Transmission IR Microscopy for the Quantitation of Biomolecular Mass In Live Cells

Abstract

Absolute quantity imaging of biomolecules on a single cell level is critical for measurement assurance in biosciences and bioindustries. While infrared (IR) transmission microscopy is a powerful label-free imaging modality capable of chemical quantification, its applicability to hydrated biological samples remains challenging due to the strong water absorption. We overcome this challenge by applying a solvent absorption compensation (SAC) technique to a home-built quantum cascade laser IR microscope. SAC-IR microscopy improves the chemical sensitivity considerably by adjusting the incident light intensity to pre-compensate the IR absorption by water while retaining the full dynamic range. We demonstrate the label-free chemical imaging of key biomolecules of a cell, such as protein, fatty acid, and nucleic acid, with sub-cellular spatial resolution. By imaging live fibroblast cells over twelve hours, we monitor the mass change of the three molecular species of single cells at various phases, including cell division. While the current live-cell imaging demonstration involved three wavenumbers, more wavenumber images could measure more biomolecules in live cells with higher accuracy. As a label-free method to measure absolute quantities of various molecules in a cell, SAC-IR microscopy can potentially become a standard chemical characterization tool for live cells in biology, medicine, and biotechnology.