Co-localized line-field confocal optical coherence tomography (LC-OCT) and confocal Raman microspectroscopy for ex vivo analysis of skin tissues

Line-field confocal optical coherence tomography (LC-OCT) is an optical technology developed for in vivo skin imaging. Combining the principles of OCT and reflectance confocal microscopy, LC-OCT generates high-resolution three-dimensional (3D) images with an isotropic spatial resolution of 1.3 micron and up to 500 microns in depth. Confocal Raman microspectroscopy is a label-free optical technique which allows for point measurement of the molecular content of a sample with micrometer resolution. While LC-OCT provides morphological information, Raman spectroscopy brings chemical information but lacks image guidance for targeting specific points of interest in the sample. Combining the two modalities would therefore provide complementary information and guidance for Raman measurements. We present a method to co-localize LC-OCT and Raman acquisitions for ex vivo applications. This co-localization approach allows acquisition of Raman spectra at specific locations targeted in a 3D LC-OCT image, with an accuracy of +/- 20 mu m. The co-localization method was developed using a LC-OCT device designed for ex vivo imaging and a custom Raman system. The principle of co-localization relies on both the use of a specific sample holder that can be positioned under each device with high repeatability and of a coordinate-based calibration between the two devices. Co-localization was validated using pig skin samples containing tattoo ink of known composition. LC-OCT images allowed to target specific regions in the samples where the presence of tattoo ink was revealed by detection of a specific Raman signature.