Fluorescence guide
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A guide to how fluorescence and tandem dyes work.
Download our Fluorescence guide here or read the first chapter below.
Updated August 24, 2023.
Fluorophores and fluorescence detection are central to many research applications. Many fixed and live cell imaging techniques rely on immunofluorescence or fluorescent proteins. Fluorescence-based techniques can also be used in many common laboratory assays, including flow cytometry, ELISA, western blot, immunohistochemistry (IHC) and immunocytochemistry (ICC).
Fluorescent detection methods have several advantages over other analytical techniques, including high sensitivity, quantitative analysis, and the potential to carry out multiplex analysis using a range of available lasers, optical filters, and fluorophores.
This guide will enhance your understanding of how to use fluorescence in your research and advise you on choosing the right fluorophores and fluorescent proteins for your experiments. It begins with a basic introduction to fluorescence and how it works, followed by discussions of more advanced fluorescence mechanisms, such as fluorescence resonance energy transfer (FRET) and time-resolved fluorescence (TRF). Finally, the guide covers essential reagents for fluorescence-based experimentation, including fluorophores, tandem dyes, and fluorescent proteins.
What is fluorescence?
Fluorescence is a light signal detected when a chemical compound called - fluorophore absorbs energy at a specific wavelength, causing it to become excited. The fluorophore then emits light at a longer wavelength as it relaxes and returns to its ground state.
Fluorescence is a three-stage process, as detailed in Figure 1. In the first step (1), the fluorophore is irradiated with electromagnetic light produced by a laser and passed through optical filters to create a very specific wavelength, matching the signature excitation wavelength for the molecule. The light provides the right amount of energy for an electron in the molecule to jump from its ground state (Gs) to an excited state (Es). In the second step (2), the electron absorbs the light and reaches an excited state.
Figure 1. The three stages of the fluorescence event.
In the final step (3), the electron undergoes vibrational relaxation to the lowest vibrational state within the excited electronic state, followed by relaxation to its ground state. As the electron returns to the ground state, energy is released in the form of a photon.
The amount of energy released and the resulting wavelength of the emitted photon is characteristic of the fluorophore and determines its color and signature emission spectrum. The wavelength of the emitted light is longer than the exciting light because the electron undergoes vibrational relaxation, which emits energy as heat before fluorescence. The difference in wavelength between the exciting light and the emitted light is called the Stokes shift.
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).
- 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.
- On absorption of this energy, the electron moves to an excitation state at the next energy level (Es).
- 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.
- 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.