Stable Isotope Probing-nanoFTIR for Quantitation of Cellular Metabolism and Observation of Growth-Dependent Spectral Features.

This study utilizes nanoscale Fourier transform infrared spectroscopy (nanoFTIR) to perform stable isotope probing (SIP) on individual bacteria cells cultured in the presence of 13C-labelled glucose. SIP-nanoFTIR simultaneously quantifies single-cell metabolism through infrared spectroscopy and acquires cellular morphological information via atomic force microscopy. The redshift of the amide I peak corresponds to the isotopic enrichment of newly synthesized proteins. These observations of single-cell translational activity are comparable to those of conventional methods, examining bulk cell numbers. Observing cells cultured under conditions of limited carbon, SIP- nanoFTIR is used to identify environmentally-induced changes in metabolic heterogeneity and cellular morphology. Individuals outcompeting their neighboring cells will likely play a disproportionately large role in shaping population dynamics during adverse conditions or environmental fluctuations. Additionally, SIP-nanoFTIR enables the spectroscopic differentiation of specific cellular growth phases. During cellular replication, subcellular isotope distribution becomes more homogenous, which is reflected in the spectroscopic features dependent on the extent of 13C-13C mode coupling or to specific isotopic symmetries within protein secondary structures. As SIP-nanoFTIR captures single-cell metabolism, environmentally-induced cellular processes, and subcellular isotope localization, this technique offers widespread applications across a variety of disciplines including microbial ecology, biophysics, biopharmaceuticals, medicinal science, and cancer research.

Cyanine Dye Coupling Mediates Self-assembly of a pH Sensitive Peptide into Novel 3D Architectures.

Synthetic multichromophore systems are of great importance in artificial light harvesting devices, organic optoelectronics, tumor imaging and therapy. Here, we introduce a promising strategy for the construction of self-assembled peptide templated dye stacks based on coupling of a de novo designed pH sensitive peptide with a cyanine dye Cy5 at its N-terminus. Microscopic techniques, in particular cryogenic TEM (cryo-TEM) and cryo-electron tomography technique (cryo-ET), reveal two types of highly ordered three-dimensional assembly structures on the micrometer scale. Unbranched compact layered rods are observed at pH 7.4 and two-dimensional membrane-like assemblies at pH 3.4, both species displaying spectral features of H-aggregates. Molecular dynamics simulations reveal that the coupling of Cy5 moieties promotes the formation of both ultrastructures, whereas the protonation states of acidic and basic amino acid side chains dictates their ultimate three-dimensional organization.

Infrared nanoscopy and tomography of intracellular structures.

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.