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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-1α (MIP-1α/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 .

In Vivo Imaging and Quantitative Analysis of Leukocyte Directional Migration and Polarization in Inflamed Tissue