What is flow cytometry?

The flow system

Light scatter

Fluorescence measurement

Multiparametric measurement

Advantages of flow cytometry

Disadvantages of flow cytometry

Sample preparation

Applications

Data display

Safety

Further reading

Self Assessment

Introductory Contents Further reading Units 	Introduction  The flow cytometer 	Data analysis 	Immunofluorescence 	Some clinical applications  DNA and the cell cycle Other applications Cell proliferation Cell death Assignment 1 Assignment 2

The flow system

The heart of the flow cytometer is the flow chamber in which the cells are hydrodynamically focused at the point of measurement. Figure 1.1 shows a diagrammatic representation of a flow chamber. Water or a saline solution (called the sheath fluid) is forced through the chamber under pressure. The sample (consisting of a suspension of single cells or other particles) is injected into the centre of the stream of sheath fluid.

When conditions are right, sample fluid flows in a central core that does not mix with the sheath fluid. The sample stream is also narrowed in diameter, due to the focussing effect of the flowing sheath fluid (called the Bernoulli effect).

The flow chamber is often referred to as the flow cell. In this Unit, the designation, chamber, has been used to avoid confusion between the flow cell and the biological cells that flow through it.

The point at which the light is focused is called the interrogation point. This point is usually inside the flow chamber, but, in some cell sorters, the cells may be interrogated as the stream emerges from the chamber.















Light scatter

As the cells pass through a laser beam, they scatter light. This light is measured

The laser light scattered by a particle depends on its size and other properties. Scattered light may be used to distinguish

An example of the latter is shown in Figure 1.5.











Figure 1.2. Forward light scatter is the laser light scattered over a narrow angle past the blocker bar.

Forward Scatter

The amount of light scattered over a narrow angle in the forward direction is detected in the forward scatter channel (FS, FSC or FALS). The primary laser beam is blocked using a blocker bar (sometimes called an obscuration bar).

The intensity of forward scatter is proportional to the size, shape and optical homogeneity of cells (or other particles), size being the predominant parameter.

Forward scatter tends to be more sensitive to surface properties of particles (e.g., cell ruffling) than side scatter. It can be used to distinguish live from dead cells.


Continued

Figure 1.1. Diagrammatic representation of a flow chamber. The sheath fluid is forced through the cell under pressure. The sample, consisting of a suspension of single cells, is injected into the centre of the sheath stream. The sample stream is focussed hydrodynamically, so that the cells all pass through a small volume in which the laser light is focused. Typically the inside of the flow chamber might be 200 µm wide; the sample stream, at the point of measurement, might be around 10 µm (similar to the diameter of a cell). Diagram modified from that of J. Paul Robinson, Purdue Cytometry Laboratories, USA.