the flow rate (cm3/s), m represents the viscosity (0.01 Poise at room tem- perature), B represents the total plate separation (0.02 cm), and W repre- sents the width (0.2 cm). The wall shear rate, gw (s21), was calculated as gw 5 6Q/(B2W). Distortions in the flow field, because of the sidewalls, were minimal for the 1:10 aspect ratio of the gap separation to the cham- ber width and for the imaging of events only in the middle of the chamber away from the sidewalls. Digital images were captured with a high speed digital camera (MotionCorder Analyzer; Eastman Kodak Co.) at imaging rates of 30–240 frames per second (fps). Images from the high speed cam- era were played back at slow motion (5 fps) or frame-by-frame and re- corded on videotape. Control of the videotape recorder was performed using LabVIEW and IMAQ Vision Software (National Instruments) with serial communication to a Pentium-based workstation, while images were digitized via an on-board capture card (model PCI-IMAQ 1408; National Instruments). During video playback, some images were contrast en- hanced with background subtraction and grey scale stretching using an Argus 20 Image Processor (Hamamatsu Corp.). Analysis of Neutrophil–Platelet Interactions When unstimulated neutrophils were perfused over spread platelet sin- glets, three general types of interaction occurred: (type 1) membrane tether formation during neutrophil translation downstream without ro- tation (see Fig. 1 a); (type 2) neutrophil rolling over the platelet without observable membrane tether formation and with an observed neutrophil rotation; or (type 3) pausing without an observable membrane tether formation or rolling. Type 1 interactions were analyzed by determina- tion of cell center positions in successive video frames from the digitized images using Scion Image software. The position of the cell center was determined within one pixel accuracy (6 0.09 mm/pixel) at each instant. During type 1 events, these tethers were extremely thin and short-lived, and often crossed the focal plane of the DIC image. Thus, the growing tethers were sometimes difficult to completely visualize in the image plane because manual refocusing was not possible in real-time during the adhesive events. However, when the microscope was readjusted to Schmidtke and Diamond Neutrophil Tethering under Flow 721 Figure 1. Neutrophil tether- ing to platelets under flow. (a) Schematic of a flowing neutrophil colliding with a spread platelet and forming an elongated membrane tether. (b) Formation of a neutrophil–platelet tether at gw 5 200 s21. Digitized im- ages taken from a video sequence of a neutrophil approaching an adherent platelet (t 5 0–0.0167 s), cap- ture (t 5 0.0292 s), growth of tether (t 5 0.0625–0.1750 s), and release (t 5 0.2125 s). The arrows on the left in the frames at t 5 0.0625 and t 5 0.1750 point to a bright sur- face feature on the neutro- phil, demonstrating that the cell is translating and not ro- tating as the tether grows. Video capture rate, 240 fps (time resolution 5 4.5 ms). (c) A single membrane tether was observed between a tear- drop-shaped neutrophil and an adherent platelet (flow from right to left). Bar, 10 mm.