Disclosed is a method for obtaining feedback to drive a servo system for aligning and maintaining alignment in optical systems that bring light to an in vivo skin sample, for adjusting the focus of the optical system, and for adjusting the net depth of focus of the optical system within the in vivo system under characterization. The invention additionally provides a method for obtaining feedback to drive a servo system for aligning and maintaining alignment in optical systems that collect light from an in vivo skin sample, and for adjusting the focus of the optical system. These methods comprise adjusting the angle of incidence of electromagnetic radiation and/or providing a shielding lens to block scattered incident light, or otherwise limiting the field of view of the Raman scattered radiation collection system to exclude optical surfaces of the excitation delivery portion of the optical system. Also provided is a method for identifying spectroscopic depth markers in tissues. In one embodiment, the method comprises discriminating between Raman signals originating on outer portions of skin from signals originating from substances deeper within the skin or other tissues. In another embodiment, the invention provides a method for detecting skin abnormalities and for assessing the aging of skin and related tissues. The method comprises characterizing the content of the skin using Raman spectroscopy. In one embodiment, the collagen, elastin, and or keratin content of the skin is characterized.