Recently, the multivariate curve resolution-alternating least squares (MCR-ALS) method, also known as self-modeling curve resolution or non-negative matrix factorization,35 has been developed and applied to the spectral decomposition of Raman spectra.25,29,31,36–41 It has also been exploited in many kinds of spectroscopy, high performance liquid chromatography,42,43 gas chromatograph/mass spectrometry,44 UV-VIS,45 near-infrared,46,47 FT-IR,48,49 fluorescence imaging,50 etc. In the MCR method, the experimental data is approximated by a linear combination of several spectral components.51,52 The decomposition of superposed spectral data sets is done with ALS calculation, under appropriate model constraints, such as non-negativity of spectral profiles and their concentrations. Due to these constraints, this method easily provides physically interpretable spectral components, without any a priori information on chemical components in the sample specimen such as a living cell. Using this advantage, we have successfully applied the MCR-ALS method to the analysis of molecular component distribution imaging (MCDI) in living cells, whose raw spectra contain a number of unknown spectral components and are hard to interpret without a priori information. We have developed an in-house software for the MCR-ALS of numerous Raman spectral data. In the following, we first show the capability of the MCR-ALS method by using a model system consisting of two different types of crystal polymorphs of titanium oxide (), anatase and rutile. MCDI of polymorphs is successfully obtained. Then, we show the MCR-ALS analysis of the time- and space-resolved Raman spectra of a dividing fission yeast cell. Unexpected dynamic changes of major cellular molecular components (lipids, proteins, and polysaccharides) during the cell cycle have been elucidated. Finally, we show the results of the study of intracellular water in a living budding yeast cell. We have successfully resolved so far unknown organelle-specific water structures by the MCR-ALS method. Highly important and otherwise unobtainable MCDI information has thus been obtained for the two living cell systems in vivo.