Figure 2

Imaging and recording of Ca²⁺ sparks. (A) Series of confocal images obtained at a temporal resolution of 41.5 ms. Ca²⁺ sparks were identified as highly localized elevations in the cytosolic Ca²⁺ concentration of the smooth muscle cell (outlined in the left-most image).(B) Image and fluorescent trace obtained from a typical 5s scan at a temporal resolution of 1.92 ms shows spontaneous Ca²⁺ sparks occurring with high frequency. Numbers beside transients indicate sparks. Sparks were identified and frequency was calculated using custom-designed software. The sparks marked 'x' and 'y' are shown in (C) and (D) with an expanded resolution. Amplitude (Peak - Base intensity), rise time (Peak - Base time) and time to half decay (Half decay - Peak time) were obtained from individual Ca²⁺ sparks. (C) An example showing identification of sparks and calculation of spark parameters by the software for a Ca²⁺ spark rising from the baseline. (D) An example showing identification of sparks and calculation of spark parameters by the software for a Ca²⁺ spark "pair", where the second spark rises before the first returns to baseline. Spark amplitude, rise time and decay time are calculated only from the first and not the second spark.