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Biology subjects

Greenbaum, A.

Publications and source records attributed to Greenbaum, A..

4 recordsLinked to original sources

Enhancing Light-Sheet Fluorescence Microscopy Illumination Beams through Deep Design Optimization

Light sheet fluorescence microscopy (LSFM) provides the benefit of optical sectioning coupled with rapid acquisition times for imaging of tissue-cleared specimen. This allows for high-resolution 3D imaging of large tissue volumes. Inherently to LSFM, the quality of the imaging heavily relies on the characteristics of the illumination beam, with the notion that the illumination beam only illuminates a thin section that is being imaged. Therefore, substantial efforts are dedicated to identifying slender, non-diffracting beam profiles that can yield uniform and high-contrast images. An ongoing debate concerns the employment of the most optimal illumination beam; Gaussian, Bessel, Airy patterns and/or others. Comparisons among different beam profiles is challenging as their optimization objective is often different. Given that our large imaging datasets ([~]0.5TB images per sample) is already analyzed using deep learning models, we envisioned a different approach to this problem by hypothesizing that we can tailor the illumination beam to boost the deep learning models performance. We achieve this by integrating the physical LSFM illumination model after passing through a variable phase mask into the training of a cell detection network. Here we report that the joint optimization continuously updates the phase mask, improving the image quality for better cell detection. Our methods efficacy is demonstrated through both simulations and experiments, revealing substantial enhancements in imaging quality compared to traditional Gaussian light sheet. We offer valuable insights for designing microscopy systems through a computational approach that exhibits significant potential for advancing optics design that relies on deep learning models for analysis of imaging datasets.

scientific communication and education↗

Transgenic porcine model reveals two roles for LGR5 during lung development and homeostasis

Cells expressing LGR5 play a pivotal role in homeostasis, repair, and regeneration in multiple organs including skin and gastrointestinal tract, yet little is known about their role in the lung. Findings from mice, a widely used animal model, suggest that lung LGR5 expression differs from that of humans. In this work, using a new transgenic pig model, we identify two main populations of LGR5+ cells in the lung that are conserved in human, but not mouse lungs. Using RNA sequencing, 3D imaging and organoid models, we determine that in the fetal lung, epithelial LGR5 expression is transient in a subpopulation of SOX9+/ETV+/SFTPC+ progenitor lung tip cells. In contrast, epithelial LGR5 expression is absent from postnatal lung, but is reactivated in bronchioalveolar organoids derived from basal airway cells. We also describe a separate population of mesenchymal LGR5+ cells that surrounds developing and mature airways, lies adjacent to airway basal cells, and is closely associated with nerve fibers. Transcriptionally, mesenchymal LGR5+ cells include a subset of peribronchial fibroblasts (PBF) that express unique patterns of SHH, FGF, WNT and TGF-{beta} signaling pathway genes. These results support distinct roles for LGR5+ cells in the lung and describe a physiologically relevant animal model for further studies on the function of these cells in repair and regeneration.

cell biology↗

COMBINe: Automated Detection and Classification of Neurons and Astrocytes in Tissue Cleared Mouse Brains

Tissue clearing renders entire organs transparent to enable combination with light sheet fluorescence microscopy and accelerate whole tissue imaging. Yet, challenges remain in analyzing the large resulting 3D datasets that consist of terabytes of images and information on millions of labeled cells. Previous work has established pipelines for automated analysis of tissue cleared mouse brains. However, they have focused on single color channels and/or detection of nuclear localized signals, in relatively low-resolution images. To address this gap, we present an automated workflow to map labeled neurons and astrocytes in the genetically distinct Mosaic Analysis with Double Markers (MADM) mouse forebrains. We named the workflow COMBINe (Cell detectiOn in Mouse BraIN) as it combines modules from multiple pipelines. With RetinaNet in its core, we quantitatively analyzed the regional and subregional effects of MADM-based deletion of the Epidermal growth factor receptor on neuronal and astrocyte populations in the mouse forebrain.

neuroscience↗

Identification of peripheral neural circuits that regulate heart rate using optogenetic and viral vector strategies

Heart rate is under the precise control of the autonomic nervous system. However, the wiring of peripheral neural circuits that regulate heart rate is poorly understood. Here, we developed a clearing-imaging-analysis pipeline to visualize innervation of intact hearts in 3D and employed a multi-technique approach to map parasympathetic and sympathetic neural circuits that control heart rate in mice. We anatomically and functionally identify cholinergic neurons and noradrenergic neurons in an intrinsic cardiac ganglion and the stellate ganglia, respectively, that project to the sinoatrial node. We also report that the heart rate response to optogenetic versus electrical stimulation of the vagus nerve displays different temporal characteristics and that vagal afferents enhance parasympathetic and reduce sympathetic tone to the heart via central mechanisms. Our findings provide new insights into neural regulation of heart rate, and our methodology to study cardiac circuits can be readily used to interrogate neural control of other visceral organs.

neuroscience↗