Monitoring Early Molecular Responses to Temperature Stimuli-Circadian rhythms in cyanobacterial cells and the Kai in vitro protein oscillator can be entrained by temperature changes (9,10,24,25). Using our FCS-based method to analyze the oscillatory system, we attempted to observe changes of the KaiB interaction profile in response to temperature stimuli. Phase responses to short stimulus pulses provide basic analytic **data** for system analysis; these experiments require many samples and **data** points obtained over time. Furthermore, transition states between the old and new phases contain useful information that helps elucidate the dynamics of the system. Monitoring the transition states requires a series of **data** with high time resolution. FCS is a good tool for this kind of experiment. We exposed the Kai protein oscillator to a high temperature pulse and evaluated the phase response of the rhythmic KaiB interactions. First, we prepared aliquots of the standard mixture, and each aliquot was exposed to 50°C for 30 min after 12, 16, 20, 24, 28, or 32 h of incubation time. The relatively high temperature did not affect the amplitude of the oscillation, suggesting that the components of the reaction mixtures were not denatured during the experiment (Fig. 5 and supplemental Fig.  S3). We detected phase shifts in the circadian rhythm when the samples were exposed to the high temperature after 20 or 28 h of incubation (Fig. 5, a and c). Aliquots exposed to the pulse after 20 h of incubation showed a phase advance of ϳ2 h compared with the control sample, whereas the pulse delivered after 28 h of incubation delayed the phase by ϳ2 h. A temperature pulse after incubation for 12, 16, 24, or 32 h did not induce any detectable phase changes ( Fig. 5b and supplemental Fig. S3).