5.3 Raman spectroscopy

Raman spectroscopy is a powerful tool for analyzing molecular composition and structure. It uses inelastic light scattering to provide unique molecular fingerprints, offering high specificity and minimal sample prep. This technique is especially useful for non-destructive, label-free analysis in aqueous environments.

In biophotonics, Raman spectroscopy shines in cancer diagnostics, drug delivery monitoring, tissue engineering, and single-cell analysis. It can identify cancerous cells, track drug distribution, assess engineered tissues, and probe individual cell contents, making it a versatile and valuable technique in various biomedical applications.

Raman Spectroscopy Principles and Applications

Principles and Advantages

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• Raman spectroscopy provides non-destructive, label-free molecular composition and structure information based on inelastic scattering of monochromatic light
• Raman effect arises from incident photon-molecule interactions, causing a shift in the energy of scattered photons
• Identifies and quantifies specific molecules, monitors chemical reactions, and studies the structure and dynamics of biological systems
• Offers high specificity, minimal sample preparation, and the ability to analyze samples in aqueous environments (water-based solutions)

Applications in Biophotonics

• Applied to various fields such as cancer diagnostics, drug delivery, tissue engineering, and single-cell analysis
  • Cancer diagnostics: Identifies cancerous cells based on their unique Raman signatures (biochemical fingerprints)
  • Drug delivery: Monitors the distribution and effectiveness of drug molecules in biological systems (nanoparticle-based drug carriers)
  • Tissue engineering: Assesses the composition and quality of engineered tissues and scaffolds (collagen, hydroxyapatite)
  • Single-cell analysis: Probes the molecular content and heterogeneity of individual cells (stem cells, immune cells)

The Raman Effect and Scattering

Raman Effect and Scattering Types

• Raman effect is the inelastic scattering of photons by molecules, resulting in a change in the energy and wavelength of the scattered light
• Raman scattering occurs when incident photons interact with molecules, inducing a change in the molecule's polarizability and causing transitions between vibrational or rotational energy levels
• Stokes Raman scattering: Scattered photon has lower energy than the incident photon (red-shifted)
• Anti-Stokes Raman scattering: Scattered photon has higher energy than the incident photon (blue-shifted)

Factors Affecting Raman Scattering Intensity

• Intensity of Raman scattering is proportional to the change in polarizability of the molecule during vibration
  • Polarizability: Ease with which the electron cloud of a molecule can be distorted by an external electric field (laser light)
• Raman shift, the difference in energy between incident and scattered photons, is characteristic of specific molecular vibrations and used for molecular identification
  • Raman shift is typically expressed in wavenumbers (cm⁻¹), which is the reciprocal of the wavelength

Interpreting Raman Spectra

Raman Spectrum Components

• Raman spectrum plots the intensity of scattered light as a function of the Raman shift, expressed in wavenumbers (cm⁻¹)
• Each peak in the Raman spectrum corresponds to a specific molecular vibration, providing a unique fingerprint of the molecule
  • Example peaks: C-H stretching (~2900 cm⁻¹), C=C stretching (~1600 cm⁻¹), C-C stretching (~1000 cm⁻¹)
• Peak position is determined by the energy difference between the vibrational states involved in the scattering process
• Peak intensity is related to the number of molecules in the sample and the polarizability of the specific vibration

Extracting Molecular Information from Raman Spectra

• Raman spectra provide information about the presence of functional groups, molecular symmetry, and the local chemical environment of molecules
  • Functional groups: Specific atomic arrangements within a molecule (hydroxyl -OH, carbonyl -C=O)
  • Molecular symmetry: Spatial arrangement of atoms in a molecule (symmetric, asymmetric)
  • Local chemical environment: Interactions between a molecule and its surroundings (hydrogen bonding, solvent effects)
• Comparing Raman spectra of unknown samples with reference spectra enables identification of the composition and structure of the sample
  • Reference spectra: Raman spectra of known pure compounds (amino acids, lipids, nucleic acids)

Raman Spectroscopy vs Other Techniques

Complementarity with Other Spectroscopic Techniques

• Raman spectroscopy is complementary to other spectroscopic techniques, providing different types of information about the sample
  • Infrared (IR) spectroscopy: Probes the absorption of light due to molecular vibrations
  • Fluorescence spectroscopy: Relies on the absorption and emission of light by fluorophores
• Raman spectroscopy is particularly useful for studying symmetric vibrations and non-polar molecules, which may have weak or no IR absorption
  • Symmetric vibrations: Vibrations that do not change the dipole moment of the molecule (C=C stretching)
  • Non-polar molecules: Molecules with evenly distributed charge (O₂, N₂)

Advantages of Raman Spectroscopy

• Higher spatial resolution compared to IR spectroscopy, suitable for analyzing small sample volumes and heterogeneous samples
  • Spatial resolution: Minimum distance between two points that can be distinguished as separate entities
• Not affected by photobleaching and provides information about intrinsic molecular properties without the need for labeling, unlike fluorescence spectroscopy
  • Photobleaching: Irreversible photochemical destruction of fluorophores due to prolonged exposure to excitation light
  • Label-free: No need for exogenous labels or dyes, which may interfere with the sample's native properties