Introduction

The great analytical power of current cytometers stems from their ability to quantify simultaneously many separate parameters on thousands of cells within a few minutes. Laser scanning cytometers and flow cytometers measure, respectively, one or two light scattering parameters, and, depending on the instrument, up to 12 fluorescence parameters. Only a few cellular components (e.g. pyridine-and flavin-containing nucleotides) are intrinsically fluorescent (autofluorescent), so cells are usually stained with compounds (known as fluorochromes, fluorophores or fluors) that fluoresce when they report the presence or activity of a particular cellular component. Three main approaches are used for staining:

• a fluorochrome-labelled antibody or other ligand is allowed to bind to complementary structures, either within or on the surface of cells

• a fluorescent dye, e.g. one that binds to nucleic acids, is allowed to accumulate within cells

• a cell-permeant precursor compound is converted by the activity, often enzymic, of some cell component into a form with distinct fluorescence.

Usually samples are stained with more than one fluorochrome and, depending on the number of fluorochromes used, the technique is known as two-colour, three-colour, etc. staining. At present, the main constraint on the number of different stains that can be measured simultaneously is the shortage of fluorochromes with distinct emission spectra and the difficulty of distinguishing those fluorochromes with emission spectra that overlap. To appreciate how fluorochromes can best be used in cytometric assays it is worth first considering how light and matter interact.

The interaction of light and matter

Light is a form of electromagnetic radiation with a wave motion defined by electric and magnetic vectors at right angles to each other and perpendicular to the direction of propagation.