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Highlights • SBFSEM was introduced in 2004 and has become a major tool for three-dimensional electron microscopy. • SBFSEM has become widespread in connectomics, cellular and matrix biology. • Problems with the technique are being overcome by advances in technology. • Data analysis represents a bottleneck but advances in computation and crowd-sourcing are set to improve this. Abstract Three-dimensional electron microscopy (3DEM) is an imaging field containing several powerful modalities such as serial section transmission electron microscopy and electron tomography. However, large-scale 3D studies of biological ultrastructure on a cellular scale have historically been hampered by the difficulty of available techniques. Serial block face scanning electron microscopy (SBFSEM) is a 3DEM technique, developed in 2004, which has greatly increased the reliability, availability and throughput of 3DEM. SBFSEM allows for 3D imaging at resolutions high enough to resolve membranes and small vesicles whilst having the capability to collect data with a large field of view. Since its introduction it has become a major tool for ultrastructural investigation and has been applied in the study of many biological fields, such as connectomics, cellular and matrix biology. In this review, we will discuss biological SBFSEM from a technical standpoint, with a focus on cellular applications and also subsequent image analysis techniques. [ABSTRACT FROM AUTHOR]

Extracellular matrix microfibrils are critical components of connective tissues with a wide range of mechanical and cellular signalling functions. Collagen VI is a heteromeric network-forming collagen which is expressed in tissues such as skin, lung, blood vessels and articular cartilage where it anchors cells into the matrix allowing for transduction of biochemical and mechanical signals. It is not understood how collagen VI is arranged into microfibrils or how these microfibrils are arranged into tissues. Therefore we have characterised the hierarchical organisation of collagen VI across multiple length scales. The frozen hydrated nanostructure of purified collagen VI microfibrils was reconstructed using cryo-TEM. The bead region has a compact hollow head and flexible tail regions linked by the collagenous interbead region. Serial block face SEM imaging coupled with electron tomography of the pericellular matrix (PCM) of murine articular cartilage revealed that the PCM has a meshwork-like organisation formed from globular densities ∼30 nm in diameter. These approaches can characterise structures spanning nanometer to millimeter length scales to define the nanostructure of individual collagen VI microfibrils and the micro-structural organisation of these fibrils within tissues to help in the future design of better mimetics for tissue engineering. Statement of Significance Cartilage is a connective tissue rich in extracellular matrix molecules and is tough and compressive to cushion the bones of joints. However, in adults cartilage is poorly repaired after injury and so this is an important target for tissue engineering. Many connective tissues contain collagen VI, which forms microfibrils and networks but we understand very little about these assemblies or the tissue structures they form. Therefore, we have use complementary imaging techniques to image collagen VI microfibrils from the nano-scale to the micro-scale in order to understand the structure and the assemblies it forms. These findings will help to inform the future design of scaffolds to mimic connective tissues in regenerative medicine applications. [ABSTRACT FROM AUTHOR]

Tendons transmit muscle-generated force through an extracellular matrix of aligned collagen fibrils. The force applied by the muscle at one end of a microscopic fibril has to be transmitted through the macroscopic length of the tendon by mechanisms that are poorly understood. A key element in this structure-function relationship is the collagen fibril length. During embryogenesis short fibrils are produced but they grow rapidly with maturation. There is some controversy regarding fibril length in adult tendon, with mechanical data generally supporting discontinuity while structural investigations favor continuity. This study initially set out to trace the full length of individual fibrils in adult human tendons, using serial block face-scanning electron microscopy. But even with this advanced technique the required length could not be covered. Instead a statistical approach was used on a large volume of fibrils in shorter image stacks. Only a single end was observed after tracking 67.5 mm of combined fibril lengths, in support of fibril continuity. To shed more light on this observation, the full length of a short tendon (mouse stapedius, 125 μm) was investigated and continuity of individual fibrils was confirmed. In light of these results, possible mechanisms that could reconcile the opposing findings on fibril continuity are discussed. Statement of Significance Connective tissues hold all parts of the body together and are mostly constructed from thin threads of the protein collagen (called fibrils). Connective tissues provide mechanical strength and one of the most demanding tissues in this regard are tendons, which transmit the forces generated by muscles. The length of the collagen fibrils is essential to the mechanical strength and to the type of damage the tissue may experience (slippage of short fibrils or breakage of longer ones). This in turn is important for understanding the repair processes after such damage occurs. Currently the issue of fibril length is contentious, but this study provides evidence that the fibrils are extremely long and likely continuous. [ABSTRACT FROM AUTHOR]

Cells in tendon deposit parallel arrays of collagen fibrils to form a functional tissue, but how this is achieved is unknown. The cellular mechanism is thought to involve the formation of intracellular collagen fibrils within Golgi to plasma membrane carriers. This is facilitated by the intracellular processing of procollagen to collagen by members of the tolloid and ADAMTS families of enzymes. The carriers subsequently connect to the extracellular matrix via finger-like projections of the plasma membrane, known as fibripositors. In this study we have shown, using three-dimensional electron microscopy, the alignment of fibripositors with intracellular fibrils as well as an orientated cable of actin filaments lining the cytosolic face of a fibripositor. To demonstrate a specific role for the cytoskeleton in coordinating extracellular matrix assembly, cytochalasin was used to disassemble actin filaments and nocodazole or colchicine were used to disrupt microtubules. Microtubule disruption delayed procollagen transport through the secretory pathway, but fibripositor numbers were unaffected. Actin filament disassembly resulted in rapid loss of fibripositors and a subsequent disappearance of intracellular fibrils. Procollagen secretion or processing was not affected by cytochalasin treatment, but the parallelism of extracellular collagen fibrils was altered. In this case a significant proportion of collagen fibrils were found to no longer be orientated with the long axis of the tendon. The results suggest an important role for the actin cytoskeleton in the alignment and organization of the collagenous extracellular matrix in embryonic tendon. [ABSTRACT FROM AUTHOR]

Summary Distinct mechanisms involving cell shape and mechanical force are known to influence the rate and orientation of division in cultured cells. However, uncoupling the impact of shape and force in tissues remains challenging. Combining stretching of Xenopus tissue with mathematical methods of inferring relative mechanical stress, we find separate roles for cell shape and mechanical stress in orienting and cueing division. We demonstrate that division orientation is best predicted by an axis of cell shape defined by the position of tricellular junctions (TCJs), which align with local cell stress rather than tissue-level stress. The alignment of division to cell shape requires functional cadherin and the localization of the spindle orientation protein, LGN, to TCJs but is not sensitive to relative cell stress magnitude. In contrast, proliferation rate is more directly regulated by mechanical stress, being correlated with relative isotropic stress and decoupled from cell shape when myosin II is depleted. Graphical Abstract Highlights • Tissue stretching increases division rate and reorients divisions with stretch • Division orientation is regulated by cell shape defined by tricellular junctions • Cadherin and LGN localize to tricellular junctions aligning division to cell shape • Division rate is linked to mechanical stress and can be decoupled from cell shape Nestor-Bergmann et al. use whole-tissue stretching and mathematical modeling to dissect the roles of mechanical stress and cell shape in cell division. They show that division orientation in stretched tissue is regulated indirectly by changes in cell shape, while division rate is more directly regulated by mechanical stress. [ABSTRACT FROM AUTHOR]

In development, wound healing, and cancer metastasis, vertebrate cells move through 3D interstitial matrix, responding to chemical and physical guidance cues. Protrusion at the cell front has been extensively studied, but the retraction phase of the migration cycle is not well understood. Here, we show that fast-moving cells guided by matrix cues establish positive feedback control of rear retraction by sensing membrane tension. We reveal a mechanism of rear retraction in 3D matrix and durotaxis controlled by caveolae, which form in response to low membrane tension at the cell rear. Caveolae activate RhoA-ROCK1/PKN2 signaling via the RhoA guanidine nucleotide exchange factor (GEF) Ect2 to control local F-actin organization and contractility in this subcellular region and promote translocation of the cell rear. A positive feedback loop between cytoskeletal signaling and membrane tension leads to rapid retraction to complete the migration cycle in fast-moving cells, providing directional memory to drive persistent cell migration in complex matrices. • Fast-moving cells in 3D matrix establish low membrane tension at the rear • Caveolae form in response to low membrane tension and recruit the GEF Ect2 • Ect2 activates RhoA to promote F-actin organization and rear retraction • Positive feedback between membrane tension and contractility reinforces retraction Cell migration through 3D matrix is critical to developmental and disease processes, but the mechanisms that control rear retraction are poorly understood. Hetmanski et al. show that differential membrane tension allows caveolae to form at the rear of migrating cells and activate the contractile actin cytoskeleton to promote rapid retraction. [ABSTRACT FROM AUTHOR]