| Abstract: | 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 C H 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. |
