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

TLDR

The study presents a computer‑controlled 10 µm spot laser‑scanning cytometer designed to measure multiple fluorescence and scatter parameters of unconstrained cells on microscope slides, aiming to improve precision of propidium iodide‑stained DNA quantification. The cytometer automatically scans slide areas, identifies cells exceeding fluorescence or scatter thresholds, and records integrated and peak values, bit‑pattern images, cell position, time, and segmentation indices, which are used as coordinates for multiproperty displays and allow selective counting, visualization, and kinetic re‑measurement of cells. The instrument’s capabilities were demonstrated, showing that the laser‑scanning cytometer can accurately measure multiple parameters and that its use of argon‑ion and HeNe lasers improves the precision of propidium iodide‑stained DNA quantification.

Abstract

Abstract We describe a computer‐controlled 10 μm 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.