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Confocal microscopy and multi-photon excitation microscopy of human skin in vivo

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Abstract

This paper compares tandem scanning reflected light confocal microscopy and multi-photon excitation microscopy for the observation of human skin in vivo. Tandem scanning confocal light microscopy based on a white light source can provide video-rate image acquisition from the skin surface to the epidermal-dermal junction. Multi-photon excitation is induced by a 80 MHz pulse train of femtosecond laser pulses at 780 nm wave length. This nonlinear microscopic technique is inherently suitable for deep tissue fluorescence imaging. The relative merits of these two techniques can be identified by comparing movies of optical sections obtained from the forearm skin of the same volunteer.

©2001 Optical Society of America

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Figures (5)

Fig. 1.
Fig. 1. Schematic drawing of a vertical section of human skin. The following layers are shown: stratum corneum, stratum granulosum, stratum spinosum, the basal layer, and the papillary dermis.
Fig. 2.
Fig. 2. Schematic of a two-photon deep tissue microscope.
Fig. 3.
Fig. 3. Frontal sections of human skin in vivo acquired with reflected light confocal microscopy (a-d) and acquired from multi-photon excitation microscopy with excitation at 780 nm (e-h). Images were acquired from the volar surface of the forearm at similar positions with the two techniques described in the Methods section. Images were acquired at the regions: 10 microns below surface in stratum corneum (a and e), cells of stratum spinosum (b and f), cells of basal layer (c and g), and within the dermis (d and h). Scale bars show 50 microns.
Fig. 4.
Fig. 4. (2.29 Mb) Movie of stacks of optical sections from human skin in vivo. Optical sections were acquired with reflected light confocal microscopy. Intensity is mapped into colors to enhance visualization, high intensities are yellow and red, low intensities are green and black. The horizontal length of this field is 175 µm.
Fig. 5.
Fig. 5. (1.45 Mb) Movie of stacks of optical sections from human skin in vivo. Optical sections were acquired with multi-photon excitation microscopy with excitation at 780 nm. Intensity is mapped into colors to enhance visualization; high intensities are red and yellow, low intensities are dark red. Reconstruction shows the cells 10 microns below the surface within the stratum corneum, the cells of the basal layer, and structures within the dermis. The horizontal length of this field is 175 µm.
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