Fluorescence guide

A guide to how fluorescence and tandem dyes work. 

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View our fluorochrome chart, a quick and easy guide to help you select the most appropriate fluorochromes for your next experiment.

How does fluorescence work?

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1. Electromagnetic energy from a laser set at the correct wavelength will provide the right amount of energy to an electron in the fluorescent dye molecule. This is the signature excitation wavelength for the molecule. The energy is absorbed by this electron.
2. On absorption of this energy, the electron moves to an excitation state at the next energy level (Es). 
3. Finally, this energy is released in the form of a photon (fluorescence) and the electron moves back down to the lower energy level. The amount of energy released will be determined by how far the electron drops down the energy levels which will always be the same in the same fluorescent molecule. This will determine the wavelength of the photon, and the color of the fluorescence observed giving the fluorescent dye its signature emission wavelength.

​How do tandem dyes work?

Tandem fluorescent dyes are conjugated 'dual' fluorescent molecules, for example, PE-Cy5. On the antibody, they will be close enough so that the energy can be transferred between the two. The laser excitation wavelength used will excite the donor molecule only (eg PE) – it will not be the correct wavelength to excite the acceptor molecule. The energy then released from the donor molecule will be at the correct wavelength to excite the electron in the acceptor molecule. The acceptor molecule will then release energy in the form of a photon at its signature wavelength.

So, for example, PE-Cy5 will excite at the excitation wavelength for PE (565 nm) and emit at the emission wavelength for Cy5 (670 nm).

1. Electromagnetic energy from a laser set at the correct wavelength will provide the right amount of energy to an electron in the donor fluorescent dye molecule. This is the signature excitation wavelength for the molecule. The energy is absorbed by this electron.
2. On absorption of this energy, the electron moves to an excitation state at the next energy level (Es).
3. Energy is released from this electron in the form of a photon. The electron moves back down to the lower energy level. The energy is released in the form of a photon. This then excites an electron in the tandem dye molecule which moves up to the next energy level.
4. Finally, this energy is released in the form of a photon (fluorescence) and the electron moves back down to the lower energy level. The amount of energy released will be determined by how far the electron drops down the energy levels which will always be the same in the same fluorescent molecule. This will determine the wavelength of the photon, and the color of the fluorescence observed.

Fluorochrome chart – a complete guide

Whether you have to select one or more fluorochromes, we have put together a new fluorochrome chart to make the process quick and easy. It contains aligned emission and excitation spectra for 30 of the most commonly-used fluorochromes with information about popular instrument lasers and filters depicted across the chart for easy visualization. We have included a step-by-step guide to walk you through the process.

Download the fluorochrome chart here.