Flow cytometry antibodies, kits, reagents

Chapter 2: Principles of Fluorescence

Fluorophores and Light

A fluorophore is a fluorochrome conjugated to a given macromolecule. Fluorophores are fluorescent markers used to detect the expression of proteins and nucleic acids, which functionally accept light energy (for example, from a laser) at a given wavelength and re-emit it at a longer wavelength. These two processes are called excitation and emission. Emission follows excitation extremely rapidly, commonly in nanoseconds, and is known as fluorescence. Before considering the different types of fluorophores available for flow cytometry, it is necessary to understand the principles of light absorbance and emission.

Light is a form of electromagnetic energy that travels in waves. These waves have both frequency and length, the latter of which determines the color of the light. The light that can be visualized by the human eye represents a narrow wavelength band (380–700 nm) between ultraviolet (UV) and infrared (IR) radiation (Figure 7). Sunlight, for example, contains UV and IR light that, although invisible to the eye, can still be felt as warmth on the skin and measured scientifically using photodetectors. The visible spectrum can further be subdivided according to color, often remembered by the mnemonic ROY G BV, standing for red, orange, yellow, green, blue, and violet. Red light is at the longer wavelength end (lower energy) and violet light is at the shorter wavelength end (higher energy).

The electromagnetic spectrum

Figure 7: The electromagnetic spectrum