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Main Menu - Block
- Overview
- Anatomy and Histology
- Cryo-Electron Microscopy
- Electron Microscopy
- Flow Cytometry
- Gene Targeting and Transgenics
- Immortalized Cell Line Culture
- Integrative Imaging
- Invertebrate Shared Resource
- Janelia Experimental Technology
- Mass Spectrometry
- Media Prep
- Molecular Genomics
- Primary & iPS Cell Culture
- Project Pipeline Support
- Project Technical Resources
- Quantitative Genomics
- Scientific Computing Software
- Scientific Computing Systems
- Viral Tools
- Vivarium
Abstract
Neutrophil and macrophage (Mϕ) migration underpin the inflammatory response. However, the fast velocity, multidirectional instantaneous movement, and plastic, ever-changing shape of phagocytes confound high-resolution intravital imaging. Lattice lightsheet microscopy (LLSM) captures highly dynamic cell morphology at exceptional spatiotemporal resolution. We demonstrate the first extensive application of LLSM to leukocytes in vivo, utilizing optically transparent zebrafish, leukocyte-specific reporter lines that highlighted subcellular structure, and a wounding assay for leukocyte migration. LLSM revealed details of migrating leukocyte morphology, and permitted intricate, volumetric interrogation of highly dynamic activities within their native physiological setting. Very thin, recurrent uropod extensions must now be considered a characteristic feature of migrating neutrophils. LLSM resolved trailing uropod extensions, demonstrating their surprising length, and permitting quantitative assessment of cytoskeletal contributions to their evanescent form. Imaging leukocytes in blood vessel microenvironments at LLSM's spatiotemporal resolution displayed blood-flow-induced neutrophil dynamics and demonstrated unexpected leukocyte-endothelial interactions such as leukocyte-induced endothelial deformation against the intravascular pressure. LLSM of phagocytosis and cell death provided subcellular insights and uncovered novel behaviors. Collectively, we provide high-resolution LLSM examples of leukocyte structures (filopodia lamellipodia, uropod extensions, vesicles), and activities (interstitial and intravascular migration, leukocyte rolling, phagocytosis, cell death, and cytoplasmic ballooning). Application of LLSM to intravital leukocyte imaging sets the stage for transformative studies into the cellular and subcellular complexities of phagocyte biology.
