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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 andin vivowork. 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 for work in biological samples. We examine three cell lines (Huh-7, differentiated 3T3-L1, and U2OS) and three organisms (E. coli, tardigrades, and zebrafish). Our results suggest that the information provided by multifrequency imaging is comparable to hyperspectral imaging while reducing imaging times twenty-fold. We also explore the utility of IR active probes, including global and site-specific probes, for tracking metabolic pathways and protein localization, structure, and local environment. Our findings illustrate the versatility of OPTIR, and together, provide a direction for future dynamic imaging of living cells and organisms.