The details of SRS have been previously described.13,21 The skin sample is mounted on the stage of a modified upright laser scanning microscope (BX81, Olympus, Pittsburgh, Pennsylvania) configured for transmission illumination. Two overlapping laser beams are focused on the sample with either a UPLSAPO20X air objective or a UPLSAPO60XW water immersion objective (Olympus). The 1064-nm Stokes beam source was an laser (picoTRAIN, High-Q, Watertown, Massachusetts). Part of the 1064-nm output is used to pump a tunable optical parametric oscillator (Levante Emerald, APE-Berlin, Berlin, Germany). The pump beam was tuned to 816.0, 810.6, or 781.3 nm for skin imaging, corresponding to Raman shifts of ca. 2850, 2950, or , respectively. The main contributors from skin at these vibrational frequencies can be assigned to vibrational modes from lipids, proteins, and water22 (Table 1). The pump laser was tuned to 869.2 nm to image the vibrational mode at after topical application of , ensuring that only the applied material would be visible in the SRS image stack. Image integration time was typically 4 to . The beams were scanned in two dimensions with a pair of galvanometer mirrors (Olympus fluosview FV-300). The transmitted light was collected with a 1.4-NA oil condenser lens (Nikon, Melville, New York) and detected by a large-area photodiode (FDS1010, Thorlabs, Newton, New Jersey). Images were collected parallel to the skin surface. Image stacks were obtained through sample stage movements controlled by the microscope software, with a step size of 2 μm over depth ranges of 60 to 100 μm. An image stack at a single wavelength can be acquired in 30 to 60 s.