Nano‐Infrared Imaging and Spectroscopy of Animal Cells in Liquid Environment

Infrared nano-spectroscopy (nano-IR) using scattering-type near-field optical microscopy (s-SNOM) is becoming an important tool for analyzing vibrational spectra in nanometer-scale volumes of intact samples. Recent s-SNOM experiments using ultrathin membranes have enabled nanoscopy of cells and biomolecules in liquid environments. This study reports the use of SiC membranes as a stable, biocompatible interface between cells in their medium and the atomic force microscopy (AFM) probe of the s-SNOM, ensuring broadband nano-IR experiments above 1050 cm^-1. Nano-IR images and spectra are collected from fibroblast cells grown adherently to confluence on the membranes using a broadband synchrotron IR source, probing the membrane-adjacent cellular region. Optimized tip-sample interaction allows to significantly increase the signal-to-noise-ratio in nano-IR imaging. The alteration of the tapping amplitude and set-point of the AFM cantilever allows to adjust the probing depth and suggests a nano-IR tomography approach that uses a single demodulation harmonic of the signal. The nano-IR spectra of fibroblast cells resemble their far-field spectra, but reflect a heterogeneity in the composition and structure of proteins, nucleic acids, carbohydrates, and of membrane lipid organization. The results demonstrate nano-IR probing of complex samples in liquid media and suggest ways to improve efficiency and standardization of existing approaches in vibrational nanoscopy.