🔬Nanobiotechnology Unit 6 – Nanoscale Imaging and Characterization Tools

Key Concepts and Principles

• Nanoscale imaging tools enable visualization and characterization of structures and phenomena at the nanometer scale (1-100 nm)
• Resolution, the smallest distance between two points that can be distinguished, is a crucial parameter in nanoscale imaging
• Imaging techniques can be classified as either far-field (optical microscopy) or near-field (scanning probe microscopy) based on the distance between the probe and the sample
• Contrast mechanisms, such as differences in topography, chemical composition, or electronic properties, allow for the differentiation of features in nanoscale imaging
• Signal-to-noise ratio (SNR) affects the quality and clarity of nanoscale images
  • Increasing the signal or reducing the noise can improve the SNR
• Interaction of probes (electrons, photons, or physical tips) with the sample determines the type of information obtained
• Vacuum conditions are often required for electron microscopy techniques to minimize interference from air molecules
• Sample preparation plays a critical role in obtaining high-quality nanoscale images

Types of Nanoscale Imaging Tools

• Electron microscopy techniques
  • Scanning Electron Microscopy (SEM)
  • Transmission Electron Microscopy (TEM)
• Scanning probe microscopy methods
  • Atomic Force Microscopy (AFM)
  • Scanning Tunneling Microscopy (STM)
• Optical microscopy techniques
  • Confocal microscopy
  • Super-resolution microscopy (STED, STORM, PALM)
• X-ray based techniques
  • X-ray diffraction (XRD)
  • Small-angle X-ray scattering (SAXS)
• Spectroscopic characterization tools
  • Raman spectroscopy
  • Fourier-transform infrared spectroscopy (FTIR)
• Correlative microscopy combines multiple imaging techniques to obtain complementary information about a sample

Electron Microscopy Techniques

• Electron microscopy utilizes a beam of accelerated electrons to illuminate and image the sample
• Scanning Electron Microscopy (SEM) scans a focused electron beam over the sample surface to generate high-resolution images of surface topography
  • Secondary electrons and backscattered electrons are detected to form the image
  • SEM can achieve resolutions down to a few nanometers
• Transmission Electron Microscopy (TEM) passes a beam of electrons through an ultrathin sample to create high-resolution images
  • TEM can provide information about internal structure, crystallinity, and chemical composition
  • High-resolution TEM (HRTEM) can achieve atomic resolution
• Scanning Transmission Electron Microscopy (STEM) combines the principles of SEM and TEM, allowing for high-resolution imaging and chemical analysis
• Electron microscopy requires conductive samples and may cause damage to biological specimens due to the high-energy electron beam

Scanning Probe Microscopy Methods

• Scanning probe microscopy techniques use a physical probe to scan the sample surface and measure various properties
• Atomic Force Microscopy (AFM) uses a sharp tip attached to a cantilever to map the surface topography
  • AFM measures the force between the tip and the sample surface
  • Operating modes include contact mode, non-contact mode, and tapping mode
• Scanning Tunneling Microscopy (STM) uses a conductive tip to measure the tunneling current between the tip and the sample
  • STM can provide atomic-resolution images of conductive surfaces
• Near-field Scanning Optical Microscopy (NSOM) uses a small aperture or tip to confine light to a subwavelength region, enabling high-resolution optical imaging
• Kelvin Probe Force Microscopy (KPFM) measures the local work function and surface potential of a sample
• Scanning probe techniques can be performed in various environments, including air, liquid, and vacuum

Spectroscopic Characterization Tools

• Spectroscopic techniques probe the interaction of electromagnetic radiation with matter to obtain information about chemical composition, molecular structure, and electronic properties
• Raman spectroscopy measures the inelastic scattering of monochromatic light by molecular vibrations
  • Raman spectra provide a fingerprint of the sample's molecular composition
  • Surface-enhanced Raman spectroscopy (SERS) enhances the Raman signal using plasmonic nanostructures
• Fourier-transform infrared spectroscopy (FTIR) measures the absorption of infrared light by molecular vibrations
  • FTIR can identify functional groups and chemical bonds in a sample
• X-ray photoelectron spectroscopy (XPS) measures the kinetic energy of electrons emitted from a sample upon X-ray irradiation
  • XPS provides information about the elemental composition and chemical state of the sample surface
• Energy-dispersive X-ray spectroscopy (EDS) analyzes the X-rays emitted by a sample during electron microscopy to determine its elemental composition
• Spectroscopic techniques are often combined with imaging tools to obtain spatially resolved chemical information

Sample Preparation and Handling

• Proper sample preparation is essential for obtaining high-quality nanoscale images and accurate characterization results
• Thin sectioning techniques, such as ultramicrotomy, are used to prepare samples for TEM
  • Samples are embedded in resin and cut into ultrathin sections (typically 50-100 nm) using a diamond knife
• Cryogenic sample preparation involves rapid freezing of samples to preserve their native structure
  • Cryo-EM allows for the imaging of biological samples in their near-native state
• Surface cleaning methods, such as plasma cleaning or solvent washing, remove contaminants from the sample surface prior to imaging
• Conductive coating (gold or carbon) is often applied to non-conductive samples for electron microscopy to prevent charging artifacts
• Proper sample handling, including the use of clean tools and minimizing exposure to contaminants, is crucial to maintain sample integrity

Data Analysis and Interpretation

• Nanoscale imaging and characterization techniques generate large amounts of data that require processing and interpretation
• Image processing techniques, such as background subtraction, contrast enhancement, and noise reduction, improve the quality and clarity of nanoscale images
• Quantitative analysis of nanoscale images can provide information about particle size distribution, surface roughness, and feature dimensions
  • Specialized software tools (ImageJ, Gwyddion) are used for image analysis and measurements
• Statistical analysis methods, such as averaging and correlation analysis, help to extract meaningful information from large datasets
• Interpretation of spectroscopic data involves comparing experimental spectra with reference databases and theoretical models
• Combining data from multiple imaging and characterization techniques provides a more comprehensive understanding of the sample's properties and structure
• Proper data management, including storage, backup, and sharing, is essential for reproducibility and collaboration

Applications in Nanobiotechnology

• Nanoscale imaging and characterization tools play a crucial role in advancing nanobiotechnology research and development
• Structural characterization of biomolecules, such as proteins and nucleic acids, helps to elucidate their function and interactions
  • Cryo-EM has revolutionized the field of structural biology by enabling near-atomic resolution imaging of biomolecular complexes
• Imaging of cellular structures and processes at the nanoscale provides insights into cell biology and disease mechanisms
  • Super-resolution microscopy techniques allow for the visualization of subcellular structures beyond the diffraction limit
• Characterization of nanomaterials used in biomedical applications, such as drug delivery systems and biosensors, ensures their safety and efficacy
  • AFM and SEM are used to study the morphology and surface properties of nanoparticles and nanostructured materials
• Monitoring the interaction of nanomaterials with biological systems, including cells and tissues, is essential for assessing their biocompatibility and toxicity
• Nanoscale imaging and characterization tools contribute to the development of personalized medicine by enabling the analysis of individual cells and biomolecules
  • Single-cell analysis techniques, such as Raman spectroscopy and AFM, provide information about cellular heterogeneity and response to therapies
• Integration of nanoscale imaging with other techniques, such as microfluidics and biosensing, enables the development of advanced diagnostic and therapeutic platforms