Benchtop mesoSPIM: a next-generation open-source light-sheet microscope for cleared samples.

In 2015, we launched the mesoSPIM initiative, an open-source project for making light-sheet microscopy of large cleared tissues more accessible. Meanwhile, the demand for imaging larger samples at higher speed and resolution has increased, requiring major improvements in the capabilities of such microscopes. Here, we introduce the next-generation mesoSPIM ("Benchtop") with a significantly increased field of view, improved resolution, higher throughput, more affordable cost, and simpler assembly compared to the original version. We develop an optical method for testing detection objectives that enables us to select objectives optimal for light-sheet imaging with large-sensor cameras. The improved mesoSPIM achieves high spatial resolution (1.5 µm laterally, 3.3 µm axially) across the entire field of view, magnification up to 20×, and supports sample sizes ranging from sub-mm up to several centimeters while being compatible with multiple clearing techniques. The microscope serves a broad range of applications in neuroscience, developmental biology, pathology, and even physics.

Studying the morphology, composition and function of the photoreceptor primary cilium in zebrafish.

Vision is one of our dominant senses and its loss has a profound impact on the life quality of affected individuals. Highly specialized neurons in the retina called photoreceptors convert photons into neuronal responses. This conversion of photons is mediated by light sensitive opsin proteins, which are found in the outer segments of the photoreceptors. These outer segments are highly specialized primary cilia, explaining why retinal dystrophy is a key feature of ciliopathies, a group of diseases resulting from abnormal and dysfunctional cilia. Therefore, research on ciliopathies often includes the analysis of the retina with special focus on the photoreceptor and its outer segment. In the last decade, the zebrafish has emerged as an excellent model organism to study human diseases, in particular with respect to the retina. The cone-rich retina of zebrafish resembles the fovea of the human macula and thus represents an excellent model to study human retinal diseases. Here we give detailed guidance on how to analyze the morphological and ultra-structural integrity of photoreceptors in the zebrafish using various histological and imaging techniques. We further describe how to conduct functional analysis of the retina by electroretinography and how to prepare isolated outer segment fractions for different -omic approaches. These different methods allow a comprehensive analysis of photoreceptors, helping to enhance our understanding of the molecular and structural basis of ciliary function in health and of the consequences of its dysfunction in disease.

B- and T-cell acute lymphoblastic leukemias evade chemotherapy at distinct sites in the bone marrow.

Persistence of residual disease after induction chemotherapy is a strong predictor of relapse in acute lymphoblastic leukemia (ALL). The bone marrow microenvironment may support escape from treatment. Using three-dimensional fluorescence imaging of ten primary ALL xenografts we identified sites of predilection in the bone marrow for resistance to induction with dexamethasone, vincristine and doxorubicin. We detected B-cell precursor ALL cells predominantly in the perisinusoidal space at early engraftment and after chemotherapy. The spatial distribution of T-ALL cells was more widespread with contacts to endosteum, nestin+ pericytes and sinusoids. Dispersion of T-ALL cells in the bone marrow increased under chemotherapeutic pressure. A subset of slowly dividing ALL cells was transiently detected upon shortterm chemotherapy, but not at residual disease after chemotherapy, challenging the notion that ALL cells escape treatment by direct induction of a dormant state in the niche. These lineage-dependent differences point to niche interactions that may be more specifically exploitable to improve treatment.

The Benchtop mesoSPIM: a next-generation open-source light-sheet microscope for large cleared samples.

In 2015, we launched the mesoSPIM initiative (www.mesospim.org), an open-source project for making light-sheet microscopy of large cleared tissues more accessible. Meanwhile, the demand for imaging larger samples at higher speed and resolution has increased, requiring major improvements in the capabilities of light-sheet microscopy. Here, we introduce the next-generation mesoSPIM ("Benchtop") with significantly increased field of view, improved resolution, higher throughput, more affordable cost and simpler assembly compared to the original version. We developed a new method for testing objectives, enabling us to select detection objectives optimal for light-sheet imaging with large-sensor sCMOS cameras. The new mesoSPIM achieves high spatial resolution (1.5 µm laterally, 3.3 µm axially) across the entire field of view, a magnification up to 20x, and supports sample sizes ranging from sub-mm up to several centimetres, while being compatible with multiple clearing techniques. The new microscope serves a broad range of applications in neuroscience, developmental biology, and even physics.

Photouncaging of Carboxylic Acids from Cyanine Dyes with Near-Infrared Light.

Near-infrared light (NIR; 650-900 nm) offers unparalleled advantages as a biocompatible stimulus. The development of photocages that operate in this region represents a fundamental challenge due to the low energy of the excitation light. Herein, we repurpose cyanine dyes into photocages that are available on a multigram scale in three steps and efficiently release carboxylic acids in aqueous media upon irradiation with NIR light up to 820 nm. The photouncaging process is examined using several techniques, providing evidence that it proceeds via photooxidative pathway. We demonstrate the practical utility in live HeLa cells by delivery and release of the carboxylic acid cargo, that was otherwise not uptaken by cells in its free form. In combination with modularity of the cyanine scaffold, the realization of these accessible photocages will fully unleash the potential of the emerging field of NIR-photoactivation and facilitate its widespread adoption outside the photochemistry community.

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.

Characterization of Deposits in Calcific Tendinitis of the Shoulder: Deposits Are Composed of Large Aggregates of Highly Crystalline, Rod-Like Crystals.

BACKGROUND: In the current literature, deposits in calcific tendinitis are described as amorphous masses of hydroxyapatite with a size in the range of 5 to 20 µm. Theoretically, these are too big to be phagocytized by macrophages and induce an inflammatory reaction. PURPOSE: To better characterize the deposits seen in calcific tendinitis. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: Included in the study were 6 patients with a history of at least 1 year of shoulder pain (range, 1-14 years). Shoulder arthroscopy was performed under general anesthesia, and calcium deposits from the supraspinatus tendon and biopsies from the adjacent subacromial bursa were taken. Samples were analyzed by light microscopy and immunostained for macrophages. Scanning electron microscopy and energy-dispersive x-ray (EDX) analysis were used to assess the morphology and chemical composition of the calcific deposits. RESULTS: Light microscopy showed round and bulky calcium deposits partially surrounded by activated CD68-positive macrophages within inflammatory tissue. Some hemosiderin positive mononuclear cells, indicative for (micro-) hemorrhage, were seen. Scanning electron microscopy revealed that the large calcific deposits (1-20 µm) were composed of rod-like structures. These highly crystalline rods had a size of approximately 100 nm in length and 20 nm in width. Chemical composition by EDX analysis showed that crystals were composed of mainly calcium, oxygen, and phosphorus, equaling the chemical composition of hydroxyapatite. CONCLUSION: Deposits in calcific tendinitis of the rotator cuff are not amorphous but composed of highly crystalline structures. Fragmentation of these aggregates and subsequent release of the needle-like nanocrystals might initiate the strong inflammatory reaction often seen in patients with calcifying tendinitis of the rotator cuff.

Quorum sensing governs a transmissive Legionella subpopulation at the pathogen vacuole periphery.

The Gram-negative bacterium Legionella pneumophila is the causative agent of Legionnaires' disease and replicates in amoebae and macrophages within a distinct compartment, the Legionella-containing vacuole (LCV). The facultative intracellular pathogen switches between a replicative, non-virulent and a non-replicating, virulent/transmissive phase. Here, we show on a single-cell level that at late stages of infection, individual motile (PflaA -GFP-positive) and virulent (PralF - and PsidC -GFP-positive) L. pneumophila emerge in the cluster of non-growing bacteria within an LCV. Comparative proteomics of PflaA -GFP-positive and PflaA -GFP-negative L. pneumophila subpopulations reveals distinct proteomes with flagellar proteins or cell division proteins being preferentially produced by the former or the latter, respectively. Toward the end of an infection cycle (˜ 48 h), the PflaA -GFP-positive L. pneumophila subpopulation emerges at the cluster periphery, predominantly escapes the LCV, and spreads from the bursting host cell. These processes are mediated by the Legionella quorum sensing (Lqs) system. Thus, quorum sensing regulates the emergence of a subpopulation of transmissive L. pneumophila at the LCV periphery, and phenotypic heterogeneity underlies the intravacuolar bi-phasic life cycle of L. pneumophila.

Fenretinide induces a new form of dynamin-dependent cell death in pediatric sarcoma.

Alveolar rhabdomyosarcoma (aRMS) is a highly malicious childhood malignancy characterized by specific chromosomal translocations mostly encoding the oncogenic transcription factor PAX3-FOXO1 and therefore also referred to as fusion-positive RMS (FP-RMS). Previously, we have identified fenretinide (retinoic acid p-hydroxyanilide) to affect PAX3-FOXO1 expression levels as well as FP-RMS cell viability. Here, we characterize the mode of action of fenretinide in more detail. First, we demonstrate that fenretinide-induced generation of reactive oxygen species (ROS) depends on complex II of the mitochondrial respiratory chain, since ROS scavenging as well as complexing of iron completely abolished cell death. Second, we co-treated cells with a range of pharmacological inhibitors of specific cell death pathways including z-vad (apoptosis), necrostatin-1 (necroptosis), 3-methyladenine (3-MA) (autophagy), and ferrostatin-1 (ferroptosis) together with fenretinide. Surprisingly, none of these inhibitors was able to prevent cell death. Also genetic depletion of key players in the apoptotic and necroptotic pathway (BAK, BAX, and RIPK1) confirmed the pharmacological data. Interestingly however, electron microscopy of fenretinide-treated cells revealed an excessive accumulation of cytoplasmic vacuoles, which were distinct from autophagosomes. Further flow cytometry and fluorescence microscopy experiments suggested a hyperstimulation of macropinocytosis, leading to an accumulation of enlarged early and late endosomes. Surprisingly, pharmacological inhibition as well as genetic depletion of large dynamin GTPases completely abolished fenretinide-induced vesicle formation and subsequent cell death, suggesting a new form of dynamin-dependent programmed cell death. Taken together, our data identify a new form of cell death mediated through the production of ROS by fenretinide treatment, highlighting the value of this compound for treatment of sarcoma patients including FP-RMS.

The iron chelator Deferasirox causes severe mitochondrial swelling without depolarization due to a specific effect on inner membrane permeability.

The iron chelator Deferasirox (DFX) causes severe toxicity in patients for reasons that were previously unexplained. Here, using the kidney as a clinically relevant in vivo model for toxicity together with a broad range of experimental techniques, including live cell imaging and in vitro biophysical models, we show that DFX causes partial uncoupling and dramatic swelling of mitochondria, but without depolarization or opening of the mitochondrial permeability transition pore. This effect is explained by an increase in inner mitochondrial membrane (IMM) permeability to protons, but not small molecules. The movement of water into mitochondria is prevented by altering intracellular osmotic gradients. Other clinically used iron chelators do not produce mitochondrial swelling. Thus, DFX causes organ toxicity due to an off-target effect on the IMM, which has major adverse consequences for mitochondrial volume regulation.

Celiprolol but not losartan improves the biomechanical integrity of the aorta in a mouse model of vascular Ehlers-Danlos syndrome.

AIMS: Antihypertensive drugs are included in the medical therapy of vascular Ehlers-Danlos syndrome (vEDS). The ß-blocker celiprolol has been suggested to prevent arterial damage in vEDS, but the underlying mechanism remains unclear. It is also unknown whether the widely used angiotensin II receptor type 1 antagonist losartan has a therapeutic effect in vEDS. Here, we evaluated the impact of celiprolol and losartan on the biomechanical integrity of the vEDS thoracic aorta. METHODS AND RESULTS: We established a new approach to measure the maximum tensile force at rupture of uniaxially stretched murine thoracic aortic rings. In a vEDS model, which we (re-)characterized here at molecular level, heterozygous mice showed a significant reduction in the rupture force compared to wild-type mice, reflecting the increased mortality due to aortic rupture. For the assessment of treatment effects, heterozygous mice at 4 weeks of age underwent a 4-week treatment with celiprolol, losartan, and, as a proof-of-concept drug, the matrix metalloproteinase inhibitor doxycycline. Compared to age- and sex-matched untreated heterozygous mice, treatment with doxycycline or celiprolol resulted in a significant increase of rupture force, whereas no significant change was detected upon losartan treatment. CONCLUSIONS: In a vEDS model, celiprolol or doxycycline, but not losartan, can improve the biomechanical integrity of the aortic wall, thereby potentially reducing the risk of dissection and rupture. As doxycycline is a broad-spectrum antibiotic with considerable side effects, celiprolol may be more suitable for a long-term therapy and thus rather indicated for the medication of patients with vEDS.

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.

The mesoSPIM initiative: open-source light-sheet microscopes for imaging cleared tissue.

Light-sheet microscopy is an ideal technique for imaging large cleared samples; however, the community is still lacking instruments capable of producing volumetric images of centimeter-sized cleared samples with near-isotropic resolution within minutes. Here, we introduce the mesoscale selective plane-illumination microscopy initiative, an open-hardware project for building and operating a light-sheet microscope that addresses these challenges and is compatible with any type of cleared or expanded sample ( www.mesospim.org ).

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.

Combined Structural and Functional Imaging of the Kidney Reveals Major Axial Differences in Proximal Tubule Endocytosis.

BACKGROUND: The kidney proximal convoluted tubule (PCT) reabsorbs filtered macromolecules via receptor-mediated endocytosis (RME) or nonspecific fluid phase endocytosis (FPE); endocytosis is also an entry route for disease-causing toxins. PCT cells express the protein ligand receptor megalin and have a highly developed endolysosomal system (ELS). Two PCT segments (S1 and S2) display subtle differences in cellular ultrastructure; whether these translate into differences in endocytotic function has been unknown. METHODS: To investigate potential differences in endocytic function in S1 and S2, we quantified ELS protein expression in mouse kidney PCTs using real-time quantitative polymerase chain reaction and immunostaining. We also used multiphoton microscopy to visualize uptake of fluorescently labeled ligands in both living animals and tissue cleared using a modified CLARITY approach. RESULTS: Uptake of proteins by RME occurs almost exclusively in S1. In contrast, dextran uptake by FPE takes place in both S1 and S2, suggesting that RME and FPE are discrete processes. Expression of key ELS proteins, but not megalin, showed a bimodal distribution; levels were far higher in S1, where intracellular distribution was also more polarized. Tissue clearing permitted imaging of ligand uptake at single-organelle resolution in large sections of kidney cortex. Analysis of segmented tubules confirmed that, compared with protein uptake, dextran uptake occurred over a much greater length of the PCT, although individual PCTs show marked heterogeneity in solute uptake length and three-dimensional morphology. CONCLUSIONS: Striking axial differences in ligand uptake and ELS function exist along the PCT, independent of megalin expression. These differences have important implications for understanding topographic patterns of kidney diseases and the origins of proteinuria.

The large GTPase atlastin controls ER remodeling around a pathogen vacuole.

The ubiquitous environmental bacterium Legionella pneumophila is the causative agent of Legionnaires' pneumonia and replicates in free-living protozoa and mammalian macrophages in a specific compartment, the Legionella-containing vacuole (LCV). LCVs communicate with the endosomal, retrograde and secretory vesicle trafficking pathway, and eventually tightly interact with the endoplasmic reticulum (ER). In Dictyostelium discoideum amoebae and macrophages, the ER tubule-resident large GTPase Sey1/atlastin3 (Atl3) accumulates on LCVs and promotes LCV expansion and intracellular replication of L. pneumophila. Fluorescence microscopy of D. discoideum infected with L. pneumophila indicated that Sey1 is involved in extensive ER remodeling around LCVs. An ultrastructural analysis confirmed these findings. Moreover, dominant negative Sey1_K154A compromises ER accumulation on LCVs and causes an aberrant ER morphology in uninfected D. discoideum as well as in amoebae infected with avirulent L. pneumophila that lack a functional type IV secretion system. Thus, the large, dynamin-like GTPase Sey1/Atl3 controls circumferential ER remodeling during LCV maturation.

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.

ER remodeling by the large GTPase atlastin promotes vacuolar growth of Legionella pneumophila.

The pathogenic bacterium Legionella pneumophila replicates in host cells within a distinct ER-associated compartment termed the Legionella-containing vacuole (LCV). How the dynamic ER network contributes to pathogen proliferation within the nascent LCV remains elusive. A proteomic analysis of purified LCVs identified the ER tubule-resident large GTPase atlastin3 (Atl3, yeast Sey1p) and the reticulon protein Rtn4 as conserved LCV host components. Here, we report that Sey1/Atl3 and Rtn4 localize to early LCVs and are critical for pathogen vacuole formation. Sey1 overproduction promotes intracellular growth of L. pneumophila, whereas a catalytically inactive, dominant-negative GTPase mutant protein, or Atl3 depletion, restricts pathogen replication and impairs LCV maturation. Sey1 is not required for initial recruitment of ER to PtdIns(4)P-positive LCVs but for subsequent pathogen vacuole expansion. GTP (but not GDP) catalyzes the Sey1-dependent aggregation of purified, ER-positive LCVs in vitro Thus, Sey1/Atl3-dependent ER remodeling contributes to LCV maturation and intracellular replication of L. pneumophila.