Visualization of intracellular NO production in platelets adhering to type I collagen under flow. DAF-FM–loaded platelets (5 × 10⁷/mL) and washed erythrocytes (hematocrit 42%-45%) were suspended in plasma and HEPES-Tyrode buffer containing 2 mM CaCl₂/MgCl₂ (1:1 vol/vol). The cell suspensions were perfused for 3 minutes over fibrillar type I collagen at the wall shear rate of 3000 s⁻¹ and fluorescence intensity within surface-interacting platelets was monitored for 60 seconds. (A) Representative single-frame images demonstrating fluorescence increases in the first 60 seconds of perfusion. (B) Surface-interacting platelets were monitored in real time during the first 60 seconds. The traces, obtained with the automatic Casti Imaging procedure, are representative curves of fluorescence intensity of 3 single platelets (representative example of 3 experiments). (C) The number of platelets exhibiting a fluorescence increase over a 3-minute period was enumerated in the absence (control) or presence of different concentrations of l-Arg or of L-NMMA, as indicated, and expressed as percent DAF-positive platelets of control (*P < .01 vs untreated). (D) The mean fluorescence intensity (MFI), an index of NO production in DAF-FM–loaded platelets, was quantified for 30 seconds in the absence (control) or presence of different concentrations of l-Arg or of L-NMMA, as indicated (**P < .001 vs untreated).