Combined in vivo confocal Raman spectroscopy and confocal microscopy of human skin

Abstract

In vivo confocal Raman spectroscopy is a noninvasive optical method to obtain detailed information about the molecular composition of the skin with high spatial resolution. In vivo confocal scanning laser microscopy is an imaging modality that provides optical sections of the skin without physically dissecting the tissue. A combination of both techniques in a single instrument is described. This combination allows the skin morphology to be visualized and (subsurface) structures in the skin to be targeted for Raman measurements. Novel results are presented that show detailed in vivo concentration profiles of water and of natural moisturizing factor for the stratum corneum that are directly related to the skin architecture by in vivo cross-sectional images of the skin. Targeting of skin structures is demonstrated by recording in vivo Raman spectra of sweat ducts and sebaceous glands in situ. In vivo measurements on dermal capillaries yielded high-quality Raman spectra of blood in a completely noninvasive manner. From the results of this exploratory study we conclude that the technique presented has great potential for fundamental skin research, pharmacology (percutaneous transport), clinical dermatology, and cosmetic research, as well as for noninvasive analysis of blood analytes, including glucose.

FIGURE 1

Combined confocal scanning laser microscope and confocal Raman spectrometer for in vivo study of the skin. (A) Schematic overview of the setup. Light from the Raman laser (720 nm and 850 nm) was transmitted by a short-pass filter and reflected by a custom-designed filter (transmission curve shown in Fig. 1 C), which partially reflected light at wavelengths 720 nm and 850 nm and completely reflected Raman scattered light with wavelengths >850 nm. The light was then focused to a diffraction limited spot by a microscope objective. Scattered light was collected by the same objective and reflected by the custom filter and by the short-pass filter. The scattered light was filtered by a laser rejection filter and focused onto the 100-μm core of an optical fiber, using an f = 149 mm achromat. The core of the optical fiber served as a confocal pinhole, rejecting out-of-focus light. The output of the fiber was connected to a spectrograph. Laser light from the CSLM, produced by an 830-nm diode laser, was transmitted by the custom filter and focused by the microscope objective to a diffraction limited spot, which was scanned across the focal plane. (B) Detailed view of the coupling between the CSLM and the Raman system. (C) Transmission curve of the coupling filter between the CSLM and the Raman spectrometer for parallel (P) and normal (S) polarization. Angle of incidence: 45°.

FIGURE 1

Combined confocal scanning laser microscope and confocal Raman spectrometer for in vivo study of the skin. (A) Schematic overview of the setup. Light from the Raman laser (720 nm and 850 nm) was transmitted by a short-pass filter and reflected by a custom-designed filter (transmission curve shown in Fig. 1 C), which partially reflected light at wavelengths 720 nm and 850 nm and completely reflected Raman scattered light with wavelengths >850 nm. The light was then focused to a diffraction limited spot by a microscope objective. Scattered light was collected by the same objective and reflected by the custom filter and by the short-pass filter. The scattered light was filtered by a laser rejection filter and focused onto the 100-μm core of an optical fiber, using an f = 149 mm achromat. The core of the optical fiber served as a confocal pinhole, rejecting out-of-focus light. The output of the fiber was connected to a spectrograph. Laser light from the CSLM, produced by an 830-nm diode laser, was transmitted by the custom filter and focused by the microscope objective to a diffraction limited spot, which was scanned across the focal plane. (B) Detailed view of the coupling between the CSLM and the Raman system. (C) Transmission curve of the coupling filter between the CSLM and the Raman spectrometer for parallel (P) and normal (S) polarization. Angle of incidence: 45°.

FIGURE 1

Combined confocal scanning laser microscope and confocal Raman spectrometer for in vivo study of the skin. (A) Schematic overview of the setup. Light from the Raman laser (720 nm and 850 nm) was transmitted by a short-pass filter and reflected by a custom-designed filter (transmission curve shown in Fig. 1 C), which partially reflected light at wavelengths 720 nm and 850 nm and completely reflected Raman scattered light with wavelengths >850 nm. The light was then focused to a diffraction limited spot by a microscope objective. Scattered light was collected by the same objective and reflected by the custom filter and by the short-pass filter. The scattered light was filtered by a laser rejection filter and focused onto the 100-μm core of an optical fiber, using an f = 149 mm achromat. The core of the optical fiber served as a confocal pinhole, rejecting out-of-focus light. The output of the fiber was connected to a spectrograph. Laser light from the CSLM, produced by an 830-nm diode laser, was transmitted by the custom filter and focused by the microscope objective to a diffraction limited spot, which was scanned across the focal plane. (B) Detailed view of the coupling between the CSLM and the Raman system. (C) Transmission curve of the coupling filter between the CSLM and the Raman spectrometer for parallel (P) and normal (S) polarization. Angle of incidence: 45°.

FIGURE 2

In vivo confocal images and a water concentration profile for the stratum corneum of the palm, based on combined CSLM and Raman measurements. (A) Cross section of the skin (xz-plane; SC: stratum corneum; VE: viable epidermis). Plotted in the image is the water concentration profile as determined from Raman measurements on the dashed line. The x axis represents the distance to the skin surface and applies to both the image and the graph. The y axis represents the water concentration in mass percentage (grams of water per 100 g of wet tissue). The solid line locates the plane from which image B was obtained. (B) Confocal image parallel to the skin surface (xy-plane), recorded at 145 μm below the skin surface at the boundary between SC and VE.

FIGURE 3

In vivo confocal images and a concentration profile of NMF for the stratum corneum of the palm. (A) Cross section of the skin (xz-plane; SC: stratum corneum; VE: viable epidermis). Plotted in the image is the relative amount of NMF as a function of depth, as determined from Raman measurements on the dashed line. The x axis represents the distance to the skin surface and applies to both the image and the graph. The y axis represents the relative amount of NMF, normalized to its maximum value. The solid line locates the plane from which image B was obtained. (B) Confocal image parallel to the skin surface (xy-plane) recorded 135 μm below the skin surface at the boundary between SC and VE.

FIGURE 4

In vivo confocal image and Raman spectroscopy of a sweat duct on the palm of the hand, 30 μm below the skin surface. The bright area is a sweat duct. The arrows (left) mark the spots from which the Raman spectra were obtained. The asterisk marks the prominent Raman band of lactate at 856 cm⁻¹ (see text). (a, right) Raman spectrum measured in the sweat duct. (b, right) Raman spectrum measured outside the sweat duct. (c, right) Difference spectrum (a minus b). (d, right) Fit result of spectrum a with spectrum b and spectra of NMF and sweat constituents (see text for details). (e, right) In vitro Raman spectrum of lactate. (f, right) In vitro Raman spectrum of urea.

FIGURE 5

In vivo confocal image and Raman spectroscopy of a sebaceous gland on the lower forearm, 15 μm below the skin surface. The arrows (left) mark the spots from which the Raman spectra were obtained. The asterisk marks the prominent Raman band of lipid at 1296 cm⁻¹. (a, right) Raman spectrum measured in the sebaceous gland. (b, right) Raman spectrum measured outside the gland. (c, right) Difference spectrum (a minus b). (d, right) In vitro Raman spectrum of palmitic acid.

FIGURE 6

In vivo confocal image and Raman spectrum of a dermal capillary in the skin of the lower forearm. The depth is 60 μm below the skin surface. The arrow (a, left) indicates the location from which the Raman spectrum was obtained. (a, right) In vivo Raman spectrum of blood measured directly in a dermal capillary. Signal integration time: 30 s. (b, right) In vitro spectrum of blood.