Differentiation of sphingomyelin and cholesterol by hyperspectral mid-infrared detection of single-bond vibrational modes in the fingerprint region

Lipids play a central role in a multitude of biological functions associated with cancer, obesity, diabetes, cardiovascular and neurological pathologies. However, sensing and mapping of lipid classes in living cells remains challenging. Here we introduce a label-free approach to lipid imaging, which differentiates lipid species in living cells by hyperspectral mid-infrared detection of single-bond vibrational modes within the fingerprint region. Hyperspectral fingerprint optoacoustic microscopy is shown to resolve phosphatidylcholine, sphingomyelin or cholesterol in test samples and in synthetic giant unilamellar vesicles used as models of cell membranes. Then, mapping of total cholesterol and sphingomyelin content and accumulation dynamics are demonstrated in living cells. Hyperspectral fingerprint optoacoustic microscopy demonstrates sensitivity not only in discerning lipids with substantially different chemical structures, such as cholesterol and phospholipids, but also lipids that are chemically similar, such as sphingomyelins and glycerophospholipids.