We report single-cell infrared (IR) imaging of onion (<i>Allium cepa</i>) root cells using an IR super-resolution microscope based on vibrational sum-frequency generation (VSFG). The resolution of recorded IR images was less than 2 μm and IR super-resolution was achieved by virtue of the VSFG detection. In addition, IR spectra measurements were successfully performed on distinct intra-cellular assemblies. The IR absorption intensity of the cell nuclear edge and the nucleolus in the 3055–3130 cm<sup>−1</sup> region was stronger than that from the cytoplasmic part. This is because the cell nucleus and nucleolus contain larger amounts of nucleic acid. Thus, the obtained IR spectra reflect differences in chemical composition among different cellular structures. In addition, the ability of our novel IR super-resolution microscope to obtain distinct information on both VSFG and two-photon fluorescence is demonstrated.

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