5: Supplementary Movie 4 Lung circulation with thoracic suction
Two-photon video of wild-type mouse injected with Texas Red dextran (red) marking vasculature. Unlabeled cellular shadows are observed in motion in the labeled vasculature.
6: Supplementary Movie 5 Perfusion velocities in the lung
Two-photon video of a single z plane with 1 μm beads (red) flowing through alveolar capillaries of diameter 10–15 μm. Bead tracks are marked with dragon tails. Vasculature marked with actin-CFP (blue). Time is indicated in s:ms. Bead speeds of ~0–400 μm/s with an average of ~110 μm/s were observed inside capillaries.
7: Supplementary Movie 6 Simultaneous imaging of neutrophil and bead velocities in the lung
Video-rate two-photon movie of a single z plane with i.v. 1 μm beads (red) flowing through vasculature (30 μm diameter) marked with actin-CFP (blue). Neutrophils (c-fms, green) flow through the vessels. Cell (green) and bead (red) tracks are marked with dragon tails. Bead track speed average is 297.6 μm/s with a range from 106.8 – 728.3 μm/s.
8: Supplementary Movie 7 Naïve and T cell blast migration in the lungs
Left-sided movie from an actin-CFP mouse injected with naïve CD2-RFP (red) T cells (5 × 107). Right-sided movie from a wild-type mouse injected with T cell blasts (ubiquitin-GFP, green, 5 × 107).
9: Supplementary Movie 8 Neutrophil recruitment into the lung with intratracheal MIP-2
Two-photon video of LysM-GFP marked neutrophils (green) in dextran marked vasculature (red) at baseline (left) and 70 minutes post-intratracheal MIP-2 treatment (right, 5 μg, i.t., 40 μm z stack).
10: Supplementary Movie 9 Intravascular neutrophil migratory activity in the lung
Two-photon video of a LysM-GFP neutrophil (green) crawling through dextran marked vasculature (red) at 60 minutes post-MIP-2 treatment (5 μg, i.t.). The center of mass of the intravascular neutrophil is marked with a grey sphere. Flashing yellow arrowheads indicate the alternating leading edge of the intravascular neutrophil.
11: Supplementary Movie 10 Extravascular neutrophil migratory activity in the lung
Two-photon video of LysM-GFP neutrophils (green) in dextran (red) marked vasculature in a mouse 60 min post-MIP-2 treatment (5 μg, i.t.). The first pass consists of one z plane to show cellular detail with the cell of interest, marked with a white sphere, deforming itself to move between alveolar spaces. The second pass includes 40 μm in z to provide context.
12: Supplementary Movie 11 Neutrophil recruitment into the lung after intratracheal LPS
Two-photon video of c-fms-GFP neutrophils (green) in a lung where all cells are marked by actin-CFP (blue) pre- (left panel) and 70 min post-LPS treatment (5 mg/kg, i.t., 40 μm z stack).
13: Supplementary Movie 12 Lung vascular leak after intratracheal LPS
Two-photon video of lung tissue marked with actin-CFP (blue) showing dextran leak (red) into the extravascular space 50 min post-LPS treatment (5 mg/kg, i.t., 40 μm z stack).
14: Supplementary Movie 13 Ventilator-induced lung injury and lung vascular leak
Two-photon video of lung tissue marked with actin-CFP(blue) showing dextran leak (red) into the extravascular space 50 min post-induction with high tidal volume lung injury (ventilator-induced lung injury, 40 μm z stack).
2: Supplementary Movie 1 Ventilation with thoracic suction
Real-time brightfield video of expanding and contracting alveoli during ventilation in the microscope set-up at 100× magnification.
3: Supplementary Movie 2 Video-rate intravital lung imaging
Video-rate (30 fps, single z plane) two-photon movie of actin-CFP (blue) mouse with un-averaged inspiration and expiration.
4: Supplementary Movie 3 Fifteen frame-averaged intravital lung imaging
Two-photon video of actin-CFP (blue) mouse with averaged (fifteen frames, single z plane) acquisition showing stability and intravascular cellular movement.