Fig. 6. Real-time calcium measurements in motile control and CAL-treated T. gondii. (A) Time-lapse images of calcium flux in DMSO, 1 µM CAL and 1 µM caffeine-treated parasites. Treated parasites were labeled with fluo-4 and allowed to glide. Parasites were observed by fluorescence and phase-contrast microscopy and recorded at 0.5 second intervals. Shown are selected merged images with the time elapsed between frames indicated in seconds. Bar, 5 µm. (B) Graph of absolute frame-by-frame fluorescence pixel intensity of one representative movie per condition showing fluo-4 fluorescence intensity oscillations during DMSO, CAL or caffeine-treated gliding. ^*The point at which the caffeine-treated parasite stopped gliding. (C) Normalized intensities of fluo-4 oscillations in control and CAL movies shown in B. Calcium oscillations (numbered) in CAL-treated parasites occurred at twice the frequency but half the amplitude of calcium oscillations in untreated parasites. F_t/F_o, temporal fluorescence intensity of fluo-4 divided by the fluorescence intensity at the start of each cycle.