Quantitative Determination of the Pigment Content of Single Cells by Means of a New Microspectrophotometer
Barry Commoner · Annals of the Missouri Botanical Garden · 1948
Information concerning the physiological activities of single living cells has long been a goal of biological investigation. Analytical biochemistry of tissue masses, while yielding precise chemical data, unfortunately is limited by the fact that it involves procedures tending to disrupt the integrated chemical activities which are the mark of living cells. On the other hand, integrated information can be obtained from studies of whole organisms, organs or tissue fragments, but the data necessarily represent a pooling of the varied rates and directions of chemical activities of at least many thousands of cells. It has become increasingly clear that these classical methods need to be supplemented by procedures which can produce data referable to individual cells. Thus, for example, investigation of the mechanisms involved in the enormously varied biochemical expression of presumably identical nuclei in the different cells of an organism demands data which can distinguish between biochemical activities of two neighboring cells in a tissue. Almost the only means of investigating single tissue cells-without removing them from their neighbors and injuring them to the point of death-is light. Because many of the cellular compounds of interest to the biologist have rather characteristic absorption spectra, the determination of the optical density of a living cell in light of various wave-lengths can supply data on the type and amount of certain of these substances present. Since the pioneer investigations of MacMunn (1914), spectroscopy has attracted increasing interest as a means of probing the living cell. Quantitative studies of cellular absorption spectra became possible when several German optical firms developed photographic spectrographs for use with high-power microscopes (see Dhere, 1933). More recently modern photoelectrical methods have been applied to this type of apparatus by the school of Caspersson (1940) in Sweden, and by Pollister and Ris (1947) in this country. Their work, while chiefly concerned with the determination of nucleic acids and proteins in various parts of the cell, has shown conclusively that microspectrophotometry is technically sound (with proper precautions) and admirably suited to the development of new methods for studying single cell physiology. The present paper describes a new microspectrophotometer, designed specifically for the study of the biochemical changes which occur in single living cells. As an example of its applicability to this problem, studies of the pigment content of single plant cells are also presented.