Virtual reality-empowered deep-learning analysis of brain cells.

Automated detection of specific cells in three-dimensional datasets such as whole-brain light-sheet image stacks is challenging. Here, we present DELiVR, a virtual reality-trained deep-learning pipeline for detecting c-Fos+ cells as markers for neuronal activity in cleared mouse brains. Virtual reality annotation substantially accelerated training data generation, enabling DELiVR to outperform state-of-the-art cell-segmenting approaches. Our pipeline is available in a user-friendly Docker container that runs with a standalone Fiji plugin. DELiVR features a comprehensive toolkit for data visualization and can be customized to other cell types of interest, as we did here for microglia somata, using Fiji for dataset-specific training. We applied DELiVR to investigate cancer-related brain activity, unveiling an activation pattern that distinguishes weight-stable cancer from cancers associated with weight loss. Overall, DELiVR is a robust deep-learning tool that does not require advanced coding skills to analyze whole-brain imaging data in health and disease.

Whole-body cellular mapping in mouse using standard IgG antibodies.

Whole-body imaging techniques play a vital role in exploring the interplay of physiological systems in maintaining health and driving disease. We introduce wildDISCO, a new approach for whole-body immunolabeling, optical clearing and imaging in mice, circumventing the need for transgenic reporter animals or nanobody labeling and so overcoming existing technical limitations. We identified heptakis(2,6-di-O-methyl)-ß-cyclodextrin as a potent enhancer of cholesterol extraction and membrane permeabilization, enabling deep, homogeneous penetration of standard antibodies without aggregation. WildDISCO facilitates imaging of peripheral nervous systems, lymphatic vessels and immune cells in whole mice at cellular resolution by labeling diverse endogenous proteins. Additionally, we examined rare proliferating cells and the effects of biological perturbations, as demonstrated in germ-free mice. We applied wildDISCO to map tertiary lymphoid structures in the context of breast cancer, considering both primary tumor and metastases throughout the mouse body. An atlas of high-resolution images showcasing mouse nervous, lymphatic and vascular systems is accessible at http://discotechnologies.org/wildDISCO/atlas/index.php .

Deciphering sources of PET signals in the tumor microenvironment of glioblastoma at cellular resolution.

Various cellular sources hamper interpretation of positron emission tomography (PET) biomarkers in the tumor microenvironment (TME). We developed an approach of immunomagnetic cell sorting after in vivo radiotracer injection (scRadiotracing) with three-dimensional (3D) histology to dissect the cellular allocation of PET signals in the TME. In mice with implanted glioblastoma, translocator protein (TSPO) radiotracer uptake per tumor cell was higher compared to tumor-associated microglia/macrophages (TAMs), validated by protein levels. Translation of in vitro scRadiotracing to patients with glioma immediately after tumor resection confirmed higher single-cell TSPO tracer uptake of tumor cells compared to immune cells. Across species, cellular radiotracer uptake explained the heterogeneity of individual TSPO-PET signals. In consideration of cellular tracer uptake and cell type abundance, tumor cells were the main contributor to TSPO enrichment in glioblastoma; however, proteomics identified potential PET targets highly specific for TAMs. Combining cellular tracer uptake measures with 3D histology facilitates precise allocation of PET signals and serves to validate emerging novel TAM-specific radioligands.

Immune-mediated denervation of the pineal gland underlies sleep disturbance in cardiac disease.

Disruption of the physiologic sleep-wake cycle and low melatonin levels frequently accompany cardiac disease, yet the underlying mechanism has remained enigmatic. Immunostaining of sympathetic axons in optically cleared pineal glands from humans and mice with cardiac disease revealed their substantial denervation compared with controls. Spatial, single-cell, nuclear, and bulk RNA sequencing traced this defect back to the superior cervical ganglia (SCG), which responded to cardiac disease with accumulation of inflammatory macrophages, fibrosis, and the selective loss of pineal gland-innervating neurons. Depletion of macrophages in the SCG prevented disease-associated denervation of the pineal gland and restored physiological melatonin secretion. Our data identify the mechanism by which diurnal rhythmicity in cardiac disease is disturbed and suggest a target for therapeutic intervention.

Spatial proteomics in three-dimensional intact specimens.

Spatial molecular profiling of complex tissues is essential to investigate cellular function in physiological and pathological states. However, methods for molecular analysis of large biological specimens imaged in 3D are lacking. Here, we present DISCO-MS, a technology that combines whole-organ/whole-organism clearing and imaging, deep-learning-based image analysis, robotic tissue extraction, and ultra-high-sensitivity mass spectrometry. DISCO-MS yielded proteome data indistinguishable from uncleared samples in both rodent and human tissues. We used DISCO-MS to investigate microglia activation along axonal tracts after brain injury and characterized early- and late-stage individual amyloid-beta plaques in a mouse model of Alzheimer's disease. DISCO-bot robotic sample extraction enabled us to study the regional heterogeneity of immune cells in intact mouse bodies and aortic plaques in a complete human heart. DISCO-MS enables unbiased proteome analysis of preclinical and clinical tissues after unbiased imaging of entire specimens in 3D, identifying diagnostic and therapeutic opportunities for complex diseases. VIDEO ABSTRACT.