A microscope-based flow cytophotometer

Abstract

By means of a new flow chamber, a standard fluorescence microscope with Epi illumination and 100 W mercury arc excitation has been turned into a flow cytophotometer combining high resolution and sensitivity with simplicity of operation. In the flow chamber, cells are passed in a narrow stream through the microscope focus carried by a laminar flow of water running on the open surface of a cover glass which is coupled to the oil immersion microscope objective. Two spectral components of the fluorescence, for example, resulting from specific staining of two different cellular constituents with different dyes, can be measured simultaneously in separate channels so as to produce three-dimensional histograms. The scattered light of the cells is detected in dark field by a second microscope situated opposite the primary objective. Scattered light detection is integrating with regard to scattering angle from 0° to 90°. Hence, diffraction pattern effects are eliminated and the light scatter signal is approximately proportional to cell dry weight. The Epi illumination, which implies that excitation and fluorescence collection are parfocal, greatly simplifies instrument adjustment, which is further facilitated by the fact that the cell stream can be viewed at high magnification. Cell measuring time is about 3 μs which implies a measuring rate of 3×10³ cells/s at 1% coincidence rate. Sensitivity is sufficient for measuring the DNA content of bacteria (that is, approximately 5×10⁻¹⁵ g/cell) with a coefficient of variance (CV) of about 6%. CV<1% is achieved for DNA histograms of mammalian cells. A 5 W argon laser as excitation source facilitates slit scan analysis and increases the sensitivity and measuring rate by one to two orders of magnitude.