Abstract
Although techniques such as fluorescence-based super-resolution imaging or confocal microscopy simultaneously gather both morphological and chemical data, these techniques often rely on the use of localized and chemically 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 complementing current fluorescence nanoscopy but at less interference due to the low energy of infrared radiation and the absence of labeling.
Highlights
Techniques such as fluorescence-based super-resolution imaging or confocal microscopy simultaneously gather both morphological and chemical data, these techniques often rely on the use of localized and chemically specific markers
Our present work describes a nanoscopic technique that applies the super-resolution imaging power and spectroscopic strengths of scattering-type scanning near-field optical microscopy (sSNOM) and nanoFTIR to cellular cross sections, prepared by a method well-established in electron microscopy (EM)
In sSNOM imaging mode, the surface is raster scanned at a single frequency of the quantum cascade lasers (QCLs) simultaneously obtaining atomic force microscopy (AFM)
Summary
Techniques such as fluorescence-based super-resolution imaging or confocal microscopy simultaneously gather both morphological and chemical data, these techniques often rely on the use of localized and chemically 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. The volume corresponding to the reconstructed tomogram is not constrained by the intrinsic limitation of sensitivity in z-direction and can be expanded by increasing the number of sequential crosssections This approach enables the detection and visualization of the chemical composition of individual microorganisms and their subcellular components in a non-invasive manner, without the need to include highly specific, potentially artifact-inducing or hazardous sample staining, which can be expanded to imaging of multicellular structures and tissues. IR nanoscopy is able to resolve single protein complexes of a cell as demonstrated in the present work
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