the angle made by the two beams with the envelope (Fig. 1·15) would necessarily be different. Superposition would not be maintained, since the two regions of the envelope would refract the two beams differently with varying wavelength.] This instrument records absorption spectra (with a 2 mμ spectral band width and a peak to peak photometric error of ±0.1 per cent of full scale) from 400 mμ to 700 mμ in 10 minutes.
As outlined at the beginning of this chapter, there are three different levels of possible application of visible microspectrophotometry to cytology, namely, (a) for the objective comparison of tests and stains, (b) as a means of making chemical estimations at the level of the individual cell, and (c) to obtain some insight into the nature of intracellular chemical complexes and molecular structure through absorption-curve analysis. The past decade of development and use of visible techniques has seen progress in each of these directions.
The objective measurement of tests and stains has led to standardization of a variety of methods, some of which are outlined in Table 1·2. Whether or not these are interpreted in chemical terms, the Feulgen test, the Millon reaction, the basophilia with the azures or methyl green, and the acidophilia with fast green or naphthol yellow are unusually desirable cytological techniques, because, through control with photometric measurements, these methods have been developed so that they are highly reproducible in the hands of any technician who is capable of preparing reagents carefully and of following a prescribed schedule of time and temperature. This advance alone is capable of raising to a new level of precision the whole field which involves coloring cells and tissues in physiological and experimental cytology, histology, and pathology. Closely akin to this contribution is the influence that the quantitative viewpoint must exert, in the long run, upon interpretation of such microscopic findings.
The major efforts in visible cytophotometry have been directed toward the quantitative absorption analysis of a small region of a tissue or cell, to complement the results of biochemical analysis of organs, tissues, and cell fragments and to fill in some of the gaps in the findings by ultraviolet cytophotometry. In spite of the many obvious difficulties, these results have amply justified the empirical approach to the problem of visible cytophotometry. For example, the Millon reactions were developed originally by the senior author in collaboration with a biochemist (91) with two aims in view: first, to assess the protein loss during isolation of cell nuclei for chemical analysis; and second, to