Explore the vast range of titles available.

At any given time, the ovary contains a number of follicles in distinct growth stages, each with a set of identifying characteristics. Although follicle counting and staging using histological stains on paraffin-embedded ovary sections has been the gold standard in assessing ovarian health in fertility studies, the final counts rely on extrapolation factors that diverge greatly among studies. These methods also limit our ability to investigate spatial aspects of ovary organization. Recent advances in optical tissue clearing and lightsheet microscopy have permitted comprehensive analysis of intact tissues. In this study, we set out to determine the best clearing and imaging methods to generate 3D images of the complete adult mouse ovary that could be used for accurate assessments of ovarian follicles. We found that a combination of iDISCO and CUBIC was the best method to clear the immunostained ovary. Using lightsheet microscopy, we generated 3D images of the intact ovary and performed qualitative assessments of follicles at all stages of development. This study is an important step toward developing quantitative computational models that allow rapid and accurate assessments of growing and quiescent primordial follicles, and to investigate the integrity of extrinsic ovarian components including vascular and neuronal networks. Summary Sentence The combination of iDISCO and CUBIC tissue clearing methods allows in toto imaging of the ovarian follicle composition and extrinsic vascular and neuronal networks.

Recent developments within micro-computed tomography (μCT) imaging have combined to extend our capacity to image tissue in three (3D) and four (4D) dimensions at micron and sub-micron spatial resolutions, opening the way for virtual histology, live cell imaging, subcellular imaging and correlative microscopy. Pivotal to this has been the development of methods to extend the contrast achievable for soft tissue. Herein, we review the new capabilities within the field of life sciences imaging, and consider how future developments in this field could further benefit the life sciences community.

During morphogenesis, epithelial sheets remodel into complex geometries. How cells dynamically organise their contact with neighbouring cells in these tightly packed tissues is poorly understood. We have used light-sheet microscopy of growing mouse embryonic lung explants, three-dimensional cell segmentation, and physical theory to unravel the principles behind 3D cell organisation in growing pseudostratified epithelia. We find that cells have highly irregular 3D shapes and exhibit numerous neighbour intercalations along the apical-basal axis as well as over time. Despite the fluidic nature, the cell packing configurations follow fundamental relationships previously described for apical epithelial layers, that is, Euler's polyhedron formula, Lewis’ law, and Aboav-Weaire's law, at all times and across the entire tissue thickness. This arrangement minimises the lateral cell-cell surface energy for a given cross-sectional area variability, generated primarily by the distribution and movement of nuclei. We conclude that the complex 3D cell organisation in growing epithelia emerges from simple physical principles.

Tissue clearing and 3D imaging have emerged as powerful techniques to assess the cellular and tissue-level architecture of the spinal cord. With the rapidly increasing variety and complexity of optical tissue clearing techniques, there is a critical need for optimization and streamlining of tissue-specific protocols, particularly when dealing with injury or disease states. We evaluated and combined multiple organic solvent-based techniques to develop sciDISCO: a spinal cord injury-optimized DISCO tissue clearing protocol. sciDISCO allows for the robust clearing, labeling, and 3D imaging of the intact spinal cord, as well as clearing around and through the lesion site formed after contusive spinal cord injury. In addition, we have identified alternatives for hazardous chemicals commonly used in organic solvent-based clearing including dichloromethane and dibenzyl ether. In this study, we demonstrate the compatibility of sciDISCO with multiple different labeling techniques to provide robust analysis of unique neuronal populations and morphologies in addition to cellular and tissue-level changes occurring following spinal cord injury.

Morphogenetic events during the development of the fetal ovary are crucial to the establishment of female fertility. However, the effects of structural rearrangements of the ovary and surrounding reproductive tissues on ovary morphogenesis remain largely uncharacterized. Using tissue clearing and lightsheet microscopy, we found that ovary folding correlated with regionalization into cortex and medulla. Relocation of the oviduct to the ventral aspect of the ovary led to ovary encapsulation, and mutual attachment of the ovary and oviduct to the cranial suspensory ligament likely triggered ovary folding. During this process, the rete ovarii (RO) elaborated into a convoluted tubular structure extending from the ovary into the ovarian capsule. Using genetic mouse models in which the oviduct and RO are perturbed, we found the oviduct is required for ovary encapsulation. This study reveals novel relationships among the ovary and surrounding tissues and paves the way for functional investigation of the relationship between architecture and differentiation of the mammalian ovary. In humans and other mammals, the female reproductive organs, or ovaries, develop early in life, while the young are still in their mother’s womb. Ovaries contain several different compartments, including the ovarian follicles. These are small groups of cells that produce reproductive hormones, and each follicle also has the potential to produce one egg for fertilisation. The ovaries are further surrounded by different tissues that develop alongside them. These include the oviducts, which carry fertilised eggs from the ovaries into the womb, and ligaments, which anchor the ovaries to the wall of the body cavity. During the development of ovaries, ovarian follicles are sorted into two distinct groups. The first, called medullary follicles, are lost before puberty. The second group, or cortical follicles, remain in a state of ‘suspended animation’ until puberty. After that, they act as a ‘reserve’ of eggs for the rest of the reproductive lifespan. Once each cortical follicle has produced an egg, it is not replenished. This means that proper follicle sorting is crucial for establishing female fertility, and therefore the ability to conceive. The mechanisms behind follicle sorting, however, are still poorly understood. McKey et al. set out to determine how the ovary’s structure changed during its development. In the experiments, high-resolution microscopy techniques were used to reconstruct ovaries of mice in 3D across different stages of development. This revealed that the ends of each ovary started folding towards each other just before birth, and that the folding also happened at the same time as follicle sorting. Simultaneous changes in the shape and orientation of the ligaments suggested that these tissues might direct the folding, for example by pushing or pulling on the rest of the ovary. These results suggest that the changes in ovary structure in early life are critically linked to the establishment of the ovary’s egg reserves. McKey et al. hope that this study will pave the way to a better understanding of infertility and, ultimately, better treatments.

As the general population ages, more people are affected by eye diseases, such as retinopathies. It is therefore critical to improve imaging of eye disease mouse models. Here, we demonstrate that 1) rapid, quantitative 3D and 4D (time lapse) imaging of cellular and subcellular processes in the mouse eye is feasible, with and without tissue clearing, using light-sheet fluorescent microscopy (LSFM); 2) flat-mounting retinas for confocal microscopy significantly distorts tissue morphology, confirmed by quantitative correlative LSFM-Confocal imaging of vessels; 3) LSFM readily reveals new features of even well-studied eye disease mouse models, such as the oxygen-induced retinopathy (OIR) model, including a previously unappreciated ‘knotted’ morphology to pathological vascular tufts, abnormal cell motility and altered filopodia dynamics when live-imaged. We conclude that quantitative 3D/4D LSFM imaging and analysis has the potential to advance our understanding of the eye, in particular pathological, neurovascular, degenerative processes. Eye diseases affect millions of people worldwide and can have devasting effects on people’s lives. To find new treatments, scientists need to understand more about how these diseases arise and how they progress. This is challenging and progress has been held back by limitations in current techniques for looking at the eye. Currently, the most commonly used method is called confocal imaging, which is slow and distorts the tissue. Distortion happens because confocal imaging requires that thin slices of eye tissue from mice used in experiments are flattened on slides; this makes it hard to accurately visualize three-dimensional structures in the eye. New methods are emerging that may help. One promising method is called light-sheet fluorescent microscopy (or LSFM for short). This method captures three-dimensional images of the blood vessels and cells in the eye. It is much faster than confocal imaging and allows scientists to image tissues without slicing or flattening them. This could lead to more accurate three-dimensional images of eye disease. Now, Prahst et al. show that LSFM can quickly produce highly detailed, three-dimensional images of mouse retinas, from the smallest parts of cells to the entire eye. The technique also identified new features in a well-studied model of retina damage caused by excessive oxygen exposure in young mice. Previous studies of this model suggested the disease caused blood vessels in the eye to balloon, hinting that drugs that shrink blood vessels would help. But using LSFM, Prahst et al. revealed that these blood vessels actually take on a twisted and knotted shape. This suggests that treatments that untangle the vessels rather than shrink them are needed. The experiments show that LSFM is a valuable tool for studying eye diseases, that may help scientists learn more about how these diseases arise and develop. These new insights may one day lead to better tests and treatments for eye diseases.

The number of grey values that can be displayed on monitors and be processed by the human eye is smaller than the dynamic range of image-based sensors. This makes the visualization of such data a challenge, especially with specimens where small dim structures are equally important as large bright ones, or whenever variations in intensity, such as non-homogeneous staining efficiencies or light depth penetration, becomes an issue. While simple intensity display mappings are easily possible, these fail to provide a one-shot observation that can display objects of varying intensities. In order to facilitate the visualization-based analysis of large volumetric datasets, we developed an easy-to-use ImageJ plugin enabling the compressed display of features within several magnitudes of intensities. The Display Enhancement for Visual Inspection of Large Stacks plugin (DEVILS) homogenizes the intensities by using a combination of local and global pixel operations to allow for high and low intensities to be visible simultaneously to the human eye. The plugin is based on a single, intuitively understandable parameter, features a preview mode, and uses parallelization to process multiple image planes. As output, the plugin is capable of producing a BigDataViewer-compatible dataset for fast visualization. We demonstrate the utility of the plugin for large volumetric image data.

We introduce MosaicExplorerJ, an ImageJ macro to stitch 3D tiles from terabyte-size microscopy datasets. As opposed to existing software, stitching does not require any prior information on the actual positions of the tiles, sample fiducials, or conversion of raw TIFF images, and the stitched images can be explored instantly. MosaicExplorerJ was specifically designed to process lightsheet microscopy datasets from optically cleared samples. It can handle multiple fluorescence channels, dual-side lightsheet illumination and dual-side camera detection.

The spinal cord contains a diverse array of sensory and motor circuits that are essential for normal function. Spinal cord injury (SCI) permanently disrupts neural circuits through initial mechanical damage, as well as a cascade of secondary injury events that further expand the spinal cord lesion, resulting in permanent paralysis. Tissue clearing and 3D imaging have recently emerged as promising techniques to improve our understanding of the complex neural circuitry of the spinal cord and the changes that result from damage due to SCI. However, the application of this technology for studying the intact and injured spinal cord remains limited. Here, we optimized the passive CLARITY technique (PACT) to obtain gentle and efficient clearing of the murine spinal cord without the need for specialized equipment. We demonstrate that PACT clearing enables 3D imaging of multiple fluorescent labels in the spinal cord to assess molecularly defined neuronal populations, acute inflammation, long-term tissue damage, and cell transplantation. Collectively, these procedures provide a framework for expanding the utility of tissue clearing to enhance the study of spinal cord neural circuits, as well as cellular- and tissue-level changes that occur following SCI.