PENETRATION INTO HAIR FIBER 277 Indeed, it was possible to watch the wall structures of the medulla change progressively over a matter of seconds. This showed quite clearly that, with a highly active molecule such as water, one can achieve total access to the centre of the fiber in one minute or less. This suggests that for small, active molecules there is a rapid transport system operating in the fiber. However, it does not indicate a particular or preferential pathway. Using typical combinations of uranyl acetate and lead citrate on intact fibers, these studies showed that electron contrast, identical to that achieved with cross sections, could be achieved by pretreating the fibers. This indicates that the stains have complete access to each of the structures, e.g., the delta layer of the cmc, endocuticle, cortex cmc's, and macrofibrils. The relative absence of stain contrast in the exocuticle and a-layer would appear to indicate that these structures cannot be accessed. However, it is well documented that these structures do not show electron contrast when these stains are used on cross sections (7). One is, therefore, left with the question of "Did the stain get into the structure and leave during the rinsing step?" or "Is the structure inaccessible?" It is not possible to conclude from this approach whether the reagents entered via the delta layer and/or endocuticle it is only safe to say that the markers are retained in these structures even though these are the most likely penetration routes. Whole hairs treated with aqueous silver nitrate, rinsed, and exposed to light showed extensive silver deposition in the cortical cmc's and pigment granules. This indicates that silver has entered the cortical cell. Aqueous silver nitrate showed no conclusive pattern of contrast, i.e., there was no specific attachment to the high-sulphur proteins even though the previous result shows that molecules can easily enter many of the hair's structures. One is, therefore, left with the problem of "Is the silver present in the cuticle but invisible?" Hairs immersed in aqueous silver nitrate and then made alkaline to precipitate insoluble silver hydroxide showed strong staining of the high-sulphur proteins of the cuticle and cortex. In addition, they showed very distinct precipitates in the cell membrane complex of the cuticle. This shows that both aqueous silver and hydroxide ions were present in the cmc at the same time, indicating that the cmc is a pathway from the outside of the hair to the cortex. This also serves to show that deposits will only be observed when they have either an affinity for a structure or are made to stay where they are. In the case of silver nitrate alone, it is likely that it is removed from the cmc when the fiber is rinsed. Subsequent experiments where whole hairs were treated with only aqueous silver nitrate but without rinsing, drying, and exposure to light showed small amounts of silver precipitated in the cell membrane complex of the cuticle. In addition, some small deposits were evident in the a-layer. This further indicates that it is essential to find a method to localize markers of penetration, as absence of evidence is not evidence of absence. Using techniques first invented in 1839 by Henry Fox-Talbot to produce photographs on writing paper, the technique was reapplied to hair fibers to produce, in essence, a photograph inside the hair. Sequential treatment of whole hair fibers using sodium chloride and then silver nitrate to precipitate silver chloride will show where chloride and silver ions have co-existed. Exposure to light will then produce a silver deposit that can be imaged in the TEM. Control experiments were conducted, as silver nitrate alone will produce a silver precipitate when exposed to light. The striking images consistently showed high levels of precipitate in the cuticle cmc. Apparently emanating from the cmc

278 JOURNAL OF COSMETIC SCIENCE are cascade-like structures of silver granules. It is interesting to speculate that these might show penetration pores from the cmc into the sulphur-rich proteins of the cuticle cell. The deposits in the cmc show that chloride ions from the first treatment are retained within the cmc and interact with silver ions in the subsequent silver nitrate treatment. This indicates that both reagents have used the same pathway. Care must be exercised in the interpretation of the cascades, as the absence of deposits from adjacent areas may be due to nucleation and increased crystal growth in the areas of marked deposits. Alternatively, it is indicative of a localized reaction with the sulphur-rich proteins. It is clear from serial sections that these "cascades" are three-dimensional in structure and expand progressively from the crnc into the exocuticle. It is difficult to interpret deposits in the endocuticle, as these may indicate further penetration from the cmc or penetration along the endocuticle and retention in the known small sulphur-rich domains. The same findings were made close to the hair root where the cuticle cell edge would be intact. Interestingly, no cascades were found associated with the cuticle cell membrane bor- dering the cortex, although deposits of silver were seen in both the cortical cmc's and inter-macrofibrillar material abutting the cuticle cortex boundary. When considering observations made on the cortex of hairs treated with chloride and silver, caution is needed. The visually larger precipitates in the cortical cmc's and inter-macrofibrillar material could lead one to conclude that access to these structures is greater than to the macrofibrills. However, the cmc and inter-macrofibrillar material probably have a greater amount of free space vs the macro fibrils and, therefore, would be able to precipitate or develop more silver. The reduced contrast in the macro fibril does not exclude the presence of silver one would require further proof by elemental analysis. By combining the results of the above studies it becomes very evident that all structures in the hair can be accessed by small, water-soluble molecules, although the pathway to each structure may well be different. It is highly probable that access to the cortex is gained primarily via the cmc, whereas access to the cuticle cell is gained via the cuticle cell edge. The latter permits access into both the high-sulphur and low-sulphur com- ponents of the cuticle cell. However, imaging this event is very dependant on the choice of reagent and the affinity of ions for a particular site. It is not clear what role the "cascade" observations play in penetration into the cuticle cell. They are present at both root and tip, indicating they are independent of hair damage. In this way their presence and appearance are independent of the limited "pores" described by Swift (1) in weath- ered hair. Combined with the typical silver stains, here used e, bloc, it is now obvious that the high-sulphur proteins of the cuticle are easily penetrated. These studies show that the hair can present a number of compartments of differing affinity and capacity for penetrating molecules. It appears that the cuticle cell presents a large compartment, but one which would not deliver material, beyond its enveloping membranes, into the adjacent cortex. Viewing the cuticle as a definite, and open-ended, compartment helps to account for the rapid initial loss of hair dyes from the fiber with washing. Conversely, the cuticle cell membrane complex presents a small compartment allowing the transport of molecules across the cuticle to the cortex. One can only speculate on the capillary forces that might exist within the cmc lamellae between adjacent cuticle cells. The large compartment presented by the cortex, which readily expands in the presence of water, will act as a pulling force to increase flux along the cmc. Once an aqueous molecule enters the cortex, it will again be presented with compartments of varying capacity, i.e., macrofibril, nuclear remnant, and inter-