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.