Real-time imaging of cellular and subcellular dynamics in vascularized organs requires image resolution and image registration to be simultaneously optimized without perturbing normal physiology. This problem is particularly pronounced in the lung, in which cells may transit at speeds >1 mm s−1 and in which normal respiration results in large-scale tissue movements that prevent image registration. Here we report video-rate, two-photon imaging of a physiologically intact preparation of the mouse lung that is stabilizing and nondisruptive. Using our method, we obtained evidence for differential trapping of T cells and neutrophils in mouse pulmonary capillaries, and observed neutrophil mobilization and dynamic vascular leak in response to stretch and inflammatory models of lung injury in mice. The system permits physiological measurement of motility rates of >1 mm s−1, observation of detailed cellular morphology and could be applied in the future to other organs and tissues while maintaining intact physiology.
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We thank O. Khan for his side-view rendering of the thoracic suction window and N. Amodaj for software support. This work was supported in part by US National Institutes of Health grants K08 HL082742 (M.R.L.) and PO1 HL024136 (M.F.K.), by the American Asthma Foundation and Sandler Basic Asthma Research Center (M.F.K.), by the National Blood Foundation (M.R.L.) and by the National Science Foundation Graduate Research Fellowships Program (E.E.T.).
The authors declare no competing financial interests.
Supplementary Figure 1 (PDF 153 kb)
Ventilation with thoracic suction. Real-time brightfield video of expanding and contracting alveoli during ventilation in the microscope setup at 100A magnification. (MOV 1844 kb)
Video-rate intravital lung imaging. Video-rate (30 fps, single z plane) two-photon movie of actin-CFP (blue) mouse with unaveraged inspiration and expiration. (MOV 4669 kb)
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. (MOV 189 kb)
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. (MOV 240 kb)
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-1 with an average of ~110 μm s-1 were observed inside capillaries. (MOV 1595 kb)
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-1 with a range of 106.8–728.3 μm s-1. (MOV 9035 kb)
Naive and T-cell blast migration in the lungs. Left-sided movie from an actin-CFP mouse injected with naive CD2-RFP (red) T cells (5 A 107). Right-sided movie from a wild-type mouse injected with T-cell blasts (ubiquitin-GFP, green, 5 A 107). (MOV 3539 kb)
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 min after intratracheal MIP-2 treatment (right, 5 μg, i.t., 40 μm z stack). (MOV 1933 kb)
Intravascular neutrophil migratory activity in the lung. Two-photon video of a LysM-GFP neutrophil (green) crawling through dextran marked vasculature (red) at 60 min after 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. (MOV 8651 kb)
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 after 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. (MOV 925 kb)
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) before (left) and 70 min after LPS treatment (5 mg kg-1, i.t., 40 μm z stack). (MOV 7266 kb)
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 after LPS treatment (5 mg kg-1, i.t., 40 μm z stack). (MOV 529 kb)
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 after induction with high tidal volume lung injury (ventilator-induced lung injury, 40 μm z stack). (MOV 848 kb)
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Looney, M., Thornton, E., Sen, D. et al. Stabilized imaging of immune surveillance in the mouse lung. Nat Methods 8, 91–96 (2011). https://doi.org/10.1038/nmeth.1543
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