Precise in vivo tissue micro-Raman spectroscopy with simultaneous reflectance confocal microscopy monitoring using a single laser

Raman spectroscopy provides molecular finger-printing of biological tissues. To achieve high spatial resolution, confocal Raman spectroscopy (micro-Raman) has been developed. To guide where micro-Raman is acquired, imaging guidance is necessary, especially for high scattering biological tissue. Reflectance confocal microscopy (RCM) has been integrated with micro-Raman. However, existing systems do not allow point-of-interest micro-Raman measurement with simultaneous RCM guidance. Here we demonstrate how a single laser can be used to localize and acquire micro-Raman signals within tissue with simultaneous real-time RCM imaging. Applications of this RCM-guided micro-Raman system for ex vivo samples and in vivo skin measurements are presented.