We present time-lapse Raman imaging (TLRI) of living cells as a new approach in label-free chemical imaging through non-electronic resonant, spontaneous Raman microspectroscopy. Raman hyperspectral datacubes of individual live peripheral blood lymphocytes were successively acquired. The Raman imaging time per voxel, with a volume of 0.3 fl, was 100 ms and the total image time of a 32 × 32 pixels image was less than 2 min. Multiple images of an individual cell have been obtained. A full series of TLRI images typically resulted in more than 1.6 million data points per image. We analyzed the datasets using hierarchical cluster analysis. A fingerprint of molecular changes was observed before the cell was blebbing. The molecular fingerprint was related to a gradual disappearance of the Raman signal from carotenoids. Concomitant changes occurred in the CH stretch high wavenumber region, presumably due to a change in the protein and lipid environment of carotenoids. These changes were smaller than 5% of the total signal at 2937 cm−1. We hypothesize that the lipid environment of the carotenoids changes as a result of the photophysics in the carotenoid molecules. The detectability of carotenoids was shown to be 2.3 µMper voxel, which corresponds to 415 molecules. TLRI enables high-speed chemical imaging not only in the intense high wavenumber region of the Raman spectrum, but particularly in the more informative fingerprint region between 500 and 1800 cm−1. Copyright © 2010 John Wiley & Sons, Ltd.