Current views on non-invasive in vivo determination of physiological parameters of the stratum corneum using confocal Raman microspectroscopy

Confocal Raman microspectroscopy is widely used in dermatology and cosmetology for analysis of the concentration of skin components (lipids, natural moisturizing factor molecules, water) and the penetration depth of cosmetic/medical formulations in the human stratum corneum (SC) in vivo. In recent years, it was shown that confocal Raman microspectroscopy can also be used for non-invasive in vivo depth-dependent determination of the physiological parameters of the SC, such as lamellar and lateral organization of intercellular lipids, folding properties of keratin, water mobility and hydrogen bonding states. The results showed that the strongest skin barrier function, which is primarily manifested by the orthorhombic organization of intercellular lipids, is provided at ≈20–40% SC depth, which is related to the maximal bonding state of water with surrounding components in the SC. The secondary and tertiary structures of keratin determine water binding in the SC, which is depth-dependent. This paper shows the technical possibility and advantage of confocal Raman microspectroscopy in non-invasive investigation of the skin and summarizes recent results on in vivo investigation of the human SC.

Influence of the measurement configuration on the results of Raman microspectroscopy of human hair

Increasing interest in spectroscopic studies of human hair raises the question about the accuracy of measurement of their spectra and requires optimisation of experimental facilities. An original method of obtaining transverse hair sections without using a microtome and chemical influence is proposed. The results obtained by confocal Raman microspectroscopy of human hair differently oriented with respect to the optical axis of the measuring setup are compared. It is shown that, in addition to expected changes in the spectra measured at different distances from the hair periphery in the direction to its centre, the spectra measured in the case of hair excitation perpendicular and parallel to its axis are also considerably different.

Lipidome profiling with Raman microspectroscopy identifies macrophage response to surface topographies of implant materials

Biomaterial characteristics such as surface topographies have been shown to modulate macrophage phenotypes. The standard methodologies to measure macrophage response to biomaterials are marker-based and invasive. Raman microspectroscopy (RM) is a marker-independent, noninvasive technology that allows the analysis of living cells without the need for staining or processing. In the present study, we analyzed human monocyte-derived macrophages (MDMs) using RM, revealing that macrophage activation by lipopolysaccharides (LPS), interferons (IFN), or cytokines can be identified by lipid composition, which significantly differs in M0 (resting), M1 (IFN-γ/LPS), M2a (IL-4/IL-13), and M2c (IL-10) MDMs. To identify the impact of a biomaterial on MDM phenotype and polarization, we cultured macrophages on titanium disks with varying surface topographies and analyzed the adherent MDMs with RM. We detected surface topography–induced changes in MDM biochemistry and lipid composition that were not shown by less sensitive standard methods such as cytokine expression or surface antigen analysis. Our data suggest that RM may enable a more precise classification of macrophage activation and biomaterial–macrophage interaction.