Time- and space-resolved Raman spectra of mitochondria in single living fission yeast cells have been measured under various nutrient, stress, and atmospheric conditions. A focus is placed on the behavior of the Raman band located at 1602 cm<sup>−1</sup>, which sensitively reflects the metabolic activity in mitochondria and which has been called by us the "Raman spectroscopic signature of life". Addition of nutrients increases the intensity of this band by ∼1.5 times, confirming its correlation with the metabolic activity in mitochondria. The spectra of cells cultured under 100% N<sub>2</sub>, 100% O<sub>2</sub>, and N<sub>2</sub>/O<sub>2</sub> (V<sub>N2</sub>:V<sub>O2</sub> ≅ 4:1) atmospheres have been measured for both <sup>16</sup>O<sub>2</sub> and <sup>18</sup>O<sub>2</sub>. Yeast cells have been found to lose their metabolic activity after the culture under 100% N<sub>2</sub> and 100% O<sub>2</sub> atmospheres. Cells cultured under a N<sub>2</sub>/<sup>16</sup>O<sub>2</sub> (<sup>16</sup>O<sub>2</sub> = 20%) atmosphere show strong "Raman spectroscopic signature of life". No <sup>18</sup>O isotopic shift has been found for the wavenumber 1602 cm<sup>−1</sup>, indicating that the origin of this signature is neither O<sub>2</sub> nor an O-containing small molecule. Addition of H<sub>2</sub>O<sub>2</sub> causes a quick decrease of the "Raman spectroscopic signature of life", followed by the <i>cis–trans</i> isomerization in the unsaturated phospholipid chain. The "Raman spectroscopic signature of life" has thus been proved to be a reliable real-time and <i>in vivo</i> indicator for monitoring the metabolic activity in living cells.

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