Basics of fluorescence guide
Understand fluorescence and how it enhances scientific exploration.
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 a 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.
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.
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.