Theory predicts that small angle light scattering by spherical particles of 5 to 20 p. diam is nearly proportional to volume and insensitive to particle refractive index. A flow system photometer with helium-neon laser light source measures the scattering between 0.5 and 2.0 0 from individual particles at 10 4 to 10 5 /min. Volume distributions of mammalian cells and plastic microspheres agree with other independent determinations.A large desorption cryostat with a heat leak of about 150 mW is described. With the help of an electronic temperature regulator it is capable of maintaining any temperature between 4.2 and 19.0 K to an accuracy of better than 0.5%, using only 200 g of activated charcoal.
A new flow-system instrument for quantitative analysis and sorting of microscopic particles, particularly biological cells, based on multiple measurements of physical and biochemical properties has been developed. Cells stained with fluorescent dyes in liquid suspension enter a unique flow chamber where electrical and optical sensors measure cell volume, single- or two-color fluorescence, and light scatter, and emerge in a liquid jet that is broken into uniform droplets. Sensor signals are electronically processed several ways for optimum cell discrimination and are displayed as pulse-amplitude distributions using a pulse-height analyzer. Processed signals trigger cell sorting according to preselected parametric criteria. Sorting is accomplished by electrically charging droplets containing the cells and electrostatically deflecting them into collection vessels. This instrument is described in detail with illustrative examples of experiments using polystyrene fluorescent microspheres, cultured human cells, and human leukocytes.
We have developed a system for the perfusion of a stirred suspension of multicellular spheroids during nuclear magnetic resonance spectroscopy. Measurement of the medium temperature, pH, oxygen tension, and glucose and lactate concentrations demonstrated that the macroenvironmental conditions around the spheroids during perfusion matched those in standard spinner culture flasks. Spheroids cultured in the NMR perfusion chamber for up to 48 h were virtually identical to spheroids cultured under standard conditions in terms of volume and cell number growth, the extent of central necrosis, cellular clonogenicity, and proliferative status. To avoid problems in interpreting the NMR spectra, we have used a medium containing 10% of the normal inorganic phosphate concentration; comparative growth and NMR studies showed that this medium had no effect on the results reported. 31P NMR spectroscopic analysis demonstrated that the mean pH, nucleotide triphosphate (NTP) to inorganic phosphate (Pi) ratio, the total amount of NTP, and the total energy charge were essentially constant over 8 h of analysis. Stopping the stirring of the spheroid culture during analysis resulted in depletion of the nucleotide phosphate pool in 30 min, with an accumulation of Pi and a shift to a more acid intracellular pH. This effect could be reversed if stirring was resumed within 30 min. Stopping the perfusion while maintaining stirring resulted in a deterioration of the 31P spectra until no high energy phosphates remained at 120 min and the pH fell to approximately 6. This effect was also partially reversible after 30 min of reperfusion, with recovery to a normal 31P spectrum requiring 10 h. The combination of the spheroid model system with 31P NMR spectroscopic analysis will provide a powerful tool for investigating basic questions about the regulation of tumor cell energy metabolism and viability.
A modified flow cell in a conventional flow cytometer is described. The refraction of light in the flow cell-to-air interface is analyzed. The analysis shows that much of the light that can be collected is refracted out of the collection optics. The efficiency of light collection is doubled by the addition of a plano-convex lens to the flow cell. Also, more stable operation of the flow cell is achieved with the addition of a secondary sheath.
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