Progress is reported on the development of equipment and techniques for the flow-systems analysis and characterization of protein contents and proliferating kinetics in tumor cells and the staining of cellular DNA.
A flow-system cell-analysis instrument is described in which cells from a heterogeneous population are characterized by their light-scatter patterns alone. As the cells pass at high speed through a focused helium/neon laser beam, the scatter pattern from each cell is sampled simultaneously at up to 32 angles between 0 degrees and 30 degrees with respect to the laser beam axis, and the scatter pattern for each cell is transferred to a computer. A mathematical clustering algorithm is used to determine the number of classes into which the cells can be divided, and a linear separation algorithm is used to find the boundaries between the classes. Preliminary results on exfoliated cells from gynecological specimens are presented. This technique may be useful for automated prescreening of gynecological specimens.
A flow chamber has been developed which collects about 60% of the total cell fluorescence for analysis compared to about 2.5% for conventional flow systems. The chamber, an ellipsoid of revolution, is gold-plated for increased reflectivity. Fluorochrome-stained cells enter the flow cell directly above the primary focus of the ellipsoid at the rate of 1000 cell/sec. A focused argon-ion laser beam enters the flow cell parallel to the semiminor axis and intersects the cell stream at the primary focus. Fluorescent light emanating from this point is reflected toward the secondary focus, where it exits the chamber for analysis. The high efficiency flow cytometer has been used to obtain nucleotide fluorescence distributions from samples of Micrococcus glutamicus bacteria stained with propidium iodide and of spermatozoa stained by the acriflavine-Feulgen procedure.
Progress is reported on instrumentation and biological aspects of the research. Improvements in hardware and software are described. Studies were conducted to determine optimal conditions for freezing tumor material and still maintaining a high degree of cell viability. New methods for separating erythrocytes and tumor cells were developed. Effects of fixation in various concentrations of ethanol on cell samples were studied. Studies using flow microfluorometry were conducted to determine dispersal, fixation, staining, and analysis of DNA and protein in the model in vivo tumor systems. (HLW)
In the radiation experiments, the induced turbidity was found to be a nonmonotonic function of the radiation dose given the protein, showing certain maxima and minima. This communication reports the results of some recent experiments where it was found that, if the same proteins were subjected to dry heating in vacuo, the turbidity became a nonmonotonic function of the time of exposure at any fixed temperature. This is unusual because the time associated with the turbidity maxima appears to be related to the rate of thermal inactivation at the same temperature. This was found to be true over the temperature range 140? to 210?C. Since the thermal response is formally similar to the radiation response, a number of combined heating and radiation runs were made to determine whether radiation and thermal effects were additive. Earlier radiation work was carried out with, among other proteins, ribonuclease and chymotrypsinogen. The results of bombarding and/or heating dry layers of these proteins, plus trypsin, are described below.
