Major drawbacks of direct mid-infrared spectroscopic imaging of single cells in aqueous buffer are strong water absorption, low resolution typically above 10 ìm, and Mie scattering effects. This study demonstrates how an indirect detection principle can overcome these drawbacks using the optical photothermal infrared (O-PTIR) technique for highresolution discrete wavenumber imaging and fingerprint spectroscopy of cultivated cell s as a model system in a simple liquid sample chamber. O-PTIR spectra of six leukemia- and cancer-derived cell lines showed main IR bands near 1648, 1547, 1447, 1400, 1220, and 1088 cm-1. Five spectra of approximately 260 single cells per cell type were averaged, the O-PTIR data set was divided into leukemia-derived cells (THP-1, HL 60, Jurkat, Raji) and cancer cells (HeLa, HepaRG), and partial least squares linear discriminant analysis (PLS-LDA) was applied in the spectral range 800-1800 cm-1 to train three classification models. Leukemia versus cancer cell model showed an accuracy of 90.0%, HeLa versus HepaRG cell model had an accuracy of 95.4%, and the model for the distinction of leukemia cells had an accuracy of 75.4%. IR bands in linear discriminants (LDs) of the models were correlated with second derivative spectra that resolved more than 25 subbands. IR and second derivative spectra of proteins, DNA, RNA and lipids were collected as references to confirm band assignments. O-PTIR images of single cells at 200 nm step size were acquired at 1086, 1548, and 1746 cm-1 to visualize the nucleic acid, protein, and lipid distribution, respectively. Variations in subcellular features, lipid-to-protein, and nucleic acid-to-protein ratios were identified that were consistent with biomolecular information in LDs. In conclusion, O-PTIR can provide high-quality spectra and images with submicron resolution of single cells in aqueous buffers that offer prospects in high-content screening applications.