Flow Cytometer Design

Introduction to Flow Cytometry The flow cytometer has been around for about three decades. During that time it has had significant impact on various fields of biology and medicine, including cell-cycle studies in relation to effects of drugs and radiation, immunology, ploidy determination in cancers, and studies of cellular parameters, such as intracellular pH and Ca2+ concentrations. In flow cytometry, cells are labelled with fluorescent molecules that bind specifically to the constituent(s) to be measured. For example, the DNA may be stained with propidium iodide or mithramycin, while other constituents may be labeled with (monoclonal) antibodies conjugated with some fluorescent dye such as FITC or phycoerythrin. Carried by a microscopic jet of water, the cells pass one by one through an intense beam of excitation light in the measuring region of the flow cytometer.

Each cell thereby produces a short flash of fluorescence, the intensity of which is proportional to the cellular content of the fluorescently labeled constituent. These flashes of fluorescence are collected by appropriate optics, which focus the light on a sensitive detector. The detector transforms the flashes of light into electrical pulses, which are measured and recorded by some electronics and a computer. Each cell also causes scattering of the excitation light. The intensity of this scattering is a function of the size, shape and structure of the cell. The resulting flash of scattered light is recorded by a separate detector. Thus, the cellular content of several constituents, labelled with dyes fluorescing at different wavelengths as well as size and shape or structure, are recorded for each individual cell. In contrast to other biomedical techniques that generally give averages over large numbers of cells, flow cytometry measures individual cells in large numbers. Hence this technique makes it possible to distinguish subpopulations of cells as, for example, in analyses of asynchronously growing cell cultures, where cells in the different phases of the cell cycle are readily distinguished, or in immunology, where the flow cytometer discriminates between different subsets of lymphocytes.

The flow cytometer is a remarkable and fascinating instrument from a technical point of view as well as with regard to performance. It employs a unique blend of modern technologies, including fluidics, lasers, optics, analogue and digital electronics, and computers and software. It can measure several parameters of each individual cell that is passed through its flow chamber to determine size, structure, and the precise contents of various cellular constituents. It can measure cells and other particles all the way down to submicroscopic sizes (0.1µm). The sensitivity is sufficient to detect 10-18 g of a specific substance per cell. And such measurements can be carried out with a precision of a few percent and at a rate of several thousand cells per second.

Harald B. Steen
Institute for Cancer Research
The Norwegian Radium Hospital
Oslo, Norway.

A prominent figure amongst Flow Cytometrists and microbiologists around the world, Harald is an consultant to Apogee Flow Systems and has made major contributions to the design and development of Apogee products.