Direct real-time imaging of leukocyte recruitment in vivo using spinning disk confocal intravital microscopy. (A-F) Staphylococcus aureus–infected (intradermal 1 × 108 CFU of methicillin-resistant S aureus, USA300-2406) LysM-eGFP mouse skin microvasculature was imaged in vivo. Images shown in panels A through C demonstrate neutrophils defined by LysM-eGFP expression (green), and the corresponding side-by-side images shown in panels D through F demonstrate neutrophils defined by the anti–Gr-1 antibody (5 μg IV) conjugated to Alexa Fluor 750 (yellow) at identical time points. Endothelium was visualized using an anti–PECAM-1 antibody (10 μg IV) conjugated to Alexa Fluor 647 (blue). (A,D) Images of neutrophils rolling and adhering within a vessel captured immediately after administration of live S aureus into the mouse skin. (B,E) Images of neutrophils transmigrating out of a vessel captured 60 minutes after S aureus administration. (C,F) Images of neutrophils emigrating and chemotaxing within the parenchyma 60 minutes after S aureus administration. Images were captured using an Olympus BX51 upright microscope equipped with a 10×/0.3 numeric aperture air objective. The microscope was equipped with a confocal light path (WaveFx; Quorum) based on a modified CSU-10 head (Yokogawa Electric). Laser excitation at 488, 649, and 730 nm (Cobalt) was used in rapid succession with the appropriate long-pass filters (Semrock). A 512 × 512 pixel back-thinned EMCCD camera (C9100-13; Hamamatsu) was used for fluorescence detection. Volocity Acquisition Version 6.0 software (Improvision) was used to drive the confocal microscope. Images captured using the spinning disk were processed and analyzed in Volocity with linear adjustments to the black-and-white points for improved image quality. Bars represent 60 μm. Images are representative of 3 independent experiments. (G-L) Neutrophil emigration (G), parenchymal tissue crawling velocity (H), meandering index (I), displacement over 10 minutes (J), and tissue neutrophil crawling tracks (K-L) after live S aureus administration in LysM-eGFP mice without antineutrophil antibodies and LysM-eGFP mice that received IV anti–Gr-1 (5 μg) immediately before imaging. For cell quantification, 2 independent LysM-eGFP experiments were compared with 3 independent LysM-eGFP + anti–Gr-1 antibody experiments. All procedures performed were approved by the University of Calgary Animal Care Committee and were in accordance with the Canadian Guidelines for Animal Research.
