Correlative and volumetric EM imaging of inflammatory dynamics in skin vessels

Abstract text (incl. figure legends and references)

Introduction: Serial block-face scanning electron microscopy (SBF-SEM) increases reliability, availability and throughput of 3D-EM. The ability to resolve nanoscales with a large field of view and great depths, can provide insight to many biological fields. However, analysis of cellular dynamics remains exclusive to fluorescent markers, visualized in light microscopy. Pairing both processes in a complicated, correlative (CLEM) approach is therefore necessary and new reliable workflows have to be established, taking into consideration mismatch of resolution and targeting on nano-scale level. For instance, tissue homeostasis and its balance upon inflammatory events often lead to severe changes regarding permeability of blood vessels and transmigration of neutrophils and platelets. Neutrophils may leave the vessel in a controlled process¹, whereas platelets seal the endothelial barrier, preventing vascular leakage². Interaction dynamics of both have been observed at sites of inflammation³ but to this day lacked ultrastructural investigation.
Objectives: Observe the interaction of platelets and leukocytes following an inflammation stimulus via intravital microscopy (IVM) before fixation. Relocate that specific vessel and region of interest led by artificial and natural landmarks to allow subsequent ultrastructural investigation. Adapt the fixation and staining protocol for complex, multi-layered skin samples and enable consistent SBF-SEM imaging.
M&M: A dorsal skinfold chamber frame was attached to anesthetized and depilated LysMeGFP mice, injected with immunolabelled PECAM-1 (vascular endothelium) and GPIbβ (platelets) and subjected to immune-complex mediated vasculitis. Neutrophil recruitment and inflammatory bleeding events within a designated stamp-labelled area were imaged for 90-120 min. Subsequent sample fixation and introduction of additional landmarks included perforation of the skin, allowing penetration of fluids, biopsy-punching the area of interest and glueing it on carbon-gridded coverslips. To enhance contrast it was crucial to increase the temperature to 40 °C during all incubation steps of the adapted protocol for SBF-SEM imaging. Beneath extensive Osmium staining, infiltration was performed with a mixture of Ketjenblack to account for charge accumulation during SBF-SEM imaging.
Results: The use of natural and artificial landmarks enabled precise relocation of distinct vessels and targeted trimming. IVM in response to inflammation stimuli reveals that neutrophils leave vessels through partial openings of endothelial junctions. Additionally, rare occasions of co-interaction in which an increased number of platelets follow the transmigration of neutrophils can be observed. Adaptations to the staining protocol enables volumetric serial block-face imaging to detect complete platelets transferring the basement membrane barrier.
Conclusion: Many CLEM projects remain challenging throughout all steps, reaching from sample preparation towards relocation and final imaging. The workflow presented had to be optimized step-by-step. Finally, SBF-SEM offers the ability to visualize complex, volumetric cell interactions. Combination of state-of-the-art LM tools can help guide and address relevant biological questions.

Fig.1: Representative IVM MIP and SBF-SEM volume merge of neutrophil recruitment.

¹Vestweber D (2015) How leukocytes cross the vascular endothelium

²Braun LJ et al. (2020) Platelets docking to VWF prevent leaks during leukocyte extravasation by stimulating Tie-2

³Lisman T (2018) Platelet-neutrophil interactions as drivers of inflammatory and thrombotic disease