The cosmetics industry has invested heavily in studying the penetration pathways of elements into hair fibers to understand how dyes and other hair products diffuse through the fibers. Generally, researchers approach these questions using fluorescent dyes with classical optical microscopy or confocal laser scanning microscopy. But these methods are limited by diffraction to roughly half the illumination wavelength and, for this reason, cannot reveal what happens in the fibers’ inner cell structures.Near-field scanning optical microscopy offers improved resolution by using a probe only nanometers in diameter to record the evanescent field at the sample surface. With this technique, the resolution is limited only by the probe’s radius of curvature, so researchers can investigate the inner cell structures of the fibers. Also, it may enable imaging of chemical elemental composition, which may be of interest to the cosmetics industry.In the November issue of Journal of Microscopy, researchers with Ecole Superieure de Physique et de Chimie Industrielles in Paris and with L’Oreal — Advanced Research Labs in Aulnay-sous-Bois, France, reported an investigation of dyed human hair fibers using apertureless near-field scanning optical microscopy. They demonstrated that the technique is especially well-suited for the study of changes in optical contrast caused by modifications in dye concentration.Most microscopy studies of biological samples such as human hair have been performed with either confocal microscopy or optical fiber scanning near-field microscopy using fluorescent dye molecules. “These techniques suffer from a poor resolution, from problems related to the photobleaching of the fluorescent dyes and from difficulties regarding the preparation of human hair samples with the proper fluorescent dyes,” explained researcher Yannick De Wilde. With optical fiber scanning near-field microscopy, preparation of a metal-coated, sharp optical fiber can be complicated, and the resolution is generally limited to 100 to 200 nm. With confocal microscopy, the resolution is limited by diffraction to about 300 nm.The near-field optical images (right) offered resolution of about 50 nm, revealing information related to optical properties that cannot be obtained in atomic force microscopy images such as that at left. The arrows in the AFM image indicate the remnants of nuclei (arrow 1) and borders of cortical cells (arrow 2). Reprinted with permission of the Journal of Microscopy.Apertureless scanning near-field optical microscopy offers several advantages over these techniques. First, the subwavelength probe used is a simple metal tip that is easily prepared using standard electrochemical etching. Also, the optical contrast in the images is not caused by fluorescence, but rather by local changes of the optical indices of the hair material — or of the dye concentration within the hair — located under the tip. It offers resolution as high as 20 to 30 nm and can operate at any wavelength.The major drawback of the technique, De Wilde said, is that a degree of interpretation is needed to retrieve the optical indices of the various materials responsible for the contrast between the regions in an image.De Wilde and colleague Florian Formanek used a homemade apertureless near-field scanning optical microscope in reflection mode. The instrument was based on an atomic force microscope operating in tapping mode, incorporating laser illumination of the sharp metallic tungsten tip. The tip served to scatter the field on the sample surface. They measured the field while scanning the sample laterally with a piezoelectric stage, acquiring measurements at each position.The tip, attached to the lower arm of a quartz tuning fork, had a radius of curvature of roughly 20 nm and thus allowed much higher lateral resolution than is possible with conventional optical microscopy. They maintained a constant distance between the tip and the sample surface using electronic feedback of the tuning fork voltage. The piezoelectric transducer gave them a range of 17 μm, allowing them to track important topographical variations, such as when changes in hair thickness created steps of about 5 μm. Typically, the technique offers a vertical range of only 2 to 3 μm.To illuminate the sample surface, they focused the beam from a 655-nm laser diode through a 50×, 0.6-NA objective attached to the tip. The same objective collected the field scattered by the tip and sent it to a photodiode for measurement.The scientists demonstrated the technique by acquiring optical contrasts from two sets of hair fibers, dyed with two types of molecular probes. De Wilde said that the hair fiber samples — combining the proper dye concentration and thin, undamaged sections with high surface quality and low roughness — were produced by collaborators Gustavo Luengo and Bernard Querleux at the L’Oreal labs.For comparison, they also obtained far-field images of the samples, without the tip. The far-field measurements revealed different structures in the hair fiber samples, including the cuticle, the melanin pigments and the medullar channel. However, with resolution of about 400 nm, the measurements could not resolve the inner structures of the cuticle and therefore did not allow study of the penetration pathways of dyes.The near-field images had much better resolution — about 50 nm — offering insight into those pathways. The researchers observed no optical contrast in samples with dyes diffusing deep inside the fiber, but the samples with dyes diffusing primarily at the surface level were more forthcoming. Here, diffusion appeared to take place in the cell membrane complex between the outer cells of the cuticle and/or the endocuticle.Researchers have reported an apertureless scanning near-field optical microscopy method for investigation of dyed human hair fibers. The technique offers improved resolution and can be used to probe the inner cell structures of the fibers.The investigation served primarily to demonstrate the technique’s potential. “The study was purely qualitative,” De Wilde said. “It shows that apertureless scanning near-field optical microscopy can say where changes in dye concentration occur within a hair fiber, but it does not provide any quantitative information regarding what is the dye concentration at a given location.” A quantitative study of the spatial variations of dye concentration would require detailed understandings of the optical indices of the hair materials as well as of the dyes, he added. Furthermore, the researchers would need to use hair from the same batch colored with different dyes.Contact: Yannick De Wilde, Laboratoire d’Optique CNRS UPR5-ESPCI (Laboratoire de Spectroscopie en Lumière Polarisée), Paris; e-mail: dewilde@optique.espci.fr.