Introduction of Fluorophores
Fluorophores are structures of compounds that can absorb photons of a certain energy (wavelength) and emit fluorescence. They usually contain aromatic groups or planar heterocyclic molecules with π bonds. Fluorophores can be stand-alone organic small molecules or fluorescent components of a functional system, e.g. a specialized structure in a fluorescent protein formed by the chemical modification of three amino acids called a chromophore. Chromophores are molecules that contain unsaturated groups and their associated chemical bonds that are capable of absorbing light radiation and have a leap. Some organic compounds contain unsaturated groups in their molecules that are capable of absorbing photons at certain wavelengths in the ultraviolet and visible regions with a large absorption coefficient.
Types of Fluorophores
Fluorophore molecules can generally be divided into four categories: proteins and peptides (such as fluorescent proteins), small organic molecules (such as dyes), synthetic oligomers and polymers (such as quantum dots), etc.
Fluorescent proteins containing chromophores are usually fused and expressed with target proteins in organisms through genetic manipulation, so as to be applied to the localization and movement tracking of target proteins.
Organic small-molecule fluorescent moieties can be used on their own. For example, as tracers in fluids, or as probes, indicators (when their fluorescence is influenced by environmental factors such as polarity or ion concentration), and substrates for enzymes (e.g. luciferin and luciferase). More commonly, they are covalently bound to macromolecules and used as markers for biologically active molecules (e.g. antibodies, peptides, nucleic acids). In addition, fluorophores can be used for applications such as fluorescent imaging and spectroscopic analysis by staining tissues, cells or materials.
| Type |
Fluorophores |
| Fluorescent protein |
Red fluorescent proteins (RFPs), orange fluorescent proteins (OFPs), yellow fluorescent proteins (YFPs), green fluorescent proteins (GFPs), cyan fluorescent proteins (CFPs), blue fluorescent proteins (BFPs) |
| Small organic molecules |
Xanthene derivatives (such as fluorescein, rhodamine), cyanine derivatives (such as cyanine, thiacarbocyanine), coumarin derivatives (such as coumarin), Alexa Fluor series dyes (such as Alexa Fluor 488, Alexa Fluor561, Alexa Fluor 647), Cy series dyes (such as Cy3, Cy5), etc. |
How to Choose Suitable Fluorophores?
The parameters of fluorophores can provide a reference for researchers to choose fluorophores, so as to ensure the selection of fluorophores suitable for research purposes and the most suitable experimental methods.
The initial brightness is an indication of the relative brightness of the fluorochromes and is derived from the molar extinction coefficient and quantum yield, both parameters specific to each individual fluorochrome. Fluorescent dyes with higher initial brightness are suitable for detection of lower abundance targets.
Stability is the relative percentage of initial fluorescence intensity remaining after 30 seconds of continuous illumination, obtained by observing samples placed in phosphate buffered saline. Dyes with higher photostability in the buffer system will be more suitable for related applications such as live cell imaging and detection. Among the antifade reagents, dyes with higher photostability are more suitable for imaging and detection experiments in fixed cell samples.
The staining index is a normalized value that can be used to compare the difference in fluorescence brightness of different fluorochromes in flow cytometry applications.
The maximum excitation wavelength indicates an excitation wavelength at which a peak is obtained, and the maximum emission wavelength indicates an emission wavelength at which a peak is obtained.
.
