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

Abstract 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.