Light-sheet Fluorescence Microscopy Imaging Research Articles

Megakaryocyte volume modulates bone marrow niche properties and cell migration dynamics.

All hematopoietic cells that develop in the bone marrow must cross the endothelial barrier to enter the blood circulation. Blood platelets, however, are released by bigger protrusions of huge progenitor cells, named megakaryocytes, and enter the blood stream as so-called proplatelets before fragmenting into mature platelets. Recently, a second function of megakaryocytes has been identified, as they modulate the quiescence of hematopoietic stem cells, mostly via different soluble factors. We know from light sheet fluorescence microscopy images that megakaryocytes are distributed throughout the bone marrow facing a dense vascular network. Here, we used such three-dimensional images to provide a realistic simulation template reflecting the in vivo cell-vessel distributions resulting in reliable whole-bone analysis in silico. Combining this approach with an automated image analysis pipeline, we found that megakaryocytes influence migration of neutrophils and hematopoietic stem cells, and thus act as biomechanical restrainers modulating cell mobility and extravasation. Indeed, as a consequence of increased megakaryocyte volumes in platelet-depleted mice neutrophil mobility was reduced in these animals.

Performance of convolutional neural networks for identification of bacteria in 3D microscopy datasets.

Three-dimensional microscopy is increasingly prevalent in biology due to the development of techniques such as multiphoton, spinning disk confocal, and light sheet fluorescence microscopies. These methods enable unprecedented studies of life at the microscale, but bring with them larger and more complex datasets. New image processing techniques are therefore called for to analyze the resulting images in an accurate and efficient manner. Convolutional neural networks are becoming the standard for classification of objects within images due to their accuracy and generalizability compared to traditional techniques. Their application to data derived from 3D imaging, however, is relatively new and has mostly been in areas of magnetic resonance imaging and computer tomography. It remains unclear, for images of discrete cells in variable backgrounds as are commonly encountered in fluorescence microscopy, whether convolutional neural networks provide sufficient performance to warrant their adoption, especially given the challenges of human comprehension of their classification criteria and their requirements of large training datasets. We therefore applied a 3D convolutional neural network to distinguish bacteria and non-bacterial objects in 3D light sheet fluorescence microscopy images of larval zebrafish intestines. We find that the neural network is as accurate as human experts, outperforms random forest and support vector machine classifiers, and generalizes well to a different bacterial species through the use of transfer learning. We also discuss network design considerations, and describe the dependence of accuracy on dataset size and data augmentation. We provide source code, labeled data, and descriptions of our analysis pipeline to facilitate adoption of convolutional neural network analysis for three-dimensional microscopy data.

Multiscale imaging of plant development by light-sheet fluorescence microscopy.

Light-sheet fluorescence microscopy (LSFM) methods collectively represent the major breakthrough in developmental bio-imaging of living multicellular organisms. They are becoming a mainstream approach through the development of both commercial and custom-made LSFM platforms that are adjusted to diverse biological applications. Based on high-speed acquisition rates under conditions of low light exposure and minimal photo-damage of the biological sample, these methods provide ideal means for long-term and in-depth data acquisition during organ imaging at single-cell resolution. The introduction of LSFM methods into biology extended our understanding of pattern formation and developmental progress of multicellular organisms from embryogenesis to adult body. Moreover, LSFM imaging allowed the dynamic visualization of biological processes under almost natural conditions. Here, we review the most important, recent biological applications of LSFM methods in developmental studies of established and emerging plant model species, together with up-to-date methods of data editing and evaluation for modelling of complex biological processes. Recent applications in animal models push LSFM into the forefront of current bio-imaging approaches. Since LSFM is now the single most effective method for fast imaging of multicellular organisms, allowing quantitative analyses of their long-term development, its broader use in plant developmental biology will likely bring new insights.

Restoration of Light Sheet Multi-View Data with the Huygens Fusion and Deconvolution Wizard

Abstract

Three-Dimensional Cross-Sectional Light-Sheet Microscopy Imaging of the Inflamed Mouse Gut

Three-Dimensional Cross-Sectional Light-Sheet Microscopy Imaging of the Inflamed Mouse Gut

Cardiac Light-Sheet Fluorescent Microscopy for Multi-Scale and Rapid Imaging of Architecture and Function.

Light Sheet Fluorescence Microscopy (LSFM) enables multi-dimensional and multi-scale imaging via illuminating specimens with a separate thin sheet of laser. It allows rapid plane illumination for reduced photo-damage and superior axial resolution and contrast. We hereby demonstrate cardiac LSFM (c-LSFM) imaging to assess the functional architecture of zebrafish embryos with a retrospective cardiac synchronization algorithm for four-dimensional reconstruction (3-D space + time). By combining our approach with tissue clearing techniques, we reveal the entire cardiac structures and hypertrabeculation of adult zebrafish hearts in response to doxorubicin treatment. By integrating the resolution enhancement technique with c-LSFM to increase the resolving power under a large field-of-view, we demonstrate the use of low power objective to resolve the entire architecture of large-scale neonatal mouse hearts, revealing the helical orientation of individual myocardial fibers. Therefore, our c-LSFM imaging approach provides multi-scale visualization of architecture and function to drive cardiovascular research with translational implication in congenital heart diseases.

3D Visualization of Osteogenic Capillaries in Mineralized Bone Tissue by Integrating Micro-CT with Light Sheet Fluorescence Microscopy Imaging

3D Visualization of Osteogenic Capillaries in Mineralized Bone Tissue by Integrating Micro-CT with Light Sheet Fluorescence Microscopy Imaging