Microscope-based multiparameter laser scanning cytometer yielding data comparable to flow cytometry data

We describe a computer-controlled 10 microns spot size laser scanning cytometer for making multiple wavelength fluorescence and scatter measurements of unconstrained cells on a surface such as a microscope slide. Designated areas of slides placed on a microscope stage are automatically scanned, and cells which generate above-threshold scatter or fluorescence values are found and individually processed to determine a list of measurement parameters. For each fluorescence or scatter measurement parameter, this list contains the integrated and peak values and bit pattern images of a scan window centered on the cell. The measurement time, the position of the cell on the slide, and two segmentation indices are also included in the list. Measurement time, cell position, and properties derived from the bit patterns are used interchangeably with integrated or peak measurement values as coordinates of multiproperty displays. Cells may be selected for counting, data display in various forms, or visual observation based on their meeting complex criteria among a chain of two property screens. Cells with selected properties may be viewed during an experiment or retrospectively. A designated specimen field may be repeatedly remeasured to perform kinetic cell studies. An argon ion and a HeNe- based laser instrument have been constructed and software has been written and evaluated with the specific goal of increasing the precision of propidium iodide-stained cellular DNA measurements. Some of the capabilities of the instrument and its current performance are described.