Chip-based multimodal super-resolution microscopy for histological investigations of cryopreserved tissue sections.

Histology involves the observation of structural features in tissues using a microscope. While diffraction-limited optical microscopes are commonly used in histological investigations, their resolving capabilities are insufficient to visualize details at subcellular level. Although a novel set of super-resolution optical microscopy techniques can fulfill the resolution demands in such cases, the system complexity, high operating cost, lack of multi-modality, and low-throughput imaging of these methods limit their wide adoption for histological analysis. In this study, we introduce the photonic chip as a feasible high-throughput microscopy platform for super-resolution imaging of histological samples. Using cryopreserved ultrathin tissue sections of human placenta, mouse kidney, pig heart, and zebrafish eye retina prepared by the Tokuyasu method, we demonstrate diverse imaging capabilities of the photonic chip including total internal reflection fluorescence microscopy, intensity fluctuation-based optical nanoscopy, single-molecule localization microscopy, and correlative light-electron microscopy. Our results validate the photonic chip as a feasible imaging platform for tissue sections and pave the way for the adoption of super-resolution high-throughput multimodal analysis of cryopreserved tissue samples both in research and clinical settings.

Transgenic zebrafish modeling low-molecular-weight proteinuria and lysosomal storage diseases.

Epithelial cells lining the proximal tubule of the kidney reabsorb and metabolize most of the filtered low-molecular-weight proteins through receptor-mediated endocytosis and lysosomal processing. Congenital and acquired dysfunctions of the proximal tubule are consistently reflected by the inappropriate loss of solutes including low-molecular-weight proteins in the urine. The zebrafish pronephros shares individual functional segments with the human nephron, including lrp2a/megalin-dependent endocytic transport processes of the proximal tubule. Although the zebrafish has been used as a model organism for toxicological studies and drug discovery, there is no available assay that allows large-scale assessment of proximal tubule function in larval or adult stages. Here we establish a transgenic Tg(lfabp::½vdbp-mCherry) zebrafish line expressing in the liver the N-terminal region of vitamin D-binding protein coupled to the acid-insensitive, red monomeric fluorescent protein mCherry (½vdbp-mCherry). This low-molecular-weight protein construct is secreted into the bloodstream, filtered through the glomerulus, reabsorbed by receptor-mediated endocytosis and processed in the lysosomes of proximal tubule cells of the fish. Thus, our proof-of-concept studies using zebrafish larvae knockout for lrp2a and clcn7 or exposed to known nephrotoxins (gentamicin and cisplatin) demonstrate that this transgenic line is useful to monitor low-molecular-weight proteinuria and lysosomal processing. This represents a powerful new model organism for drug screening and studies of nephrotoxicity.

A New Zebrafish Model for CACNA2D4-Dysfunction.

Purpose: Mutations in CACNA2D4, encoding the α2δ4 subunit of retinal voltage-gated calcium channels (Cav), cause a rare type of retinal dysfunction in human, mainly affecting cone vision. Here, we investigate the role of CACNA2D4 in targeting of Cav, its influence on cone-mediated signal transmission, and the cellular and subcellular changes upon loss of α2δ4 by exploiting the advantages of the cone-dominant zebrafish as model system. Methods: We identified two zebrafish CACNA2D4 paralogs (cacna2d4a and cacna2d4b), analyzed their expression by RNA in situ hybridization and introduced truncating frameshift mutations through CRISPR/Cas9-mediated mutagenesis. We analyzed retinal function and morphology of the single and double mutant lines by electroretinography, immunohistochemistry, light- and electron microscopy. Results: Knockout of cacna2d4b reduces the expression of Cacna1fa, the pore-forming subunit of retinal Cav1.4, whereas loss of cacna2d4a did not. Only knockout of both paralogs impaired cone-mediated ERG b-wave amplitude. The number of "floating" ribbons is increased in double-KO, while retinal morphology and expression of postsynaptic mGluR6b remain largely unaffected. Both Cacna1fa and Ribeyeb show ectopic punctate expression in cacna2d4b-KO and double-KO photoreceptors. Conclusions: We find that increasing the expression of Cav at the synaptic membrane is an evolutionarily conserved function of Cacna2d4b. Yet, since both paralogs participate in cone synaptic transmission, we propose partial subfunctionalization in zebrafish. Similar to human patients, our double KO zebrafish model shows mild cone dysfunction, which was not associated with signs of retinal degeneration. Therefore, cacna2d4-KO zebrafish is a suitable model to study the pathophysiological mechanisms underlying CACNA2D4 dysfunction in human.

Standard Preparation Protocol of Human Skin Samples for Transmission Electron Microscopy.

This chapter is intended to be a guide to skin studies dealing with the analysis of subcellular structures of cells and tissues by providing an exhaustive sample preparation protocol for transmission electron microscopy (TEM). Critical steps to obtain good ultrastructure are mentioned as well as notes to improve the sample collection. New ways to automatize the imaging acquisition are discussed.

TDP-43 extracted from frontotemporal lobar degeneration subject brains displays distinct aggregate assemblies and neurotoxic effects reflecting disease progression rates.

Accumulation of abnormally phosphorylated TDP-43 (pTDP-43) is the main pathology in affected neurons of people with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Morphological diversity and neuroanatomical distribution of pTDP-43 accumulations allowed classification of FTLD cases into at least four subtypes, which are correlated with clinical presentations and genetic causes. To understand the molecular basis of this heterogeneity, we developed SarkoSpin, a new method for biochemical isolation of pathological TDP-43. By combining SarkoSpin with mass spectrometry, we revealed proteins beyond TDP-43 that become abnormally insoluble in a disease subtype-specific manner. We show that pTDP-43 extracted from brain forms stable assemblies of distinct densities and morphologies that are associated with disease subtypes. Importantly, biochemically extracted pTDP-43 assemblies showed differential neurotoxicity and seeding that were correlated with disease duration of FTLD subjects. Our data are consistent with the notion that disease heterogeneity could originate from alternate pathological TDP-43 conformations, which are reminiscent of prion strains.

Direct imaging of uncoated biological samples enables correlation of super-resolution and electron microscopy data.

A simple method for imaging biological tissue samples by electron microscopy and its correlation with super-resolution light microscopy is presented. This room temperature protocol, based on protecting thin biological specimens with methylcellulose and imaging with low voltage scanning electron microscopy, circumvents complex classical electron microscopy sample preparation steps requiring dehydration, resin embedding and use of contrast agents. This technique facilitates visualization of subcellular structures e.g. synaptic clefts and synaptic vesicles in mouse brain tissue and the organization of mitochondrial cristae in the zebrafish retina. Application of immunogold protocols to these samples can determine the precise localization of synaptic proteins and, in combination with super-resolution light microscopy methods clearly pinpoints the subcellular distribution of several proteins in the tissue. The simplicity of the method, including section collection on a silicon wafer, reduces artefacts and correlates protein location with sample morphology.

Loss-of-function of the ciliopathy protein Cc2d2a disorganizes the vesicle fusion machinery at the periciliary membrane and indirectly affects Rab8-trafficking in zebrafish photoreceptors.

Ciliopathies are human disorders caused by dysfunction of primary cilia, ubiquitous organelles involved in transduction of environmental signals such as light sensation in photoreceptors. Concentration of signal detection proteins such as opsins in the ciliary membrane is achieved by RabGTPase-regulated polarized vesicle trafficking and by a selective barrier at the ciliary base, the transition zone (TZ). Dysfunction of the TZ protein CC2D2A causes Joubert/Meckel syndromes in humans and loss of ciliary protein localization in animal models, including opsins in retinal photoreceptors. The link between the TZ and upstream vesicle trafficking has been little explored to date. Moreover, the role of the small GTPase Rab8 in opsin-carrier vesicle (OCV) trafficking has been recently questioned in a mouse model. Using correlative light and electron microscopy and live imaging in zebrafish photoreceptors, we provide the first live characterization of Rab8-mediated trafficking in photoreceptors in vivo. Our results support a possibly redundant role for both Rab8a/b paralogs in OCV trafficking, based on co-localization of Rab8 and opsins in vesicular structures, and joint movement of Rab8-tagged particles with opsin. We further investigate the role of the TZ protein Cc2d2a in Rab8-mediated trafficking using cc2d2a zebrafish mutants and identify a requirement for Cc2d2a in the latest step of OCV trafficking, namely vesicle fusion. Progressive accumulation of opsin-containing vesicles in the apical portion of photoreceptors lacking Cc2d2a is caused by disorganization of the vesicle fusion machinery at the periciliary membrane with mislocalization and loss of the t-SNAREs SNAP25 and Syntaxin3 and of the exocyst component Exoc4. We further observe secondary defects on upstream Rab8-trafficking with cytoplasmic accumulation of Rab8. Taken together, our results support participation of Rab8 in OCV trafficking and identify a novel role for the TZ protein Cc2d2a in fusion of incoming ciliary-directed vesicles, through organization of the vesicle fusion machinery at the periciliary membrane.

Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina.

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and scanning electron microscopy. The sub-diffraction limit resolution capabilities of super-resolution light microscopes allow improving the accuracy of the correlated data. Briefly, 110 nanometer thick cryo-sections are transferred to a silicon wafer and, after immunofluorescence staining, are imaged by super-resolution light microscopy. Subsequently, the sections are preserved in methylcellulose and platinum shadowed prior to imaging in a scanning electron microscope (SEM). The images from these two microscopy modalities are easily merged using tissue landmarks with open source software. Here we describe the adapted method for the larval zebrafish retina. However, this method is also applicable to other types of tissues and organisms. We demonstrate that the complementary information obtained by this correlation is able to resolve the expression of mitochondrial proteins in relation with the membranes and cristae of mitochondria as well as to other compartments of the cell.

Efficient preparation of Arabidopsis pollen tubes for ultrastructural analysis using chemical and cryo-fixation.

BACKGROUND: The pollen tube (PT) serves as a model system for investigating plant cell growth and morphogenesis. Ultrastructural studies are indispensable to complement data from physiological and genetic analyses, yet an effective method is lacking for PTs of the model plant Arabidopsis thaliana. METHODS: Here, we present reliable approaches for ultrastructural studies of Arabidopsis PTs, as well as an efficient technique for immunogold detection of cell wall epitopes. Using different fixation and embedding strategies, we show the amount of PT ultrastructural details that can be obtained by the different methods. RESULTS: Dozens of cross-sections can be obtained simultaneously by the approach, which facilitates and shortens the time for evaluation. In addition to in vitro-grown PTs, our study follows the route of PTs from germination, growth along the pistil, to the penetration of the dense stylar tissue, which requires considerable mechanical forces. To this end, PTs have different strategies from growing between cells but also between the protoplast and the cell wall and even within each other, where they share a partly common cell wall. The separation of PT cell walls in an outer and an inner layer reported for many plant species is less clear in Arabidopsis PTs, where these cell wall substructures are connected by a distinct transition zone. CONCLUSIONS: The major advancement of this method is the effective production of a large number of longitudinal and cross-sections that permits obtaining a detailed and representative picture of pollen tube structures in an unprecedented way. This is particularly important when comparing PTs of wild type and mutants to identify even subtle alterations in cytoarchitecture. Arabidopsis is an excellent plant for genetic manipulation, yet the PTs, several-times smaller compared to tobacco or lily, represent a technical challenge. This study reveals a method to overcome this problem and make Arabidopsis PTs more amenable to a combination of genetic and ultrastructural analyses.

Topographic contrast of ultrathin cryo-sections for correlative super-resolution light and electron microscopy.

Fluorescence microscopy reveals molecular expression at nanometer resolution but lacks ultrastructural context information. This deficit often hinders a clear interpretation of results. Electron microscopy provides this contextual subcellular detail, but protein identification can often be problematic. Correlative light and electron microscopy produces complimentary information that expands our knowledge of protein expression in cells and tissue. Inherent methodological difficulties are however encountered when combining these two very different microscopy technologies. We present a quick, simple and reproducible method for protein localization by conventional and super-resolution light microscopy combined with platinum shadowing and scanning electron microscopy to obtain topographic contrast from the surface of ultrathin cryo-sections. We demonstrate protein distribution at nuclear pores and at mitochondrial and plasma membranes in the extended topographical landscape of tissue.

Remote Ischemic Preconditioning: A Novel Strategy in Rescuing Older Livers From Ischemia-reperfusion Injury in a Rodent Model.

OBJECTIVES: The aim of this study was to determine whether remote ischemic preconditioning (RIPC) protects aged liver against ischemia reperfusion (IR). SUMMARY OF BACKGROUND DATA: The demands for liver surgery in an aging population are growing. Clamping of vessels to prevent blood loss is integral to liver surgery, but the resulting IR injury (IRI) augments postoperative complications. More so, sensitivity to hepatic IRI increases with age; however, no strategies have been developed that specifically protect old liver. RIPC, a novel protective approach, was performed distant to the surgical site. Whether RIPC may also protect old liver from IRI is unknown. METHODS: RIPC to the femoral vascular bundle was compared against direct ischemic preconditioning (IPC) and the standard of care intermittent clamping (IC) using a model of partial hepatic ischemia in mice aged 20 to 24 months. Liver injury was measured 6 hours after reperfusion. Protective signaling (serotonin-Vegf-Il10/Mmp8 axis, Kupffer cell polarization) was assessed immediately after preconditioning. Neutralizing antibody was used to test the role of Vegf. Hepatic vasculature was examined by electron microscopy. RESULTS: RIPC was superior over other strategies in protecting old liver from IRI, with standard IPC approaches being ineffective. RIPC induced the strongest elevations in circulating Vegf, and Vegf inhibition dampened protective signaling and abrogated the protective effects. RIPC was further associated with improvements in vascular functionality. CONCLUSIONS: RIPC is highly effective in protecting old liver from ischemic insults, mainly owing to its ability to induce circulating Vegf. These findings warrant efforts toward clinical translation.

Rapid dephosphorylation of the renal sodium chloride cotransporter in response to oral potassium intake in mice.

A dietary potassium load induces a rapid kaliuresis and natriuresis, which may occur even before plasma potassium and aldosterone (aldo) levels increase. Here we sought to gain insight into underlying molecular mechanisms contributing to this response. After gastric gavage of 2% potassium, the plasma potassium concentrations rose rapidly (0.25 h), followed by a significant rise of plasma aldo (0.5 h) in mice. Enhanced urinary potassium and sodium excretion was detectable as early as spot urines could be collected (about 0.5 h). The functional changes were accompanied by a rapid and sustained (0.25-6 h) dephosphorylation of the NaCl cotransporter (NCC) and a late (6 h) upregulation of proteolytically activated epithelial sodium channels. The rapid effects on NCC were independent from the coadministered anion. NCC dephosphorylation was also aldo-independent, as indicated by experiments in aldo-deficient mice. The observed urinary sodium loss relates to NCC, as it was markedly diminished in NCC-deficient mice. Thus, downregulation of NCC likely explains the natriuretic effect of an acute oral potassium load in mice. This may improve renal potassium excretion by increasing the amount of intraluminal sodium that can be exchanged against potassium in the aldo-sensitive distal nephron.