Fig. 2. Dual loading of colonic myocytes with Fluo-4 and TMRE allows simultaneous observation of [Ca²⁺]_c and _m, leaving shorter wavelengths to be used for UV-photolysis of caged compounds. (A) In spite of overlapping excitation and emission profiles (i), cross-talk between Fluo-4 and TMRE fluorescence signals was minimized using a custom-made dichroic mirror (black line, ii), dual-bandpass excitation (dark-blue line, ii) and emission (light-blue line, ii) filters and exciting at 475 and 560 nm (ii). (B) Virtual absence of overlap between Fluo-4 and TMRE channels in cells loaded with either Fluo-4 alone (10 µM, i) or, in another cell, with TMRE alone (10 nM, ii). Each cell was excited at 475 nm (left-hand panel) and 560 nm (right-hand panel). Pseudocolored images represent emission intensity from a 12-bit range (calibration in top-left corners). (C) Mitochondrial localization of TMRE (10 nM, i) was confirmed by its co-localization with the mitochondria-specific dye MitoTracker Green (25 nM, ii). (iii) Overlay of red TMRE on green MitoTracker images produces yellow regions in which the two indicators are co-localized. (iv) A bright-field image of the cell is also shown. (D) Separation of [Ca²⁺]_c and _m measurements in a voltage-clamped myocyte co-loaded with Fluo-4 (green) and TMRE (red). Plasmalemma depolarization (500 ms, –70 to +10 mV, ii) induced a large increase in Fluo-4 fluorescence with no change in TMRE fluorescence (i); subsequent CCCP (1 µM) treatment caused a large decrease in TMRE fluorescence with no decrease in Fluo-4 fluorescence. Scale bars: 10 µm.