4D imaging of neutrophil transmigration. αSMA-RFPcherry ×Lys-EGFP-ki mice exhibiting endogenously labeled pericytes (RFPcherry, red) and neutrophils (EGFP, green) were subjected to EC junctional labeling using a directly conjugated Alexa Fluor 647 nonblocking mAb to PECAM-1 mAb (blue). (A) Composition of six 3D-reconstructed confocal images of the cremasteric microcirculation 120 and 240 min after intrascrotal injection of TNF. a, arteriole; c, capillary; cp, capillary pericyte; pcv, postcapillary venule. Bar, 100 µm. (B) Still images of a cremaster muscle postcapillary venule showing the development of an inflammatory reaction at the indicated time points after stimulation with TNF. Bar, 10 µm. (C) The images in the top panels are 3D reconstructions of half postcapillary venules showing a luminal neutrophil at an early stage of breaching the endothelium (cell 1) and a neutrophil that has already crossed the endothelium and is between the endothelium and pericyte layer (cell 2) at 3 h after TNF stimulation. The middle and bottom panels show the position of the indicated neutrophils from the luminal side (left hand side) or from 2-µm cross-sections of venules (right hand side) along the indicated lines demonstrating the position of the leukocyte relative to ECs and the pericyte layer. Bar, 10 µm. (D) Using the 3D real-time imaging model to analyze neutrophil transmigration through venular walls, several distinct steps were observed as illustrated in the diagram: (1) TEM, (2) motility between endothelium and the pericyte sheath (abluminal crawling), (3) migration through gaps between adjacent pericytes, and (4) interstitial migration. Images are representative of at least six separate experiments.