☀️Photochemistry Unit 7 – Fluorescence and Phosphorescence

What's the Big Deal?

• Fluorescence and phosphorescence are fundamental photochemical processes that have revolutionized various fields including biology, chemistry, and materials science
• Enable highly sensitive and selective detection of molecules and materials at low concentrations (parts per billion or trillion)
• Allow visualization of biological processes in living cells and organisms with high spatial and temporal resolution
• Provide a powerful tool for studying the structure, dynamics, and interactions of molecules and materials at the nanoscale level
  • Fluorescence resonance energy transfer (FRET) measures distances between molecules on the scale of 1-10 nanometers
  • Fluorescence anisotropy measures rotational diffusion and conformational changes of molecules on the nanosecond timescale
• Enable development of novel materials with unique optical properties for applications such as displays, lighting, and solar energy harvesting (quantum dots, organic light-emitting diodes)
• Play a critical role in medical diagnostics, drug discovery, and environmental monitoring by enabling detection of disease biomarkers, screening of drug candidates, and sensing of pollutants and toxins

Key Concepts Unpacked

• Fluorescence occurs when a molecule absorbs a photon and then emits a photon of lower energy (longer wavelength) after relaxing to the lowest vibrational level of the excited electronic state
  • Governed by the Franck-Condon principle, which states that electronic transitions are much faster than nuclear motions
  • Typically occurs on the nanosecond timescale ($10^{-9}$ seconds)
• Phosphorescence occurs when a molecule undergoes intersystem crossing from the excited singlet state to the excited triplet state, and then emits a photon upon relaxation to the ground state
  • Requires a change in electron spin multiplicity, which is a forbidden transition and thus much slower than fluorescence
  • Can persist for microseconds to seconds or even longer
• Quantum yield ($\Phi$) is a key parameter that quantifies the efficiency of fluorescence or phosphorescence
  • Defined as the ratio of the number of photons emitted to the number of photons absorbed
  • Ranges from 0 (no emission) to 1 (100% efficient emission)
• Stokes shift is the difference between the wavelengths of maximum absorption and maximum emission
  • Arises from energy loss due to vibrational relaxation and solvent reorganization
  • Allows selective detection of emission without interference from excitation light
• Quenching refers to any process that decreases the fluorescence or phosphorescence intensity of a molecule
  • Can occur through various mechanisms such as collisional deactivation, energy transfer, charge transfer, or formation of non-fluorescent complexes
  • Used to study molecular interactions, measure distances, and sense analytes

How It Actually Works

• Absorption of a photon excites an electron from the ground state to a higher energy orbital, creating an excited electronic state
• The excited state undergoes rapid vibrational relaxation ($10^{-12}$ seconds) to the lowest vibrational level of the excited electronic state
• In fluorescence, the electron then relaxes back to the ground state by emitting a photon of lower energy than the absorbed photon
  • The probability of emission is determined by the oscillator strength and the rate of competing non-radiative processes
• In phosphorescence, the excited electron first undergoes intersystem crossing to the excited triplet state
  • This requires a flip of the electron spin, which is normally forbidden by the spin selection rule
  • Facilitated by spin-orbit coupling, which mixes singlet and triplet states
• The electron then relaxes from the triplet state to the ground state by emitting a photon
  • This process is much slower than fluorescence due to the forbidden nature of the transition
• The emission spectrum is typically a mirror image of the absorption spectrum, shifted to longer wavelengths due to the Stokes shift
• The lifetime of the excited state determines the duration of fluorescence or phosphorescence
  • Fluorescence lifetimes are typically in the nanosecond range
  • Phosphorescence lifetimes can range from microseconds to seconds or longer

Real-World Applications

• Fluorescent proteins (green fluorescent protein, GFP) are used to label and track proteins in living cells and organisms
  • Enables visualization of protein localization, movement, and interactions in real-time
  • Awarded the Nobel Prize in Chemistry in 2008
• Fluorescence microscopy allows high-resolution imaging of biological samples with minimal invasiveness
  • Confocal microscopy uses a pinhole to eliminate out-of-focus light and achieve optical sectioning
  • Super-resolution techniques (STED, PALM, STORM) overcome the diffraction limit and achieve nanometer-scale resolution
• Flow cytometry uses fluorescence to sort and analyze cells based on their physical and chemical properties
  • Enables rapid and quantitative measurement of cell size, granularity, and expression of surface markers
  • Used for clinical diagnostics, immunology, and cancer research
• Fluorescent probes and sensors are used to detect and quantify various analytes in biological and environmental samples
  • Calcium indicators (Fura-2, Indo-1) measure intracellular calcium signaling
  • pH indicators (BCECF, SNARF) measure intracellular and extracellular pH
  • Metal ion sensors (calcein, FluoZin) detect and quantify metal ions such as zinc, copper, and mercury
• Phosphorescent materials are used in organic light-emitting diodes (OLEDs) for displays and lighting
  • Harvest both singlet and triplet excitons to achieve high efficiency and brightness
  • Enable flexible, thin, and transparent devices with wide color gamut and high contrast ratio

Lab Techniques and Experiments

• Fluorescence spectroscopy measures the emission spectrum of a sample as a function of excitation wavelength
  • Provides information on the electronic structure, vibrational modes, and solvent interactions of the fluorophore
  • Used to study protein folding, ligand binding, and enzyme kinetics
• Time-resolved fluorescence measures the decay of fluorescence intensity over time after excitation with a short pulse of light
  • Provides information on the excited state lifetime and the presence of multiple emitting species
  • Used to study molecular rotational diffusion, energy transfer, and conformational dynamics
• Fluorescence anisotropy measures the polarization of emission relative to the polarization of excitation
  • Provides information on the size, shape, and flexibility of molecules
  • Used to study protein-protein interactions, membrane fluidity, and viscosity
• Fluorescence correlation spectroscopy (FCS) measures the fluctuations in fluorescence intensity due to diffusion of molecules through a small observation volume
  • Provides information on the concentration, diffusion coefficient, and molecular interactions of fluorescent species
  • Used to study protein aggregation, enzyme catalysis, and membrane dynamics
• Phosphorescence spectroscopy measures the emission spectrum and lifetime of phosphorescent materials
  • Provides information on the triplet state energy, radiative and non-radiative decay rates, and quenching processes
  • Used to study oxygen sensing, singlet fission, and triplet-triplet annihilation

Comparing Fluorescence and Phosphorescence

• Fluorescence and phosphorescence are both forms of photoluminescence, but differ in their electronic transitions and timescales
• Fluorescence involves transitions between states of the same spin multiplicity (usually singlet-singlet)
  • Allowed by the spin selection rule, and thus occurs rapidly ($10^{-9}$ seconds)
  • Typically results in a small Stokes shift and narrow emission spectrum
• Phosphorescence involves transitions between states of different spin multiplicity (usually triplet-singlet)
  • Forbidden by the spin selection rule, and thus occurs slowly ($10^{-6}$ to $10^{2}$ seconds)
  • Typically results in a large Stokes shift and broad emission spectrum
• Fluorescence is more common in organic molecules with rigid, conjugated structures and low atomic number atoms
  • Quenched by collisions with solvent molecules and other quenchers
  • Sensitive to environmental factors such as pH, polarity, and viscosity
• Phosphorescence is more common in organometallic complexes and materials with heavy atoms that facilitate intersystem crossing
  • Quenched by oxygen and other triplet state quenchers
  • Sensitive to temperature and magnetic fields
• Fluorescence and phosphorescence can be combined in some materials to achieve unique properties
  • Thermally activated delayed fluorescence (TADF) materials harvest triplet excitons for fluorescence at room temperature
  • Persistent phosphors store excitation energy in long-lived triplet states and emit slowly over time

Mind-Blowing Facts and Discoveries

• The fluorescence of chlorophyll in plants is what makes them appear green, and is used to drive photosynthesis
  • The quantum yield of chlorophyll fluorescence is very low (~0.1%) because most of the absorbed energy is used for photochemistry
  • Measuring chlorophyll fluorescence can provide information on the health and stress level of plants
• The fluorescence of some deep-sea creatures, such as the crystal jellyfish Aequorea victoria, is what led to the discovery and development of green fluorescent protein (GFP)
  • GFP consists of a beta-barrel structure with a chromophore formed from three amino acids (Ser65-Tyr66-Gly67) in the center
  • The chromophore undergoes an autocatalytic cyclization and oxidation reaction to become fluorescent
• The phosphorescence of some minerals, such as zinc sulfide and strontium aluminate, can persist for hours or even days after exposure to light
  • This phenomenon, known as persistent luminescence or afterglow, is used in glow-in-the-dark materials and emergency signage
  • The long phosphorescence is due to the trapping of electrons and holes in defect sites within the crystal lattice
• The phosphorescence of oxygen is what causes the red glow in the night sky known as airglow
  • Oxygen molecules in the upper atmosphere absorb ultraviolet radiation from the sun and undergo intersystem crossing to the excited triplet state
  • The triplet oxygen then emits in the near-infrared region around 1.27 micrometers, which is not visible to the human eye but can be detected by satellites
• The phosphorescence of some organometallic complexes, such as tris(2,2'-bipyridine)ruthenium(II), is used in solar energy conversion and photocatalysis
  • These complexes absorb visible light and undergo intersystem crossing to long-lived triplet states with high redox potentials
  • The triplet states can then transfer electrons to or from other molecules to drive chemical reactions or generate electrical current

Tricky Bits and Common Mistakes

• Confusing fluorescence and phosphorescence based on their emission colors or lifetimes
  • The color of emission depends on the energy gap between the excited and ground states, not on the type of transition
  • The lifetime of emission depends on the rates of radiative and non-radiative decay, which can vary widely for different molecules and materials
• Assuming that all molecules are fluorescent or phosphorescent
  • Many molecules, such as alkanes and simple aromatics, do not exhibit significant photoluminescence due to their low absorption cross-sections or high rates of non-radiative decay
  • Some molecules, such as azobenzenes and stilbenes, undergo photoisomerization or photochemical reactions instead of emitting light
• Neglecting the effects of solvent, pH, and other environmental factors on fluorescence and phosphorescence
  • Polar solvents can stabilize the excited state and cause a red shift in emission, while non-polar solvents can cause a blue shift
  • Acidic or basic conditions can change the protonation state of the fluorophore and alter its absorption and emission spectra
  • Viscous or rigid media can restrict molecular motion and increase the fluorescence quantum yield and lifetime
• Ignoring the possibility of self-quenching, inner filter effects, and other artifacts in fluorescence measurements
  • High concentrations of fluorophores can lead to self-absorption of emission and a decrease in apparent quantum yield
  • Strongly absorbing samples can attenuate the excitation light and cause a nonlinear relationship between concentration and intensity
  • Scattering, background fluorescence, and detector saturation can also distort the measured spectra and lifetimes
• Misinterpreting the results of fluorescence quenching experiments
  • Quenching can occur through static (complex formation) or dynamic (collisional) mechanisms, which have different effects on the emission spectra and lifetimes
  • The quenching efficiency depends on the accessibility and reactivity of the fluorophore, which can be affected by its location, orientation, and environment
  • Quenching can also be caused by trivial processes such as absorption of excitation light by the quencher (inner filter effect) or direct excitation of the quencher (sensitized emission)