5.2 Fluorescence spectroscopy

Fluorescence spectroscopy is a powerful tool for studying biological systems. It measures light emission from molecules after they absorb energy, allowing scientists to detect and analyze specific biomolecules at incredibly low concentrations.

This technique offers high sensitivity and specificity, making it ideal for investigating protein interactions, enzyme activity, and cellular processes. By understanding the principles of fluorescence, researchers can gain valuable insights into complex biological systems and their functions.

Fluorescence Spectroscopy Principles

Fundamental Principles and Applications

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• Fluorescence spectroscopy measures the emission of light from a sample after it has absorbed light at a specific wavelength
  • Based on the principle of fluorescence, where certain molecules (fluorophores) absorb light at one wavelength and emit light at a longer wavelength
  • Involves the excitation of electrons in the fluorophore from the ground state to a higher energy state, followed by the relaxation of the electrons back to the ground state, resulting in the emission of a photon of lower energy (longer wavelength) than the excitation photon
• Highly sensitive and specific technique allows for the detection and quantification of fluorescent molecules at very low concentrations (down to the single-molecule level)
• Provides information about the local environment of the fluorophore
  • pH, polarity, and the presence of quenchers or enhancers can be used to study biological processes and interactions

Biophotonics Applications

• Studying protein interactions
• Monitoring enzyme activity
• Detecting specific biomolecules (DNA, RNA, proteins)
• Imaging biological systems (cells, tissues, organs)

Jablonski Diagram and Processes

Jablonski Diagram Components

• Graphical representation of the electronic states of a molecule and the transitions between these states, including the processes of excitation, emission, and non-radiative relaxation
• Consists of the ground state (S0), excited singlet states (S1, S2, etc.), and excited triplet states (T1, T2, etc.)
  • Each electronic state has multiple vibrational levels, represented by horizontal lines within each state

Excitation and Vibrational Relaxation

• Excitation occurs when a photon of appropriate energy is absorbed by the molecule, causing an electron to transition from the ground state to a higher excited singlet state (S0 to S1 or S2)
  • Very fast process, occurring on the order of 10^-15 seconds
• After excitation, the molecule undergoes rapid vibrational relaxation (10^-12 to 10^-10 seconds) to the lowest vibrational level of the excited singlet state (S1)
  • Non-radiative process involves the dissipation of energy as heat to the surrounding environment

Fluorescence and Non-Radiative Relaxation

• From the lowest excited singlet state, the molecule can return to the ground state through two main processes: fluorescence (emission) and non-radiative relaxation
• Fluorescence occurs when the electron in the excited singlet state returns to the ground state, emitting a photon of lower energy (longer wavelength) than the excitation photon
  • Slower than excitation, occurring on the order of 10^-9 to 10^-7 seconds
• Non-radiative relaxation processes, such as internal conversion and intersystem crossing, compete with fluorescence
  • Internal conversion involves the transition between two electronic states of the same spin multiplicity (S1 to S0)
  • Intersystem crossing involves the transition between states of different spin multiplicity (S1 to T1)

Analyzing Fluorescence Spectra

Excitation and Emission Spectra

• Fluorescence spectra provide valuable information about the properties of fluorescent molecules and their interactions with the local environment
• Two main types of fluorescence spectra: excitation spectra and emission spectra
  • Excitation spectra show the dependence of fluorescence intensity on the excitation wavelength while monitoring the emission at a fixed wavelength
    • Provide information about the absorption properties of the fluorophore and can be used to determine the optimal excitation wavelength for a given fluorophore
  • Emission spectra show the dependence of fluorescence intensity on the emission wavelength while exciting the sample at a fixed wavelength
    • Provide information about the emission properties of the fluorophore and can be used to determine the optimal emission wavelength range for detection

Factors Influencing Fluorescence Spectra

• The shape and position of the emission spectrum can be influenced by various factors
  • Polarity of the solvent, pH, and the presence of quenchers or enhancers can be used to study the local environment of the fluorophore and its interactions with other molecules
• The Stokes shift, which is the difference between the maximum excitation and emission wavelengths, can provide information about the energy loss due to vibrational relaxation and the polarity of the fluorophore's environment
• Fluorescence lifetime, which is the average time a molecule spends in the excited state before returning to the ground state, can be determined from time-resolved fluorescence measurements
  • Sensitive to the local environment and can be used to study molecular interactions and conformational changes
• Fluorescence quenching, which is the decrease in fluorescence intensity due to the interaction of the fluorophore with other molecules (quenchers), can be used to study molecular interactions, conformational changes, and accessibility of the fluorophore to the quencher

Fluorescence Spectroscopy Applications vs Limitations

Advantages in Biological Applications

• High sensitivity enables detection of very low concentrations of fluorophores (down to the single-molecule level), suitable for studying biological systems where the concentrations of target molecules may be low
• High specificity allows for selective detection and imaging of specific molecules of interest in complex biological samples by attaching fluorescent labels
• Non-destructive measurements can be performed on living cells and tissues without causing damage, allowing for the study of biological processes in real-time
• Multiplexing enables the study of multiple molecular interactions and processes in the same sample by using multiple fluorophores with different excitation and emission spectra simultaneously
• Spatial resolution provided by fluorescence imaging techniques (confocal microscopy, super-resolution microscopy) allows for the visualization of cellular and subcellular structures

Limitations in Biological Applications

• Photobleaching can occur upon prolonged exposure to excitation light, leading to a decrease in fluorescence intensity over time
  • Limits the duration of experiments and the number of measurements that can be performed on the same sample
• Autofluorescence from biological molecules (NADH, flavins) can interfere with the detection of the target fluorophore
  • Reduces the signal-to-noise ratio and complicates data interpretation
• Phototoxicity can be caused by high-intensity excitation light generating reactive oxygen species (ROS) in biological samples
  • Affects the viability of the sample and the accuracy of the measurements
• Perturbation of the system may occur when introducing fluorescent labels or probes into a biological system
  • Can potentially alter the native structure and function of the target molecules, leading to artifacts in the measurements
• Complexity of biological systems, with many factors influencing the fluorescence properties of the molecules, can make it challenging to interpret fluorescence data and draw definitive conclusions about the underlying biological processes