Chemical imaging with Fourier transform infrared (FT-IR) spectroscopy allows the visualization of the distribution of chemical components in cells without the need for labels or added dyes. However, obtaining such images of living cells is difficult because of the strong absorption of water in the mid-infrared region. We report the use of attenuated total reflection (ATR) FT-IR spectroscopic imaging to study live human cancer cells in an aqueous environment, on a single cell level. Two complementary approaches have been used, providing flexibility with field of view and spatial resolution: (1) micro-ATR FT-IR imaging using a microscope objective with a Ge crystal, and (2) single-reflection diamond ATR-FT-IR imaging. Using both approaches, the ATR-FT-IR spectroscopic signatures allow the differentiation between several cellular organelles, e.g., the nucleus and the endoplasmic reticulum (ER). The overall cell shape can be defined by the distribution of the amide II band in the measured image, while the DNA-rich nucleus and glycogen-rich ER could be imaged using the spectral bands at 1084 cm<sup>−1</sup> and 1023 cm<sup>−1</sup>, respectively. We also demonstrate the potential of ATR-FT-IR spectroscopic imaging for unraveling the details of the dynamics of biological processes, which are not accessible from cell ensemble studies, with high molecular specificity and satisfactory spatial resolution.

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