential for effectively discerning between healthy and cancerous cells and comprehending tumoral progression. Recent advancements in microscopy techniques have significantly advanced the study of cancer cells, with Raman microspectroscopy (RM) emerging as a particularly powerful tool. Indeed, RM can provide both biochemical and spatial details at the single-cell level without the need for labels or causing disruptions to cell integrity. Moreover, RM can be correlated with other microscopy techniques, creating a synergy that offers a spectrum of complementary insights into cancer cell morphology and biology. This review aims to explore the correlation between RM and other microscopy techniques such as confocal fluoresce microscopy (CFM), atomic force microscopy (AFM), digital holography microscopy (DHM), and mass spectrometry imaging (MSI). Each of these techniques has their own strengths, providing different perspectives and parameters about cancer cell features. The correlation between information from these various analysis methods is a valuable tool for physicians and researchers, aiding in the comprehension of cancer cell morphology and biology, unraveling mechanisms underlying cancer progression, and facilitating the development of early diagnosis and/or monitoring cancer progression. [ABSTRACT FROM AUTHOR]

The experimental limitations with optics observed in many microscopy and astronomy instruments result in detrimental effects for the imaging of objects. This can be generally described mathematically as a convolution of the real object image with the point spread function that characterizes the optical system. The popular Richardson-Lucy (RL) deconvolution algorithm is widely used for the inverse process of restoring the data without these optical aberrations, often a critical step in data processing of experimental data. Here we present the versatile RedLionfish python package, that was written to make the RL deconvolution of volumetric (3D) data easier to run, very fast (by exploiting GPU computing capabilities) and with automatic handling of hardware limitations for large datasets. It can be used programmatically in Python/numpy using conda or PyPi package managers, or with a graphical user interface as a napari plugin.

Non-junctional connexin43 (Cx43) plasma membrane hemichannels have been implicated in several inflammatory diseases, particularly playing a role in ATP release that triggers activation of the inflammasome. Therapies targeting the blocking of the hemichannels to prevent the pathological release or uptake of ions and signalling molecules through its pores are of therapeutic interest. To date, there is no close-to-native, high-definition documentation of the impact of Cx43 hemichannel-mediated inflammation on cellular ultrastructure, neither is there a robust account of the ultrastructural changes that occur following treatment with selective Cx43 hemichannel blockers such as Xentry-Gap19 (XG19).A combination of same-sample correlative high-resolution three-dimensional fluorescence microscopy and soft X-ray tomography at cryogenic temperatures, enabled in the identification of novel 3D molecular interactions within the cellular milieu when comparing behaviour in healthy states and during the early onset or late stages under inflammatory conditions. Notably, our findings suggest that XG19 blockage of connexin hemichannels under pro-inflammatory conditions may be crucial in preventing the direct degradation of connexosomes by lysosomes, without affecting connexin protein translation and trafficking. We also delineated fine and gross cellular phenotypes, characteristic of inflammatory insult or road-to-recovery from inflammation, where XG19 could indirectly prevent and reverse inflammatory cytokine-induced mitochondrial swelling and cellular hypertrophy through its action on Cx43 hemichannels. Our findings suggest that XG19 might have prophylactic and therapeutic effects on the inflammatory response, in line with functional studies.

A correlative methodology for label-free chemical imaging of soft tissue has been developed, combining non-linear optical spectroscopies and mass spectrometry to achieve sub-micron spatial resolution and critically improved drug detection sensitivity. The approach was applied to visualise the kinetics of drug reservoir formation within human skin following in vitro topical treatment with a commercial diclofenac gel. Non-destructive optical spectroscopic techniques, namely stimulated Raman scattering, second harmonic generation and two photon fluorescence microscopies, were used to provide chemical and structural contrast. The same tissue sections were subsequently analysed by secondary ion mass spectrometry, which offered higher sensitivity for diclofenac detection throughout the epidermis and dermis. A method was developed to combine the optical and mass spectrometric datasets using image registration techniques. The label-free, high-resolution visualisation of tissue structure coupled with sensitive chemical detection offers a powerful method for drug biodistribution studies in the skin that impact directly on topical pharmaceutical product development. Correlative label-free chemical imaging combining non-linear optical spectroscopies and mass spectrometry enables sensitive drug detection with sub-micron spatial resolution. [Display omitted] [ABSTRACT FROM AUTHOR]

Despite extensive research efforts, cancer continues to stand as one of the leading causes of death on a global scale. To gain profound insights into the intricate mechanisms underlying cancer onset and progression, it is imperative to possess methodologies that allow the study of cancer cells at the single-cell level, focusing on critical parameters such as cell morphology, metabolism, and molecular characteristics. These insights are essential for effectively discerning between healthy and cancerous cells and comprehending tumoral progression. Recent advancements in microscopy techniques have significantly advanced the study of cancer cells, with Raman microspectroscopy (RM) emerging as a particularly powerful tool. Indeed, RM can provide both biochemical and spatial details at the single-cell level without the need for labels or causing disruptions to cell integrity. Moreover, RM can be correlated with other microscopy techniques, creating a synergy that offers a spectrum of complementary insights into cancer cell morphology and biology. This review aims to explore the correlation between RM and other microscopy techniques such as confocal fluoresce microscopy (CFM), atomic force microscopy (AFM), digital holography microscopy (DHM), and mass spectrometry imaging (MSI). Each of these techniques has their own strengths, providing different perspectives and parameters about cancer cell features. The correlation between information from these various analysis methods is a valuable tool for physicians and researchers, aiding in the comprehension of cancer cell morphology and biology, unraveling mechanisms underlying cancer progression, and facilitating the development of early diagnosis and/or monitoring cancer progression.

The correlative imaging workflow is a method of combining information and data across modes (e.g. SEM, X‐ray CT, FIB‐SEM), scales (cm to nm) and dimensions (2D–3D–4D), providing a more holistic interpretation of the research question. Often, subsurface objects of interest (e.g. inclusions, pores, cracks, defects in multilayered samples) are identified from initial exploratory nondestructive 3D tomographic imaging (e.g. X‐ray CT, XRM), and those objects need to be studied using additional techniques to obtain, for example, 2D chemical or crystallographic data. Consequently, an intermediate sample preparation step needs to be completed, where a targeted amount of sample surface material is removed, exposing and revealing the object of interest. At present, there is not one singular technique for removing varied thicknesses at high resolution and on a range of scales from cm to nm. Here, we review the manual and automated options currently available for targeted sample material removal, with a focus on those methods which are readily accessible in most laboratories. We summarise the approaches for manual grinding and polishing, automated grinding and polishing, microtome/ultramicrotome, and broad‐beam ion milling (BBIM), with further review of other more specialist techniques including serial block face electron microscopy (SBF‐SEM), and ion milling and laser approaches such as FIB‐SEM, Xe plasma FIB‐SEM, and femtosecond laser/LaserFIB. We also address factors which may influence the decision on a particular technique, including the composition, shape and size of the samples, sample mounting limitations, the amount of surface material to be removed, the accuracy and/or resolution of peripheral parts, the accuracy and/or resolution of the technique/instrumentation, and other more general factors such as accessibility to instrumentation, costs, and the time taken for experimentation. It is hoped that this study will provide researchers with a range of options for removal of specific amounts of sample surface material to reach subsurface objects of interest in both correlative and non‐correlative workflows. [ABSTRACT FROM AUTHOR]

The colocation of elemental species with host biomolecules such as lipids and metabolites may shed new light on the dysregulation of metabolic pathways and how these affect disease pathogeneses. Alkali metals have been the subject of extensive research, are implicated in various neurodegenerative and infectious diseases and are known to disrupt lipid metabolism. Desorption electrospray ionisation (DESI) is a widely used approach for molecular imaging, but previous work has shown that DESI delocalises ions such as potassium (K) and chlorine (Cl), precluding the subsequent elemental analysis of the same section of tissue. The solvent typically used for the DESI electrospray is a combination of methanol and water. Here we show that a novel solvent system, (50:50 (%v/v) MeOH:EtOH) does not delocalise elemental species and thus enables elemental mapping to be performed on the same tissue section post-DESI. Benchmarking the MeOH:EtOH electrospray solvent against the widely used MeOH:H2O electrospray solvent revealed that the MeOH:EtOH solvent yielded increased signal-to-noise ratios for selected lipids. The developed multimodal imaging workflow was applied to a lung tissue section containing a tuberculosis granuloma, showcasing its applicability to elementally rich samples displaying defined structural information. [ABSTRACT FROM AUTHOR]

• The δ11B of coralline algae skeleton offer a means to reconstruct coastal pH. • 2D maps of trace element content and δ11B were collected on Boreolithothamniom cf. soriferum. • These images were aligned using correlated multimodal imaging techniques. • We reconstruct an ocean acidification trend of −0.018 pH units yr-1 in Loch Sween. • Loch Sween switched from being a substantial sink of CO 2 to a source in ∼2008. Ocean Acidification (OA) arises from the increase in atmospheric carbon dioxide concentration following the industrial revolution. The ecological and socio-economic consequences of OA were first identified around 10–15 years ago but remain poorly understood. This is particularly true in coastal regions where local processes can have dramatic consequences on pH trends through time, obscuring and compounding the long-term effects from rising atmospheric CO 2. Here we explore the possibility of generating long records of coastal ocean pH using the skeletons of widely distributed coralline algae (CA). The skeletons of these slow growing (<1 mm/year) taxa often contain micron-scale heterogeneities, making sampling for high-resolution climate reconstructions using bulk sampling techniques difficult. Here we use laser ablation coupled to inductively coupled plasma mass spectrometers to generate high-resolution 2D images of the element/calcium ratios and boron isotope composition (δ11B) of a sample of Boreolithothamniom cf. soriferum from Loch Sween in Scotland, UK where we have been monitoring temperature since 2004 and pH during 2014. By carefully correlating the geochemical images with a scanning electron microscopy image we can segment them to remove the marginal portions of the skeleton, isolating the central growth axis to generate an age model and growth rate. The δ11B-pH is significantly elevated above the seawater pH in Loch Sween (8.4 to 8.9 vs. 7.9 to 8.1) consistent with other CA that show internal pH elevation. On a seasonal scale, internal pH is negatively correlated with temperature and also exhibits a long-term decline. By removing this temperature effect, internal pH can be correlated to seawater pH during the 2014 monitoring period allowing us to reconstruct a seawater acidification trend from 2004 to 2018 of -0.018 pH units per year, 10x higher than open ocean trends but consistent with contemporaneous monitoring efforts of UK coastal waters. Reconstructed aqueous CO 2 suggests that prior to ∼2008 Loch Sween was a sink of CO 2 but after this date, particularly during the early summer, it was a substantial CO 2 source. Comparison of reconstructed aqueous CO 2 with a record of calcification rate of our sample of Boreolithothamniom cf. soriferum suggests this acidification and associated rise in local seawater pCO 2 may have freed this sample from carbon limitation leading to a recent increase in calcification. [ABSTRACT FROM AUTHOR]

The ability to determine the precise structure of nano-objects is essential for a multitude of applications. This is particularly true of single-walled carbon nanotubes (SWCNTs), which are produced as heterogeneous samples. Current techniques used for their characterization require sophisticated instrumentation, such as atomic force microscopy (AFM), or compromise on accuracy. In this paper, we propose to use super-resolution microscopy (SRM) to accurately determine the morphology (orientation, length, and shape) of individual luminescent SWCNTs. We generate super-resolved images using three recently published SRM analytical software packages (DPR, eSRRF, and MSSR) and metrologically compare their performances to determine the morphological properties of SWCNTs. For this, ground-truth information on nanotube morphologies was obtained using polarization measurements and AFM to directly correlate the results from SRM at the single particle level. We show a more than 4-fold improvement in resolution over standard photoluminescence imaging, revealing hidden morphologies as efficiently as AFM. We finally demonstrate that DPR, and eventually eSRRF, can effectively assess SWCNT length distribution in a much faster and more accessible way than AFM. We believe that this approach can be generalized to other types of luminescent nanostructures and thus become a standard for rapid and accurate characterization of samples.

The solvent casting method was used for five types of polyvinylidene difluoride (PVDF) nanocomposite film preparation. The effect of nanofillers in PVDF nanocomposite films on the structural, phase, and friction and mechanical properties was examined and compared with that of the natural PVDF film. The surface topography of PVDF nanocomposite films was investigated using a scanning electron microscope (SEM) and correlative imaging (CPEM, combinate AFM and SEM). A selection of 2D CPEM images was used for a detailed study of the spherulitic morphologies (grains size around 6–10 μm) and surface roughness (value of 50–68 nm). The chemical interactions were evaluated by Fourier transform infrared spectroscopy (FTIR). Dominant polar γ-phase in the original PVDF, PVDF_ZnO and PVDF_ZnO/V, the most stable non-polar α-phase in the PVDF_V_CH nanocomposite film and mixture of γ and α phases in the PVDF_V and PVDF_ZnO/V_CH nanocomposite films were confirmed. Moderately hydrophilic PVDF nanocomposite films with water contact angle values (WCA) in the range of 58°–69° showed surface stability with respect to the Zeta potential values. The effect of positive or negative Zeta-potential values of nanofillers (ζn) on the resulting negative Zeta-potential values (ζ) of PVDF nanocomposite films was demonstrated. Interaction of PVDF chains with hydroxy groups of vermiculite and amino and imino groups of CH caused transformation of γ-phase to α. The friction properties were evaluated based on the wear testing and mechanical properties were evaluated from the tensile tests based on Young's modulus (E) and tensile strength (Rm) values. Used nanofillers caused decreasing of friction and mechanical properties of PVDF nanocomposite material films. [ABSTRACT FROM AUTHOR]

Dental caries is a widespread disease that proceeds by damaging superficial tooth enamel by heterogeneous dissolution. Conventional histology identifies different zones within carious lesions by their optical appearance, but fails to quantify the underlying nanoscale structural changes as a function of specific location, impeding better understanding of the demineralisation process. We employ detailed collocative analysis using different imaging modalities, resolutions and fields of view. Focused ion beam-scanning electron microscopy (FIB-SEM) reveals subsurface 3D nanostructure within milled micro-sized volumes, whilst X-ray tomography allows minimally destructive 3D imaging over large volumes. Correlative combination of these techniques reveals fine detail of enamel rods, inter-rod substance, sheaths, crystallites and voids as a function of location. The degree of enamel demineralisation within the body of the lesion, near its front, and at the surface is visualized and quantified in 3D. We thus establish the paradigm of dental 3D nano-histology as an advanced platform for quantitative evaluation of caries-induced structural modification.

Cryogenic optical localization in three dimensions (COLD) was recently shown to resolve up to four binding sites on a single protein. However, because COLD relies on intensity fluctuations that result from the blinking behavior of fluorophores, it is limited to cases where individual emitters show different brightness. This significantly lowers the measurement yield. To extend the number of resolved sites as well as the measurement yield, we employ partial labeling and combine it with polarization encoding in order to identify single fluorophores during their stochastic blinking. We then use a particle classification scheme to identify and resolve heterogenous subsets and combine them to reconstruct the three-dimensional arrangement of large molecular complexes. We showcase this method (polarCOLD) by resolving the trimer arrangement of proliferating cell nuclear antigen (PCNA) and six different sites of the hexamer protein Caseinolytic Peptidase B (ClpB) of Thermus thermophilus in its quaternary structure, both with Angstrom resolution. The combination of polarCOLD and single-particle cryogenic electron microscopy (cryoEM) promises to provide crucial insight into intrinsic heterogeneities of biomolecular structures. Furthermore, our approach is fully compatible with fluorescent protein labeling and can, thus, be used in a wide range of studies in cell and membrane biology.

To scale up production of perovskite photovoltaics, state‐of‐the‐art laboratory recipes and processes must be transferred to large‐area coating and drying systems. The development of in situ monitoring methods that provide real‐time feedback for process control is pivotal to overcome this challenge. Herein, correlative in situ multichannel imaging (IMI) obtaining reflectance, photoluminescence intensity, and central photoluminescence emission wavelength images on areas larger than 100 cm2 with subsecond temporal resolution using a simple, cost‐effective setup is demonstrated. Installed on top of a drying channel with controllable laminar air flow and substrate temperature, IMI is shown to consistently monitor solution film drying, perovskite nucleation, and perovskite crystallization. If the processing parameters differ, IMI reveals characteristic changes in large‐area perovskite formation dynamics already before the final annealing step. Moreover, when IMI is used to study >130 blade‐coated devices processed at the same parameters, about 90% of low‐performing devices contain coating inhomogeneities detected by IMI. The results demonstrate that IMI should be of value for real‐time 2D monitoring and feedback control in industrial‐scale, high‐throughput fabrication such as roll‐to‐roll printing. [ABSTRACT FROM AUTHOR]

Information transfer at synapses occurs when vesicles fuse with the plasma membrane to release neurotransmitters, which then bind to receptors at the postsynaptic membrane. The process of neurotransmitter release varies dramatically between different synapses, but little is known about how this heterogeneity emerges. The development of super-resolution microscopy has revealed that synaptic proteins are precisely organised within and between the two parts of the synapse and that this precise spatiotemporal organisation fine-tunes neurotransmission. However, it remains unclear if variability in release probability could be attributed to the nanoscale organisation of one or several proteins of the release machinery. To begin to address this question, we have developed a pipeline for correlative functional and super-resolution microscopy, taking advantage of recent technological advancements enabling multicolour imaging. Here we demonstrate the combination of live imaging of SypHy-RGECO, a unique dual reporter that simultaneously measures presynaptic calcium influx and neurotransmitter release, with post hoc immunolabelling and multicolour single molecule localisation microscopy, to investigate the structure-function relationship at individual presynaptic boutons.

The colocation of elemental species with host biomolecules such as lipids and metabolites may shed new light on the dysregulation of metabolic pathways and how these affect disease pathogeneses. Alkali metals have been the subject of extensive research, are implicated in various neurodegenerative and infectious diseases and are known to disrupt lipid metabolism. Desorption electrospray ionisation (DESI) is a widely used approach for molecular imaging, but previous work has shown that DESI delocalises ions such as potassium (K) and chlorine (Cl), precluding the subsequent elemental analysis of the same section of tissue. The solvent typically used for the DESI electrospray is a combination of methanol and water. Here we show that a novel solvent system, (50:50 (%v/v) MeOH:EtOH) does not delocalise elemental species and thus enables elemental mapping to be performed on the same tissue section post-DESI. Benchmarking the MeOH:EtOH electrospray solvent against the widely used MeOH:H2O electrospray solvent revealed that the MeOH:EtOH solvent yielded increased signal-to-noise ratios for selected lipids. The developed multimodal imaging workflow was applied to a lung tissue section containing a tuberculosis granuloma, showcasing its applicability to elementally rich samples displaying defined structural information.

High-throughput dynamic imaging of cells and organelles is essential for understanding complex cellular responses. We report Mantis, a high-throughput 4D microscope that integrates two complementary, gentle, live-cell imaging technologies: remote-refocus label-free microscopy and oblique light-sheet fluorescence microscopy. Additionally, we report shrimPy (Smart High-throughput Robust Imaging and Measurement in Python), an open-source software for high-throughput imaging, deconvolution, and single-cell phenotyping of 4D data. Using Mantis and shrimPy, we achieved high-content correlative imaging of molecular dynamics and the physical architecture of 20 cell lines every 15 min over 7.5 h. This platform also facilitated detailed measurements of the impacts of viral infection on the architecture of host cells and host proteins. The Mantis platform can enable high-throughput profiling of intracellular dynamics, long-term imaging and analysis of cellular responses to perturbations, and live-cell optical screens to dissect gene regulatory networks., (© The Author(s) 2024. Published by Oxford University Press on behalf of National Academy of Sciences.)

In this study, we investigated the potential and limitations of using joint X‐ray and time‐of‐flight (TOF) neutron imaging for mapping the 3‐dimensional organic carbon distribution in soil. This approach is viable because neutron and X‐ray beams have complementary attenuation properties. Soil minerals consist to a large part of silicon and aluminium, and elements that are relatively translucent to neutrons but attenuate X‐rays. In contrast, attenuation of neutrons is strong for hydrogen, which is abundant in soil organic matter (SOM), while hydrogen barely attenuates X‐rays. In theory, TOF neutron imaging does further more allow the imaging of Bragg edges, which correspond to d‐spacings in minerals. This could help to distinguish between SOM and clay minerals, the mineral group in soil that is most strongly associated with hydrogen atoms. We collected TOF neutron image data at the IMAT beamline at the ISIS facility and synchrotron X‐ray image data at the I12 beamline at the Diamond Light source, both located within the Rutherford Appleton Laboratory, Harwell, UK. The white beam (the full energy spectrum) neutron image clearly showed variations in neutron attenuation within soil aggregates at approximately constant X‐ray attenuations. This indicates a constant bulk density with varying organic matter and/or clay content. Unfortunately, the combination of TOF neutron and X‐ray imaging was not suited to allow for a distinction between SOM and clay minerals at the voxel scale. While such a distinction is possible in theory, it is prevented by technical limitations. One of the main reasons is that the neutron frequencies available at modern neutron sources are too large to capture the main d‐spacings of clay minerals. As a result, inference to voxel scale SOM concentrations is presently not feasible. Future improved neutron sources and advanced detector designs will eventually overcome the technical problems encountered here. On the positive side, combined X‐ray and TOF neutron imaging demonstrated abilities to identify quartz grains and to distinguish between plastics and plant seeds. Highlights: Full understanding of biogeochemical processes requires three‐dimensional (3‐D) maps of organic matter in soil (SOM).This study investigates a novel method to map voxel‐scale SOM contents with 3‐D resolution.The method is based a combination of X‐ray and time‐of‐flight neutron tomography.At present, technical limitations prevent distinguishing between SOM and clay mineral contents.More advanced neutron sources are required to overcome the encountered technical obstacles. [ABSTRACT FROM AUTHOR]

Tumor vasculature and angiogenesis play a crucial role in tumor progression. Their visualization is therefore of utmost importance to the community. In this proof-of-principle study, we have established a novel cross-modality imaging (CMI) pipeline to characterize exactly the same murine tumors across scales and penetration depths, using orthotopic models of melanoma cancer. This allowed the acquisition of a comprehensive set of vascular parameters for a single tumor. The workflow visualizes capillaries at different length scales, puts them into the context of the overall tumor vessel network and allows quantification and comparison of vessel densities and morphologies by different modalities. The workflow adds information about hypoxia and blood flow rates. The CMI approach includes well-established technologies such as magnetic resonance imaging (MRI), positron emission tomography (PET), computed tomography (CT), and ultrasound (US), and modalities that are recent entrants into preclinical discovery such as optical coherence tomography (OCT) and high-resolution episcopic microscopy (HREM). This novel CMI platform establishes the feasibility of combining these technologies using an extensive image processing pipeline. Despite the challenges pertaining to the integration of microscopic and macroscopic data across spatial resolutions, we also established an open-source pipeline for the semi-automated co-registration of the diverse multiscale datasets, which enables truly correlative vascular imaging. Although focused on tumor vasculature, our CMI platform can be used to tackle a multitude of research questions in cancer biology. [ABSTRACT FROM AUTHOR]

The purpose of this work is to develop an integrated imaging approach to characterize without labeling at the sub-cellular level the formation of lipid body droplets (LBs) in microalgae undergoing nitrogen starvation. First conventional optical microscopy approaches, gas chromatography, and turbidimetry measurements allowed to monitor the biomass and the total lipid content in the oleaginous microalgae Parachlorella kesslerii during the starvation process. Then a local analysis of the LBs was proposed using an innovative infrared nanospectroscopy technique called atomic force microscopy-based infrared spectroscopy (AFM-IR). This label-free technique assessed the formation of LBs and allowed to look into the LB composition thanks to the acquisition of local infrared spectra. Last correlative measurements using fluorescence microscopy and AFM-IR were performed to investigate the subcellular reorganization of LB and the chloroplasts. [ABSTRACT FROM AUTHOR]

Imaging of actin filaments is crucial due to the integral role that they play in many cellular functions such as intracellular transport, membrane remodelling and cell motility. Visualizing actin filaments has so far relied on fluorescence microscopy and electron microscopy/tomography. The former lacks the capacity to capture the overall local ultrastructure, while the latter requires rigorous sample preparation that can lead to potential artefacts, and only delivers relatively small volumes of imaging data at the thinnest areas of a cell. In this work, a correlative approach utilizing in situ super‐resolution fluorescence imaging and cryo X‐ray tomography was used to image bundles of actin filaments deep inside cells under near‐native conditions. In this case, fluorescence 3D imaging localized the actin bundles within the intracellular space, while X‐ray tomograms of the same areas provided detailed views of the local ultrastructure. Using this new approach, actin trails connecting vesicles in the perinuclear area and hotspots of actin presence within and around multivesicular bodies were observed. The characteristic prevalence of filamentous actin in cytoplasmic extensions was also documented. [ABSTRACT FROM AUTHOR]

Splenosis, commonly occurs incidentally and locates to bowel surfaces, parietal peritoneum, mesentery, and diaphragm, but can potentially occur anywhere in the peritoneal cavity. Patients frequently have a history of splenectomy or trauma. On the other hand, hepatic splenosis is a rare entity and may present itself clinically. Indeterminate liver lesions can pose a clinical dilemma and may lead to additional investigations, anxiety, follow-up imaging and even to invasive procedures. MRI usually performs extremely well. In difficult cases, scintigraphy can be of great value -especially with novel SPECT-CT and SPECT-MR techniques-. We describe a case of a 29-year-old lady with hepatic splenosis and the impact of hybrid imaging. [ABSTRACT FROM AUTHOR]

Micro-computed tomography (µCT) provides non-destructive three-dimensional (3D) imaging of soft tissue microstructures. Specific features in µCT images can be identified using correlated two-dimensional (2D) histology images allowing manual segmentation. However, this is very time-consuming and requires specialist knowledge of the tissue and imaging modalities involved. Using a custom-designed µCT system optimized for imaging unstained formalin-fixed paraffin-embedded soft tissues, we imaged human lung tissue at isotropic voxel sizes less than 10 µm. Tissue sections were stained with haematoxylin and eosin or cytokeratin 18 in columnar airway epithelial cells using immunofluorescence (IF), as an exemplar of this workflow. Novel utilization of tissue autofluorescence allowed automatic alignment of 2D microscopy images to the 3D µCT data using scripted co-registration and automated image warping algorithms. Warped IF images, which were accurately aligned with the µCT datasets, allowed 3D segmentation of immunoreactive tissue microstructures in the human lung. Blood vessels were segmented semi-automatically using the co-registered µCT datasets. Correlating 2D IF and 3D µCT data enables accurate identification, localization and segmentation of features in fixed soft lung tissue. Our novel correlative imaging workflow provides faster and more automated 3D segmentation of µCT datasets. This is applicable to the huge range of formalin-fixed paraffin-embedded tissues held in biobanks and archives.

Structural and Doppler velocity data collected from optical coherence tomography have already provided crucial insights into cardiac morphogenesis. X-ray microtomography and other ex vivo methods have elucidated structural details of developing hearts. However, by itself, no single imaging modality can provide comprehensive information allowing to fully decipher the inner workings of an entire developing organ. Hence, we introduce a specimen-specific correlative multimodal imaging workflow combining OCT and micro-CT imaging which is applicable for modeling of early chick heart development—a valuable model organism in cardiovascular development research. The image acquisition and processing employ common reagents, lab-based micro-CT imaging, and software that is free for academic use. Our goal is to provide a step-by-step guide on how to implement this workflow and to demonstrate why those two modalities together have the potential to provide new insight into normal cardiac development and heart malformations leading to congenital heart disease.

• The imaging protocol visualises intracellular calcium signals and their sources. • It correlates calcium spark TIRF images with DNA-PAINT images of RyR channels. • It is a powerful method for studying nanoscale structure-function relationship. Nanometre-scale cellular information obtained through super-resolution microscopies are often unaccompanied by functional information, particularly transient and diffusible signals through which life is orchestrated in the nano-micrometre spatial scale. We describe a correlative imaging protocol which allows the ubiquitous intracellular second messenger, calcium (Ca2+), to be directly visualised against nanoscale patterns of the ryanodine receptor (RyR) Ca2+ channels which give rise to these Ca2+ signals in wildtype primary cells. This was achieved by combining total internal reflection fluorescence (TIRF) imaging of the elementary Ca2+ signals, with the subsequent DNA-PAINT imaging of the RyRs. We report a straightforward image analysis protocol of feature extraction and image alignment between correlative datasets and demonstrate how such data can be used to visually identify the ensembles of Ca2+ channels that are locally activated during the genesis of cytoplasmic Ca2+ signals. [ABSTRACT FROM AUTHOR]

Molecular imaging aims to depict the molecules in living patients. However, because this aim is still far beyond reach, patchworks of different solutions need to be used to tackle this overarching goal. From the vast toolbox of imaging techniques, we focus on those recent advances in optical microscopy that image molecules and cells at the submicron to centimeter scale. Mesoscopic imaging covers the "imaging gap" between techniques such as confocal microscopy and magnetic resonance imagingthat image entire live samples but with limited resolution. Microscopy focuses on the cellular level; mesoscopy visualizes the organization of molecules and cells into tissues and organs. The correlation between these techniques allows us to combine disciplines ranging from whole body imaging to basic research of model systems. We review current developments focused on improving microscopic and mesoscopic imaging technologies and on hardware and software that push the current sensitivity and resolution boundaries. [ABSTRACT FROM AUTHOR]

Protein misfolding is common to neurodegenerative diseases (NDs) including Alzheimer's disease (AD), which is partly characterized by the self-assembly and accumulation of amyloid-beta in the brain. Lysosomes are a critical component of the proteostasis network required to degrade and recycle material from outside and within the cell and impaired proteostatic mechanisms have been implicated in NDs. We have previously established that toxic amyloid-beta oligomers are endocytosed, accumulate in lysosomes, and disrupt the endo-lysosomal system in neurons. Here, we use pioneering correlative cryo-structured illumination microscopy and cryo-soft X-ray tomography imaging techniques to reconstruct 3D cellular architecture in the native state revealing reduced X-ray density in lysosomes and increased carbon dense vesicles in oligomer treated neurons compared with untreated cells. This work provides unprecedented visual information on the changes to neuronal lysosomes inflicted by amyloid beta oligomers using advanced methods in structural cell biology. [Display omitted] • 3D reconstructions of primary hippocampal neurons by correlative cryo-SXT and cryo-SIM • Lysosomes in neurons incubated with Aβ oligomers show decreased carbon density • Lysosomes containing Aβ oligomers are enlarged • Alterations to neuronal ultrastructure may represent abnormal lysosome function Lysosomes perform critical roles in cells and their dysfunction has been associated with neurodegenerative diseases. Here, Marshall et al. use correlative X-ray and fluorescence imaging to identify lysosomes in a neuronal model for Alzheimer's disease, revealing ultrastructural changes that may have implications for the toxic effects of misfolded proteins. [ABSTRACT FROM AUTHOR]

Raman spectroscopy is becoming increasingly popular as an in vitro bioimaging technique, when coupled with plasmonic substrates such as gold nanoparticles (AuNPs). Plasmonic AuNPs not only display excellent biocompatibility but can also induce the surface‐enhanced Raman scattering (SERS) effect, which can be exploited for cell labeling, as an interesting alternative to fluorescence‐based techniques. SERS bioimaging requires the use of so‐called SERS tags or SERS‐encoded AuNPs. A remaining difficulty toward the general implementation of this method is the difficulty to correlate the SERS signal (spectral intensity) with the number of SERS tags. Therefore, a general correlation method, suitable for arbitrary AuNP morphologies and Raman‐active molecules (Raman reporters or RaRs), should largely improve the quantitative character of SERS as an imaging technique. We propose a protocol, with an associated app (SERSTEM), which enables the user to determine the average SERS intensity per nanoparticle from transmission electron microscopy (TEM) and SERS data. As a proof of concept, we demonstrated the method for Au nanostars and nanorods, carrying four different RaRs, and implemented the SERSTEM app, which is publicly available from an open‐source platform. [ABSTRACT FROM AUTHOR]

The functionality of supramolecular nanostructures can be expanded if systems containing multiple components are designed to either self-sort or mix into coassemblies. This is critical to gain the ability to craft self-assembling materials that integrate functions, and our understanding of this process is in its early stages. In this work, we have utilized three different peptide amphiphiles with the capacity to form β-sheets within supramolecular nanostructures and found binary systems that self-sort and others that form coassemblies. This was measured using atomic force microscopy to reveal the nanoscale morphology of assemblies and confocal laser scanning microscopy to determine the distribution of fluorescently labeled monomers. We discovered that PA assemblies with opposite supramolecular chirality self-sorted into chemically distinct nanostructures. In contrast, the PA molecules that formed a mixture of right-handed, left-handed, and flat nanostructures on their own were able to coassemble with the other PA molecules. We attribute this phenomenon to the energy barrier associated with changing the handedness of a β-sheet twist in a coassembly of two different PA molecules. This observation could be useful for designing biomolecular nanostructures with dual bioactivity or interpenetrating networks of PA supramolecular assemblies.

By limiting protein exchange between the soma and the axon, the axon initial segment (AIS) enables the segregation of specific proteins and hence the differentiation of the somatodendritic compartment and the axonal compartment. Electron microscopy and super-resolution fluorescence imaging have provided important insights in the ultrastructure of the AIS. Yet, the full extent of its filtering properties is not fully delineated. In particular, it is unclear whether and how the AIS opposes the free exchange of soluble proteins. Here we describe a robust framework to combine whole-cell photobleaching and retrospective high-resolution imaging in developing neurons. With this assay, we found that cytoplasmic soluble proteins that are not excluded from the axon upon expression over tens of hours exhibit a strong mobility reduction at the AIS – i.e., are indeed compartmentalized – when monitored over tens of minutes. This form of compartmentalization is developmentally regulated, requires intact F-actin and may be correlated with the composition and ultrastructure of the submembranous spectrin cytoskeleton. Using computational modeling, we provide evidence that both neuronal morphology and the AIS regulate this compartmentalization but act on distinct time scales, with the AIS having a more pronounced effect on fast exchanges. Our results thus suggest that the rate of protein accumulation in the soma may impact to what extent and over which timescales freely moving molecules can be segregated from the axon. This in turn has important implications for our understanding of compartment-specific signaling in neurons.

Alzheimer’s disease (AD) is a neurodegenerative disorder which is characterized by increasing dementia. It is accompanied by the development of extracellular β-amyloid plaques and neurofibrillary tangles in the gray matter of the brain. Histology is the gold standard for the visualization of this pathology, but also has intrinsic shortcomings. Fully three-dimensional analysis and quantitative metrics of alterations in the tissue structure require a complementary approach. In this work we use x-ray phase-contrast tomography to obtain three-dimensional reconstructions of human hippocampal tissue affected by AD. Due to intrinsic electron density differences, tissue components and structures such as the granule cells of the dentate gyrus, blood vessels, or mineralized plaques can be identified and segmented in large volumes. Based on correlative histology, protein (tau, β-amyloid) and elemental content (iron, calcium) can be attributed to certain morphological features occurring in the entire volume. In the vicinity of senile plaques, an accumulation of microglia in combination with a loss of neuronal cells can be observed.

Soil is a heterogeneous mixture of various organic and inorganic parent materials. Major soil functions are driven by their quality, quantity and spatial arrangement, resulting in soil structure. Physical protection of organic matter (OM) in this soil structure is considered as a vital mechanism for stabilizing organic carbon turnover, an important soil function in times of climate change. Herein, we present a technique for the correlative analysis of 2D imaging visible light near-infrared spectroscopy and 3D X-ray computed microtomography (μCT) to investigate the interplay of biogeochemical properties and soil structure in undisturbed soil samples. Samples from the same substrate but different soil management and depth (no-tilled topsoil, tilled topsoil and subsoil) were compared in order to evaluate this method in a diversely structured soil. Imaging spectroscopy is generally used to qualitatively and quantitatively identify OM with high spatial resolution, whereas 3D X-ray μCT provides high-resolution information on pore characteristics. The unique combination of these techniques revealed that, in undisturbed samples, OM can be found mainly at greater distances from macropores and close to biopores. However, alterations were observed because of disturbances by tillage. The correlative application of imaging infrared spectroscopic and X-ray μCT analysis provided new insights into the biochemical processes affected by soil structural changes.

By limiting protein exchange between the soma and the axon, the axon initial segment (AIS) enables the segregation of specific proteins and hence the differentiation of the somatodendritic compartment and the axonal compartment. Electron microscopy and super-resolution fluorescence imaging have provided important insights in the ultrastructure of the AIS. Yet, the full extent of its filtering properties is not fully delineated. In particular, it is unclear whether and how the AIS opposes the free exchange of soluble proteins. Here we describe a robust framework to combine whole-cell photobleaching and retrospective high-resolution imaging in developing neurons. With this assay, we found that cytoplasmic soluble proteins that are not excluded from the axon upon expression over tens of hours exhibit a strong mobility reduction at the AIS – i.e., are indeed compartmentalized – when monitored over tens of minutes. This form of compartmentalization is developmentally regulated, requires intact F-actin and may be correlated with the composition and ultrastructure of the submembranous spectrin cytoskeleton. Using computational modeling, we provide evidence that both neuronal morphology and the AIS regulate this compartmentalization but act on distinct time scales, with the AIS having a more pronounced effect on fast exchanges. Our results thus suggest that the rate of protein accumulation in the soma may impact to what extent and over which timescales freely moving molecules can be segregated from the axon. This in turn has important implications for our understanding of compartment-specific signaling in neurons. [ABSTRACT FROM AUTHOR]

Recent improvements in correlative light and electron microscopy (CLEM) technology have led to dramatic improvements in the ability to observe tissues and cells. Fluorescence labeling has been used to visualize the localization of molecules of interest through immunostaining or genetic modification strategies for the identification of the molecular signatures of biological specimens. Newer technologies such as tissue clearing have expanded the field of observation available for fluorescence labeling; however, the area of correlative observation available for electron microscopy (EM) remains restricted. In this study, we developed a large-area CLEM imaging procedure to show specific molecular localization in large-scale EM sections of mouse and marmoset brain. Target molecules were labeled with antibodies and sequentially visualized in cryostat sections using fluorescence and gold particles. Fluorescence images were obtained by light microscopy immediately after antibody staining. Immunostained sections were postfixed for EM, and silver-enhanced sections were dehydrated in a graded ethanol series and embedded in resin. Ultrathin sections for EM were prepared from fully polymerized resin blocks, collected on silicon wafers, and observed by multibeam scanning electron microscopy (SEM). Multibeam SEM has made rapid, large-area observation at high resolution possible, paving the way for the analysis of detailed structures using the CLEM approach. Here, we describe detailed methods for large-area CLEM in various tissues of both rodents and primates.

Recent improvements in correlative light and electron microscopy (CLEM) technology have led to dramatic improvements in the ability to observe tissues and cells. Fluorescence labeling has been used to visualize the localization of molecules of interest through immunostaining or genetic modification strategies for the identification of the molecular signatures of biological specimens. Newer technologies such as tissue clearing have expanded the field of observation available for fluorescence labeling; however, the area of correlative observation available for electron microscopy (EM) remains restricted. In this study, we developed a large-area CLEM imaging procedure to show specific molecular localization in large-scale EM sections of mouse and marmoset brain. Target molecules were labeled with antibodies and sequentially visualized in cryostat sections using fluorescence and gold particles. Fluorescence images were obtained by light microscopy immediately after antibody staining. Immunostained sections were postfixed for EM, and silver-enhanced sections were dehydrated in a graded ethanol series and embedded in resin. Ultrathin sections for EM were prepared from fully polymerized resin blocks, collected on silicon wafers, and observed by multibeam scanning electron microscopy (SEM). Multibeam SEM has made rapid, large-area observation at high resolution possible, paving the way for the analysis of detailed structures using the CLEM approach. Here, we describe detailed methods for large-area CLEM in various tissues of both rodents and primates. [ABSTRACT FROM AUTHOR]