Frontline Science: Dynamic cellular and subcellular features of migrating leukocytes revealed by in vivo lattice lightsheet microscopy

Abstract

Neutrophil and macrophage (M) 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.

Live Imaging of Heart Injury in Larval Zebrafish Reveals a Multi-Stage Model of Neutrophil and Macrophage Migration

“…This live imaging directly confirmed the CHT as a major source of mobilized neutrophils and macrophages in both laser heart injury and tail transection. Others have reported the use of blood vessels (Manley et al, 2020), however our timelapse videos show this to be the primary mode of migration. We speculate that the immune adherence proteins found on endothelium might make these routes more tractable to migrating neutrophils and macrophages (von Andrian et al, 1993).…”

High-speed squeeze: Light-sheet imaging of zebrafish neutrophils

“…This study is notable in 2 respects, first, for the nuanced morphological features it reveals in full 3D volumes of leukocytes imaged as they move through tissues and vessels, which confirm and embellish the cell behaviors recorded in many previous studies using more conventional forms of in vitro and intra‐vital imaging 1,3–5 . Now, painstaking quantification 2 from sets of up to 30 LLSM movies also delivers metrics not previously possible for the evaluation of cell volumes, uropod extensions, and other cell features. Second, by successfully setting up a system for intra‐vital LLSM on zebrafish larvae—a technological feat—their high‐quality imaging and text‐book depiction of classical neutrophil behaviors showcase the capabilities inherent in LLSM and presage its use for a wide range of future studies on leukocytes and other cell types.…”

“…A new study by Manley et al. uses cutting‐edge, lattice light‐sheet microscopy (LLSM) to re‐examine neutrophil migration and intravascular behavior in zebrafish larvae 2 . This study is notable in 2 respects, first, for the nuanced morphological features it reveals in full 3D volumes of leukocytes imaged as they move through tissues and vessels, which confirm and embellish the cell behaviors recorded in many previous studies using more conventional forms of in vitro and intra‐vital imaging 1,3–5 .…”

“…By making the most of the high‐speed and high‐resolution capabilities of LLSM, compared to confocal imaging, Manley and colleagues 2 tracked the fast moving and multi‐directional contortions of neutrophils, and in some cases Mϕs, during interstitial, wound‐induced migration in transgenic zebrafish larvae labeled with membrane and cytoplasmic fluorescent reporters. These findings shore up current knowledge about immune cell behaviors, derived earlier from imaging cultured cells or in some cases with conventional intra‐vital imaging in transgenic zebrafish larvae or mice 1,6 .…”

LLAMA: a robust and scalable machine learning pipeline for analysis of cell surface projections in large scale 4D microscopy data

“…This imaging now enables live, 3D fluorescence imaging of sufficient speed, duration and temporal-spatial resolution to adequately capture and record exquisite details of dynamic cell surface projections. LLSM studies have recorded novel features of ruffling, macropinocytic cups, filopodia and other surface projections in amoeba and vertebrate immune cells (Condon et al, 2018; Fritz-Laylin et al, 2017; Manley et al, 2020; Veltman et al, 2016). LLSM recordings in 3D extending over many hours can capture thousands of cell surface protrusions., routinely resulting in terabyte-scale datasets that cannot be interrogated manually or with traditional segmentation, nor with techniques such as thresholding and automatic spot detection, which require careful calibration or manual editing (Heddleston & Chew, 2016).…”