3.2 The colour of the emitted light depends on the fluorescent molecule

The light emitted by a fluorescent molecule is a different colour to the light that it absorbs. This is because electrons in their excited state lose a tiny amount of energy before they return to the ground state. Fluorescent molecules absorb light at specific wavelengths in the visible spectrum, which is depicted in Figure 10. If you examine the image, you will notice that the wavelength of light increases as the spectrum progresses from blue to red, while the energy of the light decreases. Therefore, the light used to excite a fluorescent molecule (e.g. blue or violet light) has more energy and a shorter wavelength than the light the molecule emits (often green, yellow, or red).

This shift in colour (known as the Stokes shift) is crucial in fluorescence microscopy. It allows researchers to clearly distinguish between the excitation light and the emitted fluorescence, enabling accurate visualisation of specific targets within cells and tissues.

Figure 10 The visible spectrum, showing the colours with their corresponding wavelength in nanometres (10^–9 m).

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An illustration of the visible spectrum of light, showing the colours with their corresponding wavelength in nanometres (10–9 m). A coloured horizontal bar indicates the colours visible at various wavelengths. An oscillating black line below the bar illustrates that the wavelength of light increases as the spectrum progresses from violet through blue to red, but the energy of the light decreases.

If you are interested in the colours for the respective wavelength (in nanometres) shown in the horizontal bar, these are (from left to right): violet around 350; purple around 400; blue around 450; green around 500; yellow around 550; orange around 600; red around 650.

Figure 10 The visible spectrum, showing the colours with their corresponding wavelength in nanometres (10^–9 m).

The wavelength of light absorbed by a fluorescent molecule is characteristic of that particular molecule, as is the wavelength of light it emits. The absorption and emission of specific colours by different fluorescent molecules allows researchers to investigate many different aspects of cell biology at the same time within a single biological sample.

In the next section, you will learn how different structures inside cells can be stained differently to obtain the images you will encounter throughout the rest of the course. But before that you will embark on a ‘Journey into a cell’, an immersive exploration that showcases the remarkable capabilities of both fluorescence and electron microscopy.