In vivo imaging and quantitative analysis of leukocyte directional migration and polarization in inflamed tissue

Directional migration of transmigrated leukocytes to the site of injury is a central event in the inflammatory response. Here, we present an in vivo chemotaxis assay enabling the visualization and quantitative analysis of subtype-specific directional motility and polarization of leukocytes in their natural 3D microenvironment. Our technique comprises the combination of i) semi-automated in situ microinjection of chemoattractants or bacteria as local chemotactic stimulus, ii) in vivo near-infrared reflected-light oblique transillumination (RLOT) microscopy for the visualization of leukocyte motility and morphology, and iii) in vivo fluorescence microscopy for the visualization of different leukocyte subpopulations or fluorescence-labeled bacteria. Leukocyte motility parameters are quantified off-line in digitized video sequences using computer-assisted single cell tracking. Here, we show that perivenular microinjection of chemoattractants [macrophage inflammatory protein-1alpha (MIP-1alpha/Ccl3), platelet-activating factor (PAF)] or E. coli into the murine cremaster muscle induces target-oriented intravascular adhesion and transmigration as well as polarization and directional interstitial migration of leukocytes towards the locally administered stimuli. Moreover, we describe a crucial role of Rho kinase for the regulation of directional motility and polarization of transmigrated leukocytes in vivo. Finally, combining in vivo RLOT and fluorescence microscopy in Cx3CR1(gfp/gfp) mice (mice exhibiting green fluorescent protein-labeled monocytes), we are able to demonstrate differences in the migratory behavior of monocytes and neutrophils.Taken together, we propose a novel approach for investigating the mechanisms and spatiotemporal dynamics of subtype-specific motility and polarization of leukocytes during their directional interstitial migration in vivo

Morphology and interdiffusion behavior of evaporated metal films on crystalline diindenoperylene films

We present a transmission electron microscopy (TEM)/Rutherford backscattering spectrometry (RBS)/x-ray-diffraction (XRD) study of Au evaporated on crystalline organic thin films of diindenoperylene (DIP). Cross-sectional TEM shows that the preparation conditions of the Au film (evaporation rate and substrate temperature) strongly determine the interfacial morphology. In situ XRD during annealing reveals that the organic layer is thermally stable up to about 150 degreesC, a temperature sufficient for most electronic applications. The x-ray measurements show that the "as-grown" Au layer exhibits a large mosaicity of around 10degrees. Upon annealing above approximate to120 degreesC the Au film starts to reorder and shows sharp (111)-diffraction features. In addition, temperature dependent RBS measurements indicate that the Au/DIP interface is thermally essentially stable against diffusion of Au in the DIP layer up to approximate to100 degreesC on the time scale of hours, dependent on the Au thickness. (C) 2003 American Institute of Physics