Imaging innate immunity.

Innate immunity is the first line of defense against infectious intruders and also plays a major role in the development of sterile inflammation. Direct microscopic imaging of the involved immune cells, especially neutrophil granulocytes, monocytes, and macrophages, has been performed since more than 150 years, and we still obtain novel insights on a frequent basis. Initially, intravital microscopy was limited to small-sized animal species, which were often invertebrates. In this review, we will discuss recent results on the biology of neutrophils and macrophages that have been obtained using confocal and two-photon microscopy of individual cells or subcellular structures as well as light-sheet microscopy of entire organs. This includes the role of these cells in infection defense and sterile inflammation in mammalian disease models relevant for human patients. We discuss their protective but also disease-enhancing activities during tumor growth and ischemia-reperfusion damage of the heart and brain. Finally, we provide two visions, one experimental and one applied, how our knowledge on the function of innate immune cells might be further enhanced and also be used in novel ways for disease diagnostics in the future.

Rapid and fully automated blood vasculature analysis in 3D light-sheet image volumes of different organs.

Light-sheet fluorescence microscopy (LSFM) can produce high-resolution tomograms of tissue vasculature with high accuracy. However, data processing and analysis is laborious due to the size of the datasets. Here, we introduce VesselExpress, an automated software that reliably analyzes six characteristic vascular network parameters including vessel diameter in LSFM data on average computing hardware. VesselExpress is ∼100 times faster than other existing vessel analysis tools, requires no user interaction, and integrates batch processing and parallelization. Employing an innovative dual Frangi filter approach, we show that obesity induces a large-scale modulation of brain vasculature in mice and that seven other major organs differ strongly in their 3D vascular makeup. Hence, VesselExpress transforms LSFM from an observational to an analytical working tool.

ComplexEye: a multi-lens array microscope for high-throughput embedded immune cell migration analysis.

Autonomous migration is essential for the function of immune cells such as neutrophils and plays an important role in numerous diseases. The ability to routinely measure or target it would offer a wealth of clinical applications. Video microscopy of live cells is ideal for migration analysis, but cannot be performed at sufficiently high-throughput (HT). Here we introduce ComplexEye, an array microscope with 16 independent aberration-corrected glass lenses spaced at the pitch of a 96-well plate to produce high-resolution movies of migrating cells. With the system, we enable HT migration analysis of immune cells in 96- and 384-well plates with very energy-efficient performance. We demonstrate that the system can measure multiple clinical samples simultaneously. Furthermore, we screen 1000 compounds and identify 17 modifiers of migration in human neutrophils in just 4 days, a task that requires 60-times longer with a conventional video microscope. ComplexEye thus opens the field of phenotypic HT migration screens and enables routine migration analysis for the clinical setting.

High-resolution three-dimensional imaging for precise staging in melanoma.

INTRODUCTION: Many cancer guidelines include sentinel lymph node (SLN) staging to identify microscopic metastatic disease. Current SLN analysis of melanoma patients is effective but has the substantial drawback that only a small representative portion of the node is sampled, whereas most of the tissue is discarded. This might explain the high clinical false-negative rate of current SLN diagnosis in melanoma. Furthermore, the quantitative assessment of metastatic load and microanatomical localisation might yield prognosis with higher precision. Thus, methods to analyse entire SLNs with cellular resolution apart from tedious sequential physical sectioning are required. PATIENTS AND METHODS: Eleven melanoma patients eligible to undergo SLN biopsy were included in this prospective study. SLNs were fixed, optically cleared, whole-mount stained and imaged using light sheet fluorescence microscopy (LSFM). Subsequently, compatible and unbiased gold standard histopathological assessment allowed regular patient staging. This enabled intrasample comparison of LSFM and histological findings. In addition, the development of an algorithm, RAYhance, enabled easy-to-handle display of LSFM data in a browsable histologic slide-like fashion. RESULTS: We comprehensively quantify total tumour volume while simultaneously visualising cellular and anatomical hallmarks of the associated SLN architecture. In a first-in-human study of 21 SLN of melanoma patients, LSFM not only confirmed all metastases identified by routine histopathological assessment but also additionally revealed metastases not detected by routine histology alone. This already led to additional therapeutic options for one patient. CONCLUSION: Our three-dimensional digital pathology approach can increase sensitivity and accuracy of SLN metastasis detection and potentially alleviate the need for conventional histopathological assessment in the future. GERMAN CLINICAL TRIALS REGISTER: (DRKS00015737).

Loss of the Extracellular Matrix Molecule Tenascin-C Leads to Absence of Reactive Gliosis and Promotes Anti-inflammatory Cytokine Expression in an Autoimmune Glaucoma Mouse Model.

Previous studies demonstrated that retinal damage correlates with a massive remodeling of extracellular matrix (ECM) molecules and reactive gliosis. However, the functional significance of the ECM in retinal neurodegeneration is still unknown. In the present study, we used an intraocular pressure (IOP) independent experimental autoimmune glaucoma (EAG) mouse model to examine the role of the ECM glycoprotein tenascin-C (Tnc). Wild type (WT ONA) and Tnc knockout (KO ONA) mice were immunized with an optic nerve antigen (ONA) homogenate and control groups (CO) obtained sodium chloride (WT CO, KO CO). IOP was measured weekly and electroretinographies were recorded at the end of the study. Ten weeks after immunization, we analyzed retinal ganglion cells (RGCs), glial cells, and the expression of different cytokines in retina and optic nerve tissue in all four groups. IOP and retinal function were comparable in all groups. Although RGC loss was less severe in KO ONA, WT as well as KO mice displayed a significant cell loss after immunization. Compared to KO ONA, less ßIII-tubulin+ axons, and downregulated oligodendrocyte markers were noted in WT ONA optic nerves. In retina and optic nerve, we found an enhanced GFAP+ staining area of astrocytes in immunized WT. A significantly higher number of retinal Iba1+ microglia was found in WT ONA, while a lower number of Iba1+ cells was observed in KO ONA. Furthermore, an increased expression of the glial markers Gfap, Iba1, Nos2, and Cd68 was detected in retinal and optic nerve tissue of WT ONA, whereas comparable levels were observed in KO ONA. In addition, pro-inflammatory Tnfa expression was upregulated in WT ONA, but downregulated in KO ONA. Vice versa, a significantly increased anti-inflammatory Tgfb1 expression was measured in KO ONA animals. We conclude that Tnc plays an important role in glial and inflammatory response during retinal neurodegeneration. Our results provide evidence that Tnc is involved in glaucomatous damage by regulating retinal glial activation and cytokine release. Thus, this transgenic EAG mouse model for the first time offers the possibility to investigate IOP-independent glaucomatous damage in direct relation to ECM remodeling.