What is Fluorescence Spectroscopy?
Fluorescence spectroscopy analyzes the fluorescence of molecules based on their fluorescent properties.
Fluorescence is the luminescence that occurs when a molecule is excited by a photon to an electronically excited state in order to return to the ground state.
Fluorescence spectroscopy uses a beam of light to excite electrons in certain compound molecules and cause them to emit light. Light passes through a monochromator and enters a detector where it is detected, which is used to measure and identify molecules or changes in molecules.
Introduction to Fluorescence Steady-State Spectroscopy and Lifetime Testing
Fluorescence broadly refers to the phenomenon of luminescence, the light emitted by molecules. There are several types of glow.
Photoluminescence is the emission of photons excited by light energy or photons.
Chemiluminescence, defined as the luminescence of photons excited by chemical energy, includes bioluminescence, as seen in fireflies and many marine life.
Electroluminescence is the emission of photons when electrical energy or a strong electric field stimulates photons, such as in some lighting applications.
Specifically, fluorescence is a type of photoluminescence where light puts electrons in an excited state. The excited state rapidly loses thermal energy to the environment through vibration, and then emits photons from the lowest singlet excited state. This photon emission process competes with other nonradiative processes, including energy transfer and heat loss.
When the term "fluorescence" is used, the same measurement method generally applies to any of the above classes of luminescence.
What is Fluorescence Spectroscopy?
Fluorescence excitation and emission spectra mirroring each other. Fluorescence spectroscopy uses emission and excitation to see how a sample is changing.
Fig. 1: A fluorescence excitation spectrum (blue) and an emission spectrum (purple) are mirror images of each other
Steady-state fluorescence spectroscopy is when a molecule fluoresces when excited by a constant light source, and the emitted photons or intensity is detected as a function of wavelength. Fluorescence emission spectrum is when the excitation wavelength is fixed, the emission wavelength is scanned, and the relationship between the intensity and the emission wavelength is obtained.
The fluorescence excitation spectrum is when the emission wavelength is fixed, by changing the excitation monochromator wavelength, scanning the intensity at different wavelengths. In this way, the spectrum provides information about the sample and absorption wavelengths to select the optimal single emission wavelength for detection of emission. It is similar to absorption spectroscopy, but is a more sensitive technique in terms of detection limits and molecular specificity. Excitation spectra are specific to a single emission wavelength/species, relative to the absorption spectra of all absorbing species in the measurement solution or sample. The emission and excitation spectra of a given fluorophore are mirror images of each other. Typically, emission spectra appear at higher wavelengths (lower energies) than excitation or absorption spectra
These two spectral types (emission and excitation) are used to observe how the sample changes. The spectral intensity and peak wavelength can vary with variables such as temperature, concentration, or interactions with surrounding molecules. This includes quenching molecules and molecules or materials involved in energy transfer. Some fluorophores are sensitive to properties of the solvent environment, such as pH, polarity, and certain ionic concentrations.
What types of molecules or materials exhibit fluorescence?
Fluorescence emission spectra of some common fluorophores. Fluorophores play the central role in fluorescence spectroscopy and make this type of spectroscopy a highly sensitive technique.
Fig. 2: Fluorescence emission spectra of some common fluorophores across the UV and visible spectrum
Fluorescent molecules and materials come in all shapes and sizes. Some are inherently fluorescent, such as chlorophyll and the amino acid residues tryptophan (Trp), phenylalanine (Phe) and tyrosine (Tyr). Others are molecules synthesized as stable organic dyes or tags that can be added to other non-fluorescent systems. Generally, organic fluorescent molecules have structural features such as aromatic rings and π-conjugated electrons. Depending on their size and structure, organic dyes emit light in wavelengths ranging from the ultraviolet to the near-infrared.
Below are some random common fluorophores whose emission can be seen spanning the UV-Vis range. For some rare earth elements, such as lanthanides, due to the higher electron orbitals being filled, electronic transitions due to charge transfer from metal ligands occur between 4f-5d or even 4f-4f orbitals. (Bunzli, 1989) There are many molecules that emit light in nature, such as some amino acids, chlorophyll and natural pigments. Still others are carefully designed for special uses in fluorescence spectroscopy
Examples of fluorescent molecules and materials:
Amino acids (Trp, Phe, Tyr)
Base pair derivatives (2-AP, 3-MI, 6-MI, 6-MAP, pyrrole-c, tC)
chlorophyll
Fluorescent proteins (FPs)
Organic dyes (fluorescein, rhodamine, n-aminocoumarins and their derivatives)
Rare Earth Elements (Lanthanides)
semiconductor
quantum dots
single-walled carbon nanotubes
Solar battery
Pigment, Electroplating
phosphor
More…
Other molecules and materials such as fluorescent proteins, semiconductors, phosphors, and rare earth elements are commonly used fluorescent samples. Polymers containing conjugated aromatic hydrocarbons or dienes often also have fluorescent properties. Of course, scientists have also been working on the creation of new materials.
