LLSM imaging reveals tent pole ruffles. Macrophages stably expressing GFP-LifeAct incubated in medium containing LPS were imaged using LLSM. (A) Single frame from LLSM recording (Video 3) visualized by tilting the FOV on the x axis and using the Amira Blue-Green LUT. Boxed region shows 11-s intervals illustrating a full ruffling event. Tent poles at the ruffle margins are marked with red and white arrows. (B) A second example using a 3D-surface render of GFP-LifeAct (Video 4) to show details of tent poles within the F-actin sheath. Poles indicated by the red and white arrows switch sides during crossover. (C) 3D rendering of x,y and x,z ruffle event (Video 5). The marginal tent poles are overlaid with red and green. The ruffle veil is overlaid blue, and in the upper panel, it is extracted from the x,y plane to illustrate the linear ruffle circularization and constriction phases. Bars: 10 µm (main panels); 5 µm (inset panels). Time stamps, min:s. (D) Quantification of ruffle type from Videos 1 and 2. Data are represented as mean ± SEM; n = 6 cells with a total of 133 tent pole–associated and 27 non–tent pole–associated ruffles. (E) Formation of ruffles in the same location (hotspots) from a 30-min period in Videos 1 and 2, represented as a frequency distribution. Frequency distribution is calculated as a percentage from n = 10 cells, containing 104 hotspots and 345 ruffles. (F) Quantification of mean ruffle duration and mean time to crossover of tent poles. Data are represented as a min/max box whisker plot calculated using n = 115 events.