Video-rate two-photon imaging of mouse footpad - a promising model for studying leukocyte recruitment dynamics during inflammation

Abstract

Leukocyte recruitment is a key host defense mechanism to infection and a salient feature of autoimmune diseases such as arthritis. The cell dynamics of these processes are difficult to study due to the challenge of tracking cells flowing in the circulation and migrating through light scattering tissues. Here, we describe a noninvasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. In the absence of inflammation, cells moved > several hundred microm/s in vessels and only rarely adhered to endothelium or entered the tissue parenchyma. In response to bacterial infection, neutrophils moved in small capillaries at reduced speeds of (14-45 microm/min) and rolled in larger vessels at 5-60 microm/min. Within minutes of adoptive transfer, neutrophils entered the connective tissue and crawled with a median velocity of 7.3 microm/min. 2P imaging has excellent spatiotemporal resolution and is a promising in vivo approach to study the cellular basis of inflammation.

Fig. 1

(A) Footpad preparation for 2P imaging. An anesthetized mouse’s hind paw was secured with Vetbound tissue glue to the bottom of a heated (37 °C) imaging chamber, covered with PBS and imaged with a 20 × 0.95 NA Olympus water dipping objective. The mouse is resting on a 37 °C warming pad to prevent hypothermia. (B) 3D reconstruction of mouse footpad tissue. The image shows a rendered tissue volume with the dimensions x = 225 μm, y = 200 μm and z = 150 μm. Rhodamine dextran labeled blood vessels appear red, CD11c-YFP expressing cells appear green and connective tissue appears blue. Scale bar equals 40 μm. (C) Time-lapse images of neutrophils homing to inflamed footpad. Each time point represents a maximum intensity projection of 31-z-planes (75 μm). The scale bar represents 40 μm. Yellow arrows indicate a CFSE-labeled neutrophil (green) with “torpedo like morphology” moving in a small capillary (red). White arrows show a representative neutrophil migrating in the tissue parenchyma. (D) Measuring high-speed cell movement in microvessels. A continuous 1-second video-rate record (single plane) showing a CFSE labeled neutrophil flowing through a vessel at 300 μm/s. Each dot is the cell captured in successive frames. By measuring the distance traveled between frames (see asterisk, each $\raisebox{1ex}{$1$}\!\left/ \!\raisebox{-1ex}{$30$}\right.$ of a second apart) cell velocity can be calculated. Scale bar equals 20 μm.