A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescentchemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with several π bonds.[1]
Fluorophores are sometimes used alone, as a tracer in fluids, as a dye for staining of certain structures, as a substrate of enzymes, or as a probe or indicator (when its fluorescence is affected by environmental aspects such as polarity or ions). More generally they are covalently bonded to macromolecules, serving as a markers (or dyes, or tags, or reporters) for affine or bioactive reagents (antibodies, peptides, nucleic acids). Fluorophores are notably used to stain tissues, cells, or materials in a variety of analytical methods, such as fluorescent imaging and spectroscopy.
Fluorescein, via its amine-reactive isothiocyanate derivative fluorescein isothiocyanate (FITC), has been one of the most popular fluorophores. From antibody labeling, the applications have spread to nucleic acids thanks to carboxyfluorescein. Other historically common fluorophores are derivatives of rhodamine (TRITC), coumarin, and cyanine.[2] Newer generations of fluorophores, many of which are proprietary, often perform better, being more photostable, brighter, or less pH-sensitive than traditional dyes with comparable excitation and emission.[3][4]
Fluorescence
The fluorophore absorbs light energy of a specific wavelength and re-emits light at a longer wavelength. The absorbed wavelengths, energy transfer efficiency, and time before emission depend on both the fluorophore structure and its chemical environment, since the molecule in its excited state interacts with surrounding molecules. Wavelengths of maximum absorption (≈ excitation) and emission (for example, Absorption/Emission = 485 nm/517 nm) are the typical terms used to refer to a given fluorophore, but the whole spectrum may be important to consider. The excitation wavelength spectrum may be a very narrow or broader band, or it may be all beyond a cutoff level. The emission spectrum is usually sharper than the excitation spectrum, and it is of a longer wavelength and correspondingly lower energy. Excitation energies range from ultraviolet through the visible spectrum, and emission energies may continue from visible light into the near infrared region.
The main characteristics of fluorophores are:
Maximum excitation and emission wavelength (expressed in nanometers (nm)): corresponds to the peak in the excitation and emission spectra (usually one peak each).
Molar absorption coefficient (in mol−1cm−1): links the quantity of absorbed light, at a given wavelength, to the concentration of fluorophore in solution.
Quantum yield: efficiency of the energy transferred from incident light to emitted fluorescence (the number of emitted photons per absorbed photons).
Lifetime (in picoseconds): duration of the excited state of a fluorophore before returning to its ground state. It refers to the time taken for a population of excited fluorophores to decay to 1/e (≈0.368) of the original amount.
Stokes shift: the difference between the maximum excitation and maximum emission wavelengths.
Dark fraction: the proportion of the molecules not active in fluorescence emission. For quantum dots, prolonged single-molecule microscopy showed that 20-90% of all particles never emit fluorescence.[5] On the other hand, conjugated polymer nanoparticles (Pdots) show almost no dark fraction in their fluorescence.[6]Fluorescent proteins can have a dark fraction from protein misfolding or defective chromophore formation.[7]
These characteristics drive other properties, including photobleaching or photoresistance (loss of fluorescence upon continuous light excitation). Other parameters should be considered, as the polarity of the fluorophore molecule, the fluorophore size and shape (i.e. for polarization fluorescence pattern), and other factors can change the behavior of fluorophores.
Most fluorophores are organic small molecules of 20–100 atoms (200–1000 Dalton; the molecular weight may be higher depending on grafted modifications and conjugated molecules), but there are also much larger natural fluorophores that are proteins: green fluorescent protein (GFP) is 27 kDa, and several phycobiliproteins (PE, APC...) are ≈240kDa. As of 2020, the smallest known fluorophore was claimed to be 3-hydroxyisonicotinaldehyde, a compound of 14 atoms and only 123 Da.[8]
Fluorescence particles like quantum dots (2–10 nm diameter, 100–100,000 atoms) are also considered fluorophores.[9]
The size of the fluorophore might sterically hinder the tagged molecule and affect the fluorescence polarity.
Families
Fluorescence of different substances under UV light. Green is a fluorescein, red is Rhodamine B, yellow is Rhodamine 6G, blue is quinine, purple is a mixture of quinine and rhodamine 6g. Solutions are about 0.001% concentration in water.
Fluorophore molecules could be either utilized alone, or serve as a fluorescent motif of a functional system. Based on molecular complexity and synthetic methods, fluorophore molecules could be generally classified into four categories: proteins and peptides, small organic compounds, synthetic oligomers and polymers, and multi-component systems.[10][11]
Fluorescent proteins GFP, YFP, and RFP (green, yellow, and red, respectively) can be attached to other specific proteins to form a fusion protein, synthesized in cells after transfection of a suitable plasmid carrier.
Non-protein organic fluorophores belong to following major chemical families:
These fluorophores fluoresce due to delocalized electrons which can jump a band and stabilize the energy absorbed. For example, benzene, one of the simplest aromatic hydrocarbons, is excited at 254 nm and emits at 300 nm.[12] This discriminates fluorophores from quantum dots, which are fluorescent semiconductor nanoparticles.
Additionally, various functional groups can be present to alter their properties, such as solubility, or confer special properties, such as boronic acid which binds to sugars or multiple carboxyl groups to bind to certain cations. When the dye contains an electron-donating and an electron-accepting group at opposite ends of the aromatic system, this dye will probably be sensitive to the environment's polarity (solvatochromic), hence called environment-sensitive. Often dyes are used inside cells, which are impermeable to charged molecules; as a result of this, the carboxyl groups are converted into an ester, which is removed by esterases inside the cells, e.g., fura-2AM and fluorescein-diacetate.
The following dye families are trademark groups, and do not necessarily share structural similarities.
Bovine Pulmonary Artery Endothelial cell nuclei stained blue with DAPI, mitochondria stained red with MitoTracker Red CMXRos, and F-actin stained green with Alexa Fluor 488 phalloidin and imaged on a fluorescent microscope.
StayGold and mStayGold are advanced fluorescent proteins that have significantly contributed to the field of live-cell imaging. StayGold, known for its high photostability and brightness, was originally designed as a dimeric fluorescent protein, which, while effective, posed challenges related to the aggregation and labelling accuracy.[15] To address these limitations, mStayGold was engineered as a monomeric variant, enhancing its utility in precise protein labeling. mStayGold exhibits superior photostability, maintaining fluorescence under high irradiance conditions and demonstrates increased brightness compared to its former variant StayGold. Additionally, it matures faster, allowing for quicker imaging post-transfection. These advancements make mStayGold a versatile tool for a variety of applications, including single molecule tracking and high resolution imaging of dynamic cellular processes, thereby expanding the capabilities of fluorescent protein in biological research.[16]
^Lakowicz, JR (2006). Principles of fluorescence spectroscopy (3rd ed.). Springer. p. 954. ISBN978-0-387-31278-1.
^Pons T, Medintz IL, Farrell D, Wang X, Grimes AF, English DS, Berti L, Mattoussi H (2011). "Single-molecule colocalization studies shed light on the idea of fully emitting versus dark single quantum dots". Small. 7 (14): 2101–2108. doi:10.1002/smll.201100802. PMID21710484.
^Li Z, Zhao X, Huang C, Gong X (2019). "Recent advances in green fabrication of luminescent solar concentrators using nontoxic quantum dots as fluorophores". J. Mater. Chem. C. 7 (40): 12373–12387. doi:10.1039/C9TC03520F. S2CID203003761.
^Juan Carlos Stockert, Alfonso Blázquez-Castro (2017). "Chapter 4 Fluorescent Labels". Fluorescence Microscopy in Life Sciences. Bentham Science Publishers. pp. 96–134. ISBN978-1-68108-519-7. Retrieved 24 December 2017.
^Sirbu, Dumitru; Luli, Saimir; Leslie, Jack; Oakley, Fiona; Benniston, Andrew C. (2019). "Enhanced in vivo Optical Imaging of the Inflammatory Response to Acute Liver Injury in C57BL/6 Mice Using a Highly Bright Near-Infrared BODIPY Dye". ChemMedChem. 14 (10): 995–999. doi:10.1002/cmdc.201900181. ISSN1860-7187. PMID30920173. S2CID85544665.
^Taki, Masayasu (2013). "Chapter 5. Imaging and sensing of cadmium in cells". In Astrid Sigel; Helmut Sigel; Roland K. O. Sigel (eds.). Cadmium: From Toxicology to Essentiality. Metal Ions in Life Sciences. Vol. 11. Springer. pp. 99–115. doi:10.1007/978-94-007-5179-8_5. PMID23430772.
