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Abstract
Infrared nano-spectroscopy (nano-IR) using a scattering-type near-field optical microscope (s-SNOM) is becoming an important tool to obtain vibrational spectra from nanometer-scaled volumes in intact microscopic samples. The necessity to understand biochemical composition, structure, and function from such experiments requires nano-IR probing of cells and biomolecules in aqueous media of different complexity that ranges from pure water to protein-rich cell culture media. We report on the use of 30 nm SiC membranes as a protective, stable and biocompatible interface between grown-on, adhering cells in their medium and the s-SNOM (AFM) probe tip, and a highly IR transparent window that enables the collection of nano-IR spectra from cells and biomolecules across a wide spectral range. Nano-IR spectra were collected from fibroblast cells grown on the membranes using a broadband synchrotron IR source. The acquisition of nano-IR images at varied tip-sample interactions showed that the signal-to-noise ratio can be significantly increased when optimized parameters are used. The alteration of tapping amplitude and set-point of the s-SNOM tip allows to systematically vary the probing depth, thereby opening the possibility of an alternative approach to sensitive and efficient nano-IR tomography based on a single demodulation harmonic of the optical signal. The nano-IR spectra of the fibroblast cells resemble their far-field spectra, but give evidence of a variation in proteins, nucleic acids, carbohydrates and membrane lipid content. The observed cell structure at the nanoscale is in agreement with the known heterogeneity of the cells, such as the organization of the cell membrane and its composition in different domains. The results show that sensitive and stable nano-IR probing of complex samples in different liquid media can be achieved and provide suggestions to further improve the efficiency and standardization of existing approaches.
Supplementary materials
Title
Supporting Information
Description
Additional images of fibroblast cells in PBS buffer in Figure S1, masks used to calculate signal-to-noise ratios in nano-IR images in Figure S2, Tentative assignment of the vibrational bands observed in the nano-IR spectra of DNA and BSA water solutions in Table S1 complemented by the text description, average calculated from individual nano-IR spectra with an ATR-IR spectrum in Figure S3 complemented by the text comparison of the observed spectral bands, images and spectra of fibroblast cells on a SiN membrane in Figure S4.
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