Plasmon-enhanced fluorescence spectroscopy is a useful tool for imaging cells and other samples on a single-molecule level. This method poses several advantages over conventional fluorescence microscopy.
Andre Nantel | Shutterstock
The development of plasmon-enhanced fluorescence spectroscopy
Plasmon-enhanced fluorescence was first observed after the discovery of surface-enhanced Raman scattering. This method has recently grown in popularity due to advancements in our understanding of plasmonics and spectroscopy.
Molecular fluorescence results from the irradiation of a fluorophore using either monochromatic electromagnetic radiation or white light. Based on the state of excitation of the electrons in the fluorophore, this luminescence can be divided into fluorescence and phosphorescence.
However, when plasmonic metal nanoparticles, including gold or silver nanoparticles, are excited by monochromatic electromagnetic radiation, they show very weak photoluminescence. The oscillation of electrons by radiation excitation, known as a plasmon, can provide an enhancement in the optical field around the metal nanostructures.
How does plasmon-enhanced fluorescence spectroscopy work?
Fluorophores, such as organic dye molecules, commonly consist of aromatic rings or conjugated carbon chains. The singlet and triplet states are represented in the Jablonski diagram and vibrational and rotational sub-levels. The emission of a fluorophore consists of two variables: quantum yield and lifetime. The molecules that exhibit a very large quantum yield show very bright emission.
Following excitation, the molecules move from a high vibrational state to low vibrational state. This is accompanied by the emission of light. During plasmon enhanced fluorescence, Po, refers to the excitation power that is absorbed by the fluorophore and nanoparticles leading to its excited state.
Localized surface plasmon resonance describes the collective oscillations of electrons that lead to intense electromagnetic local fields. The coupling of this resonance wavelength to the emitter improves the quantum efficiency of absorption and emission.
Plasmon electromagnetic frequency
The enhancement of emitted light due to plasmon enhancement can range from ten to several hundred-fold. There is a certain class of nanostructures that can produce hotspots of the local electric field. These enhancing regions have been investigated and can be engineered into nanoparticle dimers and aggregates.
For example, an enhancement factor of 1340 times was achieved for the single molecule fluorescence of near- infrared dye N,N0-bis(2,6-diisopropyl phenol)-1,6,11,16-tetra-[4-(1,1,3,3-tetramethyl butyl)phenoxy]- quaterrylene-3,4:13,14-bis(dicarboximide) (TPQDI).
Surfaces for plasmon-enhanced fluorescence
The fluorescent properties of fluorescein isothiocyanate (FITC) and rhodamine 6G were first observed after being adsorbed on rough silver island films. The luminescence of these molecules is indistinguishable on smooth silver film. The researchers later discovered that the emission intensity was the strongest when the plasmon resonance wavelength of rough silver nanoparticle film overlapped with the absorption peak of the dye.
Other plasmon-enhanced fluorescence substrates include: metal films with rough surface morphology, embedded nanoparticles created with micro or nanofabrication methods, chemically synthesized nanoparticles with different morphology, shell isolated nanoparticles.
The main application of this method is the study of single molecules. Being able to study the behavior of specific molecules within certain microenvironments (such as a cancer cell membrane within the tumor microenvironment) is highly beneficial.
Plasmon-enhanced fluorescence spectroscopy can also be used to study DNA due to the specificity with which nucleotides bind to one another. DNA strands can thus be included as a tool for fabrication of the plasmon-enhanced frequency architecture.
DNA origami is a molecular self-assembly method that is used to design and build ultra-fine nanostructures using nucleic acids. In one study, DNA origami was used to arrange silver nanoparticles and then the plasmonic hotspot was manipulated to improve plasmon-enhanced fluorescence further. Similarly, the kinetics of DNA hybridization can also be studied using this method.