Label-free chemical contrast is highly desirable in biomedical imaging. Spontaneous Raman microscopy provides specific vibrational signatures of chemical bonds, but is often hindered by low sensitivity. Here we report a three-dimensional multiphoton vibrational imaging technique based on stimulated Raman scattering (SRS). The sensitivity of SRS imaging is significantly greater than that of spontaneous Raman microscopy, which is achieved by implementing high-frequency (megahertz) phase-sensitive detection. SRS microscopy has a major advantage over previous coherent Raman techniques in that it offers background-free and readily interpretable chemical contrast. We show a variety of biomedical applications, such as differentiating distributions of omega-3 fatty acids and saturated lipids in living cells, imaging of brain and skin tissues based on intrinsic lipid contrast, and monitoring drug delivery through the epidermis.

Many sight-threatening diseases have two critical phases, vessel loss followed by hypoxia-driven destructive neovascularization. These diseases include retinopathy of prematurity and diabetic retinopathy, leading causes of blindness in childhood and middle age affecting over 4 million people in the United States. We studied the influence of ω-3-and ω-6-polyunsaturated fatty acids (PUFAs) on vascular loss, vascular regrowth after injury, and hypoxia-induced pathological neovascularization in a mouse model of oxygen-induced retinopathy 1 . We show that increasing ω-3-PUFA tissue levels by dietary or genetic means decreased the avascular area of the retina by Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissionsCorrespondence should be addressed to L.E.H.S. (lois.smith@childrens.harvard.edu).. Supplementary information is available on the Nature Medicine website. COMPETING INTERESTS STATEMENTThe authors declare competing financial interests: details accompany the full-text HTML version of the paper at http:// www.nature.com/naturemedicine/. HHS Public AccessAuthor manuscript Nat Med. Author manuscript; available in PMC 2015 July 05. Published in final edited form as:Nat Med. 2007 July ; 13(7): 868-873. doi:10.1038/nm1591. Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript increasing vessel regrowth after injury, thereby reducing the hypoxic stimulus for neovascularization. The bioactive ω-3-PUFA-derived mediators neuroprotectinD1, resolvinD1 and resolvinE1 also potently protected against neovascularization. The protective effect of ω-3-PUFAs and their bioactive metabolites was mediated, in part, through suppression of tumor necrosis factor-α. This inflammatory cytokine was found in a subset of microglia that was closely associated with retinal vessels. These findings indicate that increasing the sources of ω-3-PUFA or their bioactive products reduces pathological angiogenesis. Western diets are often deficient in ω-3-PUFA, and premature infants lack the important transfer from the mother to the infant of ω-3-PUFA that normally occurs in the third trimester of pregnancy 2 . Supplementing ω-3-PUFA intake may be of benefit in preventing retinopathy.Ocular neovascularization is the most common cause of blindness in all age groups: retinopathy of prematurity in children, diabetic retinopathy in working-age adults and agerelated macular degeneration in the elderly. In principle, destructive angiogenesis in the eye can be ameliorated by either direct inhibition of neovascularization or by controlling vessel loss in order to reduce the hypoxic stimulus that drives neovascularization. Retinopathy is modeled in the mouse eye with oxygen-induced vessel loss, which precipitates hypoxiainduced retinopathy, allowing for assessment of retinal vessel loss, vessel regrowth after injury and pathological angiogenesis 1 .The role of lipids in angiogenesis is just beginning to be defined 3,4 . The major polyunsaturated fatty acids (PUFA) found in the retina a...