Fluorescence Spectroscopy

• Fluorescence spectroscopy examines how molecules absorb photons and emit light, specifically looking at fluorescence.

Essence of "-escences"

• Absorbance occurs when a photon puts a molecule into an excited state, dissipating energy as heat.
• Fluorescence happens when a photon excites a molecule, and a photon is emitted within nanoseconds.
• Phosphorescence involves a photon exciting a molecule, with a photon emitted within seconds to minutes.
• Chemiluminescence is when a chemical reaction leads to an excited state, emitting a photon (e.g., luminol + H2O2).
• Bioluminescence is chemiluminescence produced by living organisms.

Fluorescence Fundamentals

• Fluorescence measures the intensity of light emitted by fluorophores.
• Emitted light has a longer wavelength than absorbed light and radiates in all directions.
• All fluorophores absorb light, but not all light-absorbing molecules fluoresce.

Fluorescence Process

• Photon absorption leads to an excited electronic state
• The excited state then rapidly relaxes to its lowest vibrational state due to intermolecular collisions.
• Fluorescence, or photon emission, occurs from the ground vibrational state of the excited molecule to any vibrational state of the ground state molecule.
• Stokes' shift explains the emission spectrum shifting to longer wavelengths compared to the excitation spectrum.

Fluorescent Molecules

• molecules that fluoresce are rigid.
• Flexible molecules disperse energy into rotational modes.
• Dispersal of energy into rotational modes prevents the molecules from emitting a UV/visible photon.
• Rigid polycyclic aromatics contain few rotational states.
• A high-energy photon has to be emitted for relaxation to cause fluorescence

Absorption vs Emission Spectra

• Different excited states are separated by relaxation modes.
• A fluorescence spectrum mirrors the absorption spectrum, with similar peaks.

Biochemical Applications of Fluorescence

• Examples of fluorophores used in biochemistry include Ethidium, Fluorescein, ANS, and Acridine orange.
• Fluorescence intensity depends on both excitation and emission wavelengths.

Fluorescence Spectrometer

• Fluorescence is measured by counting emitted photons against a dark background.
• Measuring photons against a dark background makes fluorescence more sensitive than measuring light differences as done in absorbance.
• Xenon arc lamps are light sources that are intense and continuous from 250-700 nm.
• Because of the danger from explosion and brightness, operating xenon arc lamps should never be observed directly.

Quantitation in Fluorescence Spectroscopy

• Intensity depends on lamp intensity.
• Comparing Input to output is not possible like in absorbance, and transmission.
• Fluorescence is measured in arbitrary, instrument-specific units.
• Use "arbitrary units" or "a.u." with caution to avoid confusion with AU (absorbance units).
• Some papers use "relative fluorescent units" or RFU.
• Preparing and measuring a standard curve on the same instrument determines concentrations under identical conditions to quantity concentrations.

Quantum Yield and Quenching

• Quantum yield (Q) refers to the number of fluorescence photons emitted per absorption event (Q = F / (Io - I)).
• The Quantum yield is ≤ 1, meaning at most one photon is emitted per photon absorbed.
• Molecular environment affects Q and interactions with the solvent lead to fluorescence quenching.
• Q serves as a probe of the local environment of fluorescent species.
• At low concentrations, fluorescence intensity is proportional to incident light intensity, path length, concentration, quantum yield, and the molar extinction coefficient of the fluorophore.
• The wavelength of the incident light, the molar extinction coefficient of the fluorophore, the intensity of the incident light, and the concentration of the fluorophore all affect the intensity of observed fluorescence.

Applications of Fluorescence Spectroscopy

• Can use enzyme assays; peptidase/protease degradation assays.
• Extrinsic fluorophores and intrinsic fluorescence of biomolecules can be used for detection(e.g. Trp).
• Tryptophan is the only amino acid that has significant fluorescence
• Trp is commonly used as a "reporter" of protein conformation
• Tryptophan emission is sensitive to its environment and can also be used to monitor structural changes.
• Protein unfolding can be monitored via the Trp emission peak.
• High toxic death cap mushrooms naturally made the cyclic peptide phalloidin
• Use rhodamine phalloidin for actin and with DAPI for the nucleus to stain rat glioma

FRET

• FRET (Forster resonance energy transfer) relies on the distance between a donor and acceptor molecule
• FRET occurs when the emission spectrum of one fluorophore, the donor overlaps another molecule’s absorption spectrum.
• Energy moves directly from the donor to the acceptor without emitting a photon (the "resonance").
• FRET is monitored as a decrease of fluorescent intensity in the donor.
• Fluorescence resonance energy transfer only occurs over short distances (< 10 nm).
• Can monitor the binding of chromophores and fluorophores
• Can monitor protein complex formation
• Can also measure distances and motions on a molecular scale.

GFP

• Pacific jellyfish use bioluminescence and aequorin known as green fluorescent protein to emit light.
• Fusing a desired GFP tag is used to build a protein for visualization
• Allows real time visualization of the protein's location
• Because they do not disrupt cells, GFP fusion proteins follow the behavior of proteins inside a cell
• Green Fluorescent Protein is 26.9 kDa and has a quantum yield of 0.8
• GFP's extinction peak is 395 nm, and the emission peak is 509 nm
• Surface exposed Trp residues tend to be quenched by the solvent
• Engineered fluorescent protein variants allow labeling across the spectrum.