CONFOCAL 3D RECONSTRUCTION OF HAIR 3 Thick semi-transparent specimens (almost all biological tissues) can be vertically scanned. In-depth optical sections are then stacked for volume rendering by computer treatment. At the end of the procedure, the sample has not been destroyed. As no blurring spoils the image, the resolution is improved (0.25 !xm) compared with that obtained by conventional light microscopy (0.6 I•m). HAIR SURFACE STUDY The Tracor-Northern TSM is a real-time confocal microscope equipped with a Nipkow scanning disk. The sample is illuminated by a 100-watt mercury-arc lamp. Reflected light from the focal plane is captured by a monochrome video camera (COHU-0.02 lux) and rapidly digitized via 4-data flow processors (PC-Oeil) on a microcomputer. Image processing is carried out by PC-Oeil, which provides a 3D reconstruction based on up to 256 stacked optical sections using 3D + software (MISIS-Tracor Europa). This gives two 512 x 512 image arrays coded on 256 gray levels, which are known as the "reflection map" (Figure 1) and the "topologic map" (Figure 2). Respectively, they provide the brightness and spatial position (X, Y, and Z coordinates) of each pixel. Combining the two maps gives a "perspective view" image (Figure 3), which reproduces the cylindrical shape of the hair. Line profiles can be generated interactively and provide local surface roughness parameters (see below). Freshly-plucked or treated hair was simply attached to a microscope slide coated with double-sided adhesive tape and observed with no further preparation. A dry 100X objective was selected, providing a resolution of approximately 0.25 I•m in X, Y, and Z. 3D images containing full information were obtained by stacking 100 optical sec- tions in 0.1-1•m vertical steps. Noise reduction by frame averaging or gray processing was unnecessary. Hair was stretched between two parallel jaws moved by an electric motor, thus avoiding manipulation during the experiment. The hair was glued to the jaws using cyanoacrylate resin. Twenty optical sections in 0.5-1•m vertical steps were collected at each step of the extension for 3D reconstruction each 3D image thus obtained was made to overlap the preceding ones by interactive shifting in X and Y coordinates in order to examine Figure I. "Reflectance map" of a natural brown hair. Local changes in brightness can be evaluated on a 256 gray scale range.

4 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS : Figure 2. "Topologic map" of the same hair. This map contains the vertical position of each pixel coded on 256 gray levels from O-black = bottom to 255 white = top. Figure 3. "Perspective view" showing the curvature of hair by pooling information on both brightness and altitude. The best angle of view can be interactively selected by rotation, tilting, and shadowing. the same field of view at any given step. Fast scanning of the sequence of images gave movement virtually in real time, facilitating the perception of subtle changes during the elongation. HAIR VOLUME STUDY The Molecular Dynamics Sarastro instrument is a confocal microscope (5) based on a focused laser beam (argon-ion). Scanning is ensured by two rotating mirrors (X,Y), and image formation takes less than five seconds for a 256 x 256 pixels array. This microscope is used to acquire fluorescence signals. We selected a 60X oil-immersion objective. The contact fluid reduced surface reflection and consequently improved the quality of in-depth optical sections. Noise was reduced by image averaging (four frames). A full cylinder of hair shaft was reconstructed in three dimensions on a Silicon Graphics workstation.