Infrared Nanoscopy and Tomography of Intracellular Structures

Abstract

The few microscopic techniques that simultaneously gather morphological and chemical data often rely on the use of specific markers. To eliminate this flaw, we have developed a method of examining cellular cross sections using the imaging power of scattering-type scanning near-field optical microscopy and Fourier-transform infrared spectroscopy at a spatial resolution far beyond the diffraction limit. Herewith, nanoscale surface and volumetric chemical imaging is performed using the intrinsic contrast generated by the characteristic absorption of mid-infrared radiation by the covalent bonds. We employ infrared nanoscopy to study the subcellular structures of eukaryotic (Chlamydomonas reinhardtii) and prokaryotic (Escherichia coli) species, revealing chemically distinct regions within each cell such as the microtubular structure of the flagellum. Serial 100 nm-thick cellular cross-sections were compiled into a tomogram yielding a three-dimensional infrared image of subcellular structure distribution at 20 nm resolution. The presented methodology is able to image biological samples competing current fluorescence nanoscopy but at less interference due to the low energy of infrared radiation and the absence of labeling.

Super-resolution mid-infrared spectro-microscopy of biological applications through tapping mode and peak force tapping mode atomic force microscope

“…In fact, several s-SNOM studies have already investigated slices microtomed from the bulk sample, such as hair [93], silk [108], and cellulose materials [125]. Recently, Kanevche et al has reported s-SNOM imaging of 100 nm-thick cellular cross-sections of eukaryotic and prokaryotic cells, and a 3D tomogram with 20-nm IR resolution was composed [126]. The smooth combination of AFM-based nano-IR techniques with advanced microtome methodology will streamline the discovery of spatial-temporal correlations in life-relevant cells and tissues.…”