Fig. 7. Localisation of calcium channels using dihydropyridine-BODIPY (fDHP) and RH414 in striated muscles dissociated at room temperature (20°C; A) and at 30 °C (B). Dissociated cells were incubated with both fDHP (final concentration 10 µmol l-1) and the styryl dye RH414 (final concentration 5 µmol l-1), for 15 min at room temperature before examination. Images were obtained using laser-scanning, confocal microscopy. The wavelengths of the excitation beam were set to 488 nm and 568 nm. The emitted fluorescence from labelled cells was collected at 530 nm for green fluorescence (fDHP) and 590 nm for red fluorescence (RH414). At 20 °C cells were spherical and fDHP labelled mostly small vesicles within the cytoplasm (A) while at 30 °C there was strong fDHP labelling of the sarcolemma of the muscle feet (B). Scale bars, 2 µm. (C) Ratio of labelling intensity of green (fDHP) versus red (RH414) fluorescence in the plasma membrane of myofibres (feet) and the cell somata. Data on the intensity of fluorescence (both green and red) were collected from those areas of the membrane showing sharp and definitive RH414 labelling (red), indicating that a perpendicular optical section through the cell membrane was being analyzed. The intensity of green fluorescence (fDHP) was divided by the intensity of red fluorescence (RH414) pixel-by-pixel using NIH image software. Values are means ± S.E.M., N=20 cells. Asterisk, significant difference (P<0.05).
