Fig. 2. FLIP analysis of DiIC₁₆ diffusion in the plasma membrane overlying the sperm head. (A) Separate frames from a video sequence to illustrate repeated photobleaching of a 2.0 µm diameter area located at the tip of the anterior acrosome (broken circle, frame 46). Only four out of the six bleaches are shown (bleaches were in frames 45, 153, 233 and 326). With each successive bleach, the intensity of fluorescence over the whole head declines uniformly. (B) (i) line-profile analysis of fluorescence intensity along the x-y axis shown in A in the pre-bleach state (frame 43), immediately post-bleach (frame 46), and after 3 seconds' recovery (frame 151); (ii) same as in (i) except after four successive bleaches (recovery frame 405). Arrows at `a' and `b' mark the anterior and posterior positions of the equatorial segment. Note that fluorescence intensity is similar across the equatorial segment-postacrosome boundary. (C) Similar to A except the bleach beam is located over the postacrosome (broken circle in pre-bleach frame 20). There was no difference in fluorescence intensity along the c-d axis after six successive bleaches (frame 554), indicating free diffusion of the lipid between surface compartments. Bar, 2 µm.