Optical Photothermal Infrared Imaging Using Metabolic Probes in Biological Systems

Infrared spectroscopy is a powerful tool for identifying biomolecules. In biological systems, infrared spectra provide information on structure, reaction mechanisms, and conformational change of biomolecules. However, the promise of applying infrared imaging to biological systems has been hampered by low spatial resolution and the overwhelming water background arising from the aqueous nature of in-cell and in vivo work. Recently, optical photothermal infrared microscopy (OPTIR) has overcome these barriers and achieved both spatially and spectrally resolved images of live cells and organisms. Here, we determine the most effective modes of collection on a commercial OPTIR microscope for work in biological samples. We examine three cell lines (Huh-7, differentiated 3T3-L1, and U2OS) and three organisms (Escherichia coli, tardigrades, and zebrafish). Our results suggest that the information provided by multifrequency imaging is comparable to hyperspectral imaging while reducing imaging times 20-fold. We also explore the utility of IR active probes for OPTIR using global and site-specific noncanonical azide containing amino acid probes of proteins. We find that photoreactive IR probes are not compatible with OPTIR. We demonstrate live imaging of cells in buffers with water. ¹³C glucose metabolism monitored in live fat cells and E. coli highlights that the same probe may be used in different pathways. Further, we demonstrate that some drugs (e.g., neratinib) have IR active moieties that can be imaged by OPTIR. Our findings illustrate the versatility of OPTIR and, together, provide a direction for future dynamic imaging of living cells and organisms.