Correlative Raman Imaging: Development and Cancer Applications.

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.

Self-Sorting vs Coassembly in Peptide Amphiphile Supramolecular Nanostructures.

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 ß-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 ß-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.

Correlative light and X-ray tomography jointly unveil the critical role of connexin43 channels on inflammation-induced cellular ultrastructural alterations.

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.

Correlative cryo-soft X-ray tomography and cryo-structured illumination microscopy reveal changes to lysosomes in amyloid-ß-treated neurons.

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.

Mantis: high-throughput 4D imaging and analysis of the molecular and physical architecture of cells.

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, 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 minutes over 7.5 hours. 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.

Advanced optical imaging for the rational design of nanomedicines.

Despite the enormous potential of nanomedicines to shape the future of medicine, their clinical translation remains suboptimal. Translational challenges are present in every step of the development pipeline, from a lack of understanding of patient heterogeneity to insufficient insights on nanoparticle properties and their impact on material-cell interactions. Here, we discuss how the adoption of advanced optical microscopy techniques, such as super-resolution optical microscopies, correlative techniques, and high-content modalities, could aid the rational design of nanocarriers, by characterizing the cell, the nanomaterial, and their interaction with unprecedented spatial and/or temporal detail. In this nanomedicine arena, we will discuss how the implementation of these techniques, with their versatility and specificity, can yield high volumes of multi-parametric data; and how machine learning can aid the rapid advances in microscopy: from image acquisition to data interpretation.

Insights into protein structure using cryogenic light microscopy.

Fluorescence microscopy has witnessed many clever innovations in the last two decades, leading to new methods such as structured illumination and super-resolution microscopies. The attainable resolution in biological samples is, however, ultimately limited by residual motion within the sample or in the microscope setup. Thus, such experiments are typically performed on chemically fixed samples. Cryogenic light microscopy (Cryo-LM) has been investigated as an alternative, drawing on various preservation techniques developed for cryogenic electron microscopy (Cryo-EM). Moreover, this approach offers a powerful platform for correlative microscopy. Another key advantage of Cryo-LM is the strong reduction in photobleaching at low temperatures, facilitating the collection of orders of magnitude more photons from a single fluorophore. This results in much higher localization precision, leading to Angstrom resolution. In this review, we discuss the general development and progress of Cryo-LM with an emphasis on its application in harnessing structural information on proteins and protein complexes.

Visualisation of drug distribution in skin using correlative optical spectroscopy and mass spectrometry imaging.

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.

Volume electron microscopy reveals placental ultrastructure in 3D.

Many structures necessary for placental function can only be visualised at the ultrastructural scale. Recent technological advances have made Volume electron microscopy (volume EM) approaches much more accessible. Volume EM allows the ultrastructure of tissues, cells and organelles to be visualised in 3D. It also allows the 3D spatial relationships between these structures to be determined. This review will highlight the potential for volume EM to advance our understanding of placental ultrastructure. It will focus on the human term placenta highlighting key findings spanning the placental barrier from trans-syncytial nanopores in the syncytiotrophoblast to tunnelling nanotubes in the fetal capillary endothelium. Volume EM is advancing our understanding of placental ultrastructure, but to fully exploit its potential, it will be necessary to use it as part of multimodal and correlative workflows. The complementary strengths of these different approaches can complement volume EM and reveal the biological significance of its novel observations. The use of volume EM also highlighted how ultrastructural features might underpin pregnancy pathologies and demonstrates the need for more research in this underrepresented area.

Addressing uncertainties in correlative imaging of exogenous particles with the tissue microanatomy with synchronous imaging strategies.

Exposure to exogenous particles is of increasing concern to human health. Characterizing the concentrations, chemical species, distribution, and involvement of the stimulus with the tissue microanatomy is essential in understanding the associated biological response. However, no single imaging technique can interrogate all these features at once, which confounds and limits correlative analyses. Developments of synchronous imaging strategies, allowing multiple features to be identified simultaneously, are essential to assess spatial relationships between these key features with greater confidence. Here, we present data to first highlight complications of correlative analysis between the tissue microanatomy and elemental composition associated with imaging serial tissue sections. This is achieved by assessing both the cellular and elemental distributions in three-dimensional space using optical microscopy on serial sections and confocal X-ray fluorescence spectroscopy on bulk samples, respectively. We propose a new imaging strategy using lanthanide-tagged antibodies with X-ray fluorescence spectroscopy. Using simulations, a series of lanthanide tags were identified as candidate labels for scenarios where tissue sections are imaged. The feasibility and value of the proposed approach are shown where an exposure of Ti was identified concurrently with CD45 positive cells at sub-cellular resolutions. Significant heterogeneity in the distribution of exogenous particles and cells can be present between immediately adjacent serial sections showing a clear need of synchronous imaging methods. The proposed approach enables elemental compositions to be correlated with the tissue microanatomy in a highly multiplexed and non-destructive manner at high spatial resolutions with the opportunity for subsequent guided analysis.

First insight in element localisation in different body parts of the acanthocephalan Dentitruncus truttae using TEM and NanoSIMS.

Acanthocephalans, intestinal parasites of vertebrates, are characterised by orders of magnitude higher metal accumulation than free-living organisms, but the mechanism of such effective metal accumulation is still unknown. The aim of our study was to gain new insights into the high-resolution localization of elements in the bodies of acanthocephalans, thus taking an initial step towards elucidating metal uptake and accumulation in organisms under real environmental conditions. For the first time, nanoscale secondary ion mass spectrometry (NanoSIMS) was used for high-resolution mapping of 12 elements (C, Ca, Cu, Fe, N, Na, O, P, Pb, S, Se, and Tl) in three selected body parts (trunk spines, inner part of the proboscis receptacle and inner surface of the tegument) of Dentitruncus truttae, a parasite of brown trout (Salmo trutta) from the Krka River in Croatia. In addition, the same body parts were examined using transmission electron microscopy (TEM) and correlated with NanoSIMS images. Metal concentrations determined using HR ICP-MS confirmed higher accumulation in D. truttae than in the fish intestine. The chemical composition of the acanthocephalan body showed the highest density of C, Ca, N, Na, O, S, as important and constitutive elements in living cells in all studied structures, while Fe was predominant among trace elements. In general, higher element density was found in trunk spines and tegument, as body structures responsible for substance absorption in parasites. The results obtained with NanoSIMS and TEM-NanoSIMS correlative imaging represent pilot data for mapping of elements at nanoscale resolution in the ultrastructure of various body parts of acanthocephalans and generally provide a contribution for further application of this technique in all parasite species.

A Multimodal Desorption Electrospray Ionisation Workflow Enabling Visualisation of Lipids and Biologically Relevant Elements in a Single Tissue Section.

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.

Methods to expose subsurface objects of interest identified from 3D imaging: The intermediate sample preparation stage in the correlative microscopy workflow.

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.

OCT Meets micro-CT: A Subject-Specific Correlative Multimodal Imaging Workflow for Early Chick Heart Development Modeling.

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.

Hybrid Nanofillers Creating the Stable PVDF Nanocomposite Films and Their Effect on the Friction and Mechanical Properties.

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.

Near-Infrared Carbon Nanotube Tracking Reveals the Nanoscale Extracellular Space around Synapses.

We provide evidence of a local synaptic nanoenvironment in the brain extracellular space (ECS) lying within 500 nm of postsynaptic densities. To reveal this brain compartment, we developed a correlative imaging approach dedicated to thick brain tissue based on single-particle tracking of individual fluorescent single wall carbon nanotubes (SWCNTs) in living samples and on speckle-based HiLo microscopy of synaptic labels. We show that the extracellular space around synapses bears specific properties in terms of morphology at the nanoscale and inner diffusivity. We finally show that the ECS juxta-synaptic region changes its diffusion parameters in response to neuronal activity, indicating that this nanoenvironment might play a role in the regulation of brain activity.

Corrigendum: Correlative Live-Cell and Super-Resolution Imaging to Link Presynaptic Molecular Organisation With Function.

[This corrects the article DOI: 10.3389/fnsyn.2022.830583.].

Deciphering a hexameric protein complex with Angstrom optical resolution.

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.

Amino Acid PET Imaging with 18F-DOPA in the evaluation of Pediatric Brain Tumors.

Although MRI is the workhorse of brain tumor initial evaluation and follow-up, there is a growing amount of data recommending the incorporation of amino-acid PET imaging at different stages of the management of these patients. Recent nuclear medicine and neuro-oncology clinical practice recommendations support the use of amino-acid imaging in brain tumor imaging. Considering 18F-DOPA is FDA approved for the evaluation of parkinsonian syndromes, it could be used clinically for other valuable clinical indications such as brain tumor evaluations. This value seems to be well established in adults and has growing evidence for its use in pediatrics as well. We offer to present four pediatric brain tumor cases imaged with 18F-DOPA and review the literature.

Correlative Live-Cell and Super-Resolution Imaging to Link Presynaptic Molecular Organisation With Function.

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.