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

Unit 1. Introduction

About this Unit

This Unit answers the question, What is flow cytometry? It describes the principles underlying the technique, explains its advantages and disadvantages and outlines the major applications. The last part of the Unit introduces some of the concepts used in data analysis.

This Unit is fairly short. When you have completed it, you might want to make on a start Unit 2, which is rather longer.

Contents

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

What is flow cytometry?

As its name implies, flow cytometry is the measurement of single cells in a flow system. The flow system delivers particles singly past a point at which a measurement is made.

The particles may be:

Cells (mammalian, plant, algae, yeast, bacteria)

Nuclei

Chromosomes

Small beads (possibly fluorescent)

Light is focussed at point of measurement. The light source can be a:

Laser

Arc lamp

A laser emits an intense beam of monochromatic light, that is, light at a single wavelength. The large majority of flow cytometers use a laser emitting monochromatic blue light at 488 nm; up to seven other lasers may be fitted but few instruments have more than three. The choice of lasers is discussed further in Unit 2.

If an arc lamp is used, the desired wavelength of light must be selected using an optical filter.

As the cells pass through the focussed beam of light, we measure:

Fluorescence

The cells (or other particles) may be autofluorescent or they may be labelled with one or more of a variety of fluorescent compounds.

Light scattered by the cells

The important feature is that measurements are made on single cells, individually.

The major elements of a flow cytometer consist of the

Flow cytometry (FCM) is sometimes abbreviated to FACS (Fluorescence Activated Cell Sorting). The origin of the latter abbreviation is that some more sophisticated instruments are capable of physically separating different populations of cells, based on their fluorescent properties. It is a trade name for instruments produced by a particular manufacturer (BD).

Continued