QUANTITATIVE MICROSCOPY 503 of a few Angstrom units. What then does an assumed sensitivity of 5 A. mean for a practical application in cosmetics research ? One practical example is the study of hair. Hair as such is rather highly birefringent, which makes it difficult to measure small changes against the fairly nonuniform background. Nevertheless, it may be interesting to calculate, as a numerical example, how thin a coating of birefringent mate- rial on the surface of hair can be measured in a polarizing microscope, or how much birefringent material would have to penetrate into the interior of a hair to change its anisotropy. The total birefringence of hair would, by the way, already be affected by any penetration (even of isotropic materials) due to a change of form birefringence. For the purpose of this calculation, a polymeric hair conditioning agent shall be assumed to coat the hair. Assuming a value for the birefringence of oriented polymer material of 0.02, we can then calculate the minimum thickness necessary for such a coating to be measurable. This follows from the equation F = t (n•. -- n•), in which I' represents the retardation or optic path difference in m•, t the geometric thickness, also expressed in m•, and n•. - n• the birefringence. F•in was assumed as 5 A., or 0.5 m•, n•. - n• as 0.02. The minimum value for t then becomes 25 m•, or 250 A. The total birefringence of a structure like a human hair is the result of the contributions of the different histological components, such as the medul- lary and the cortical cells, and the total birefringence of each of these com- ponents again is the sum of their textural and their intrinsic birefringence. The intrinsic birefringence is a material constant the textural birefringence, also called form birefringence, is a function of the orientation of the in- trinsically birefringent elements and of their partial volumes, compared to the partial volume of the unoriented matrix. Any mechanical influence, such as mechanical stress which leads to elastic or plastic deformation, will affect form birefringence (23), the changes of which can be used as sensitive indicators. Swelling and thermal treatment will not only affect form birefringence but also intrinsic birefringence. FLUORESCENCE MICROSCOPY Fluorescence microscopy in its various applications combines highest sensitivity of detection with extreme specificity. It utilizes the fact that many substances become self-luminous when irradiated with light of short wavelengths, i.e., with light from the energy-rich blue and ultraviolet range of the spectrum. This irradiating or "exciting light" stimulates fluo- rescence in such substances. The fluorescent light always has a longer wavelength than the exciting light. Almost any microscope can be adapted for fluorescence microscopy. Some applications require hardly any, others demand more elaborate

504 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS accessories. First, a light source is needed which yields a powerful flux of short-wavelength light. Maximum pressure mercury arcs are necessary for all work involving immuno-fluorescence (26, 27) and for work carried out under oil immersion with other staining techniques. For medium and low power work, where fluorochromes like Acridine Orange are employed, adequate excitation can often be obtained from a high intensity incandes- cent lamp. All such light sources do, of course, not only emit the desirable short wavelengths, but also light of longer wavelengths, which would com- pletely mask any fluorescence. For this reason, all exciting light is filtered through a set of exciter filters, which transmit only the short wavelength range of the spectrum and completely absorb light of longer wavelengths. The exciting light is then concentrated on the specimen by a microscope condenser. In immuno-fluorescence this is normally a dark field condenser to give images of very high contrast, but in most other work a regular microscope condenser with all diaphragms wide open is sufficient. Fluo- rescence is stimulated in the specimen and both the remaining exciting light and the stimulated fluorescence enter the objective. To remove any of the remaining short wavelength exciting light, a barrier filter is mounted above the objective. It has a transmission curve complementary to that of the exciting filters and absorbs all light of shorter wavelengths than that of the fluorescence. The specimen, therefore, appears in brilliant luminosity against a dark background. Many organic substances have the intrinsic property to fluoresce (28) and can be traced directly in a microscopic preparation. Others can be stained selectively with fluorescing dyes, so-called fluorochromes this fluorescence is called secondary fluorescence. Finally, a fluorescing molecule can be used to tag an otherwise nonfluorescing complex, such as an antigen or anti- body. It is in this last procedure that the high sensitivity of detection inherent in fluorescence techniques can be combined with the extreme specificity of serological methods. This technique is known as the fluores- cent antibody technique, or immuno-fluorescence. It may be that the remarkable success and the exciting potential of this latter technique has diverted the interest in the field of fluorescence microscopy from those simpler staining techniques which can be used so successfully in routine work. Among the many fiuorochromes which are available for fluorescence staining, Acridine Orange has found some interesting applications. For example, under certain conditions, it permits differentiation between living and dead protoplasm (29). Acridine Orange, or tetramethyl-diamino acridine, is a basic dye. In the alkaline pH-range it is present as an un- charged base molecule, which selectively stains lipid phases with a dark, saturated green fluorescence. In the weakly alkaline, neutral or acid range, however, Acridine Orange is present in solution in the form of a univalent