Infrared (IR) microscopy and spectroscopy, while being sensitive to the samples’ chemical composition, suffer from poor lateral resolution due to diffraction. Therefore, imaging of the cellular inner structure in the IR range of the electromagnetic spectrum proves to be challenging. An approach to overcome this limitation is by using scattering-type near-field optical microscopy (sSNOM) and nano-Fourier transform infrared (nanoFTIR) spectroscopy, achieved by combining the high spatial resolution of atomic force microscopy (AFM) and the chemical sensitivity of IR absorption. This methodology was applied to various biophysical systems with increasing complexity, starting with native membrane proteins, followed by aggregating peptides and light-induced surface patterning, concluding with investigation of cells. In this work I was able to resolve the subcellular structure of C. reinhardtii and assign the IR absorption of various organelles to molecular vibrations with spatial resolution of 20 nm. The necessity and power of chemical imaging was demonstrated by scanning the nuclear area, where several nuclear bodies were distinguished in the sSNOM images while remaining hidden in the AFM topography. Finally, a stack of sSNOM images, obtained by sequential scanning of serial sections, was used to reconstruct a three-dimensional image. Thus, we demonstrate that sSNOM tomography allows visualizing three-dimensional intracellular structures at nanometer resolution where the contrast originates from molecular vibrations of chemical bonds.
