Characteristics of a Simple, High-resolution Flow Cytometer Based O a New Flow Configuration

A new flow configuration allows a flow cytometer of high resolution and stability to be assembled from an inverted fluorescence microscope with incident illumination, a pulse photometer, and a multichannel pulse height analyzer. A nozzle produces a hydrodynamically focused sample stream in a liquid jet that id directed onto a microscope cover glass in front of the microscope objective. The microscope provides a mechanically stable optical system of high numerical aperture (N.A.) (oil immersion, N.A. = 1.3) for focusing the excitation light and collection of the fluorescence light. The instrument has wide optima with regard to the various characteristics of the flow configuration, such as the rate of sample analysis and sheath flow, and the angle of incidence of the liquid jet, thus making it easy to adjust for optimal performance. DNA histograms of rat thymocytes stained with ethidium bromide and mithramycin demonstrate that all angles of incidence can be used. Large-angle incidence (70 degrees) gives the best resolution, i.e., a coefficient of variance (CV) of 0.9% of the peak of the histogram. This is only slightly better than values obtained at other angles, e.g., CV = 1.3% at vertical (0 degrees) incidence. It is concluded that instrumental resolution is equal to or better than CV = 0.9%. Linearity (proportionality between channel number and fluorescence intensity) is within 1%, and instrumental drift over a 1-h period is normally less than 1-2%.