(A) Energy diagram of SRS. If the difference frequency of the excitation beams Δω=ω_p-ω_S matches a vibrational frequency in the sample at Ω_vib, a molecule is excited from the vibrational ground state (ν=0) to a vibrational excited state (ν=1), passing through a virtual state (VS). This results in a photon in the pump field being annihilated (stimulated Raman loss) and a photon in the Stokes field being created (stimulated Raman gain) which can be probed as a contrast for microscopy. (B) Experimental setup for in vivo SRS microscopy. The Stokes beam is modulated with an electro-optic modulator (EOM), spatially overlapped with the pump beam with a dichroic mirror (DM) and aligned into a laser scanning microscope. The beams are focused by the objective lens (OL) and the common focal spot is scanned through the specimen (SP) by a galvo mirror (GM) and a resonant galvo mirror (RGM). The detected intensity of the backscattered pump beam is demodulated with a custom high speed lock-in amplifier (LIA) to provide the SRS signal to the computer (PC). The inset depicts the epi-detector assembly. The sample is excited by focusing light through a small hole in the center of the large-area epi-detector (PD). Scattering re-directs a significant portion of the forward-traveling light to illuminate the detector active area. The modulated Stokes beam is blocked by an optical filter (FI), and the transmitted pump beam is detected. (C) Raman spectra of chemical compounds imaged in this work. To image the architecture of skin, we make use of the OH-stretching vibration of water (3250 cm^-1, blue), the CH₃ stretching vibration, which primarily arises from protein (2950 cm^-1, magenta, represented by soy protein extract), and the CH₂ stretching vibration, which arises primarily from lipids (2845 cm^-1, red, represented by oleic acid). Exogenous topically applied drugs may have unique vibrational signatures, such as the polyene stretching vibration of retinol (1596 cm^-1, green), or may be labeled with deuterium and imaged using C-D stretching, in this case of d6-dimethyl sulfoxide (2125 cm^-1, yellow).

(A) Logarithmic plot of the distribution of the relative intensity of the backscattered light at the tissue surface from a focus at a depth of 100 μm into the tissue (scattering mean free path = 200 μm, anisotropy = 0.9) emitting in the forward direction with a 0.4 numerical aperture. (B) Depth profile of tissue showing sample ray trajectories colored according to the final outcome. Green traces are back-scattered to the tissue surface, red traces are scattered within the tissue until they are absorbed, and blue traces are transmitted through the 2 cm thickness of the sample. (C,D) Simulations of collection efficiency of back-scattered forward-traveling light versus detector radius for (C) different scattering mean free paths μS of the sample and (D) different numerical apertures (NA) of the excitation objective.

(A) SRS image of lipids of the stratum corneum shows intercellular spaces between hexagonal corneocytes and (B) SRS water image (3250 cm^-1) of the same region shows a homogenous distribution of water. (C) A CARS water image acquired simultaneously with (B) shows artifacts from the non-resonant background of lipids. (D) SRS lipid and (E) water images of the viable epidermis show sebaceous glands with positive and negative contrast, respectively. (F) SRS images of the viable epidermis at the CH₃ stretching vibration (2950 cm^-1) mainly highlights proteins as well as residual lipid-rich structures. A capillary with individual red blood cells (arrow) is visible. The cells are imaged without motion blur due to video rate acquisition speed. (G) SRS in vivo flow cytometry. An x-t plot acquired by line-scanning across a capillary at the position of the arrow in (F). Individual red blood cells are captured on the fly. (A-E) are acquired in epi-direction, while (F) and (G) are acquired in transmission, all with 37 ms / frame acquisition speed and 512 × 512 pixel sampling. Scale: 25 μm.

(A-C) SRS images of hair and stratum corneum in the ear of a living mouse. (D-F) Images of a hair shaft within the viable epidermis. (G-I) Images of a sebaceous gland within the viable epidermis. (J-L) Depth projection of three-dimensional image stacks of the viable epidermis. Images are acquired at the indicated Raman shifts. Contrast coming primarily from protein (2950 cm^-1) and lipid (2845 cm^-1) shows the morphology of the skin with all its structural elements and sub cellular resolution (see nuclei in D and K). Hairs are visible as solid structures in the protein image (D) and surrounded by oil secreted from the sebaceous glands in the lipid image (E). We are able to visualize with SRS that drug penetration of the topically applied trans-retinol (C) occurs along the hair shaft (F,I and L). Images are collected in transmission with 37 ms / frame acquisition speed and 512 × 512 pixel sampling. Scale: 25 μm.

(A-C) SRS images of the stratum corneum and viable epidermis tuned into CH₃ stretching vibration of proteins (2950cm^-1) showing nuclei of variable size (A and B) as well as a hair (C). (D) SRS image of DMSO penetrating the skin at the same region as (C). We find that DMSO also accumulates in the hair shaft. We used deuterium labeling to create a unique vibration of d6-DMSO at 2120 cm^-1 for specific imaging. Images are acquired in epi-direction on the forearm of a volunteer. Image acquisition time is 150 ms for (A) and (B) and 37 ms for (C) and (D), all with 512 × 512 pixel sampling. Scale: 50 μm.