CA2+ Sparks within 200 nm of the Sarcolemma of Rat Ventricular Cells: Evidence from Total Internal Reflection Fluorescence Microscopy

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Abstract
Total internal reflection fluorescence microscopy (TIRFM) was used to measure local calcium releases in resting cardiac myocytes stained with fluo-3AM. The measured fluorescence originated from regions where cells were close to, and develop adhesions to, a totally reflecting glass surface. The excitation of the fluorescent Ca2+ indicator dye by the exponentially attenuated evanescent wave penetrated approximately 200 nm into the fluid phase. In rat ventricular cells, Ca2+ waves and Ca2+ sparks were observed within the adhesions. Ca2+ sparks recorded with TIRFM compared favorably to sparks recorded under similar conditions with confocal microscopy. Computer simulation supported this assessment. It is concluded that TIRFM can provide an economical, flexible tool for detailed measurement of Ca2+-transients in the subsarcolemmal space of live cells.