Simultaneous wide-field epifluorescence and TIRF [Ca²⁺] measurements in voltage-clamped single colonic myocytes. Depolarization (−70 to +10 mV; H) activated a voltage-dependent Ca²⁺ current (I_Ca; G) to evoke a rise in [Ca²⁺] (B, C, E, and F). The rise in [Ca²⁺], which occurred in the subplasma membrane space (measured by TIRF; C and F), was more rapid in onset than that seen in the bulk cytoplasm (measured by wide-field epifluorescence; B and E). The [Ca²⁺] images (B and C) are derived from the time points indicated by the corresponding numerals in D. [Ca²⁺] changes in B and C are represented by color: blue low and red/white high [Ca²⁺]. The images (B and C) were taken before (i), during (ii and iii), and after (iv–vi)depolarization and show the resulting [Ca²⁺] changes. Changes in the fluorescence ratio with time (E and F) are derived from 2 × 2–pixel boxes (regions 1–6 in A, middle and right (expanded) panels, drawn at a 3 × 3–pixel size to facilitate visualization) and from a larger region encompassing the entire TIRF region (region 1). The latter was used to obtain an average subplasma membrane and bulk average [Ca²⁺]_c increase (I). Significantly, although the [Ca²⁺]_c increase that occurred in the bulk cytoplasm (B and E) was approximately uniform and simultaneous throughout the cell, those in the subplasma membrane space (C and F) had a wide range of amplitudes and various time courses. Note the spark-like events in region 3 toward the end of the recording. (A; left) A bright field image of the cell; see also whole cell electrode (right side). Insets in E and F show the rising phase of the transients on an expanded time base. For comparison, I shows the average subplasma membrane and bulk average [Ca²⁺]_c rise as measured in region 1 (A, right). B and C (VII) show an enlargement of ii to illustrate the localized nature of the rise in [Ca²⁺] in the subplasma membrane space.