Analytical Cytology: Methods for Studying Cellular Form and Function

By Robert C. Mellors | Go to book overview
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CHAPTER 1 Cytophotometric Analysis in the Visible Spectrum

ARTHUR W. POLLISTER, PH.D.

LEONARD ORNSTEIN, M.A.

Department of Zoölogy, Columbia University


Introduction

Of all the techniques of analytical cytology with the compound microscope, perhaps quantitative absorption analysis in the visible spectrum is destined to become most widely used. First and foremost, this tool offers the possibility of expressing in objective values the differences in intensity of natural color or of the stains and tests which have been developed by histochemists and cytochemists for localization of substances in cells (Chap. 2). Visual comparisons are of very limited use in resolving any question where variations in both concentration and cell volume are involved, but where the chemical and physical conditions are favorable for absorption analysis, it is not a difficult procedure to obtain data from which the relative amounts of absorbing substance can be computed over a wide range of concentration. Swift (111), for example, was thus able to estimate desoxyribose nucleic acid in spherical nuclei, where the highest concentration was over twenty times that of the lowest. This possibility of objective measurement of intensity should make a microspectrophotometer as essential a piece of equipment in any histological, pathological, or cytological laboratory as the spectrophotometer is in a modern biochemical laboratory.

Second, and of nearly equal importance, is the role of visible microspectrophotometry as an adjunct to biochemical and physiological studies which seek to localize chemical components and functions within the cell. Very often such studies involve an estimate of the number of cells in the analytical sample and a computation therefrom of the amount of the components in the average cell. This is an unjustifiable procedure and of dubious value unless it is supported by cytological studies of the variation within the group. For example, it was thus computed by biochemists that the "average nucleus" from a number of different tissues of an animal contained the same amount of desoxy

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