biophy

• Lesson Outline

  • Lesson 1: NMR Spectroscopy
  • Lesson 2: Electron Microscopy
  • Lesson 3: Atomic Force Microscopy
  • Lesson 4: Optical Tweezers
  • Lesson 5: Voltage, Current, and Patch Clamps
• NMR Spectroscopy


  • Odd mass nuclei have fractional spins
  • Even mass nuclei (odd number of protons and neutrons) have integral spin
  • Even mass nuclei (even number of protons and neutrons) have zero spin
  • Resonance frequency and mass are inversely related; large masses have lower resonance frequency
  • Nuclear spin is the orientation of the magnetic field generated by the protons relative to an external magnetic field

  
• Nuclear magnetic resonance spectroscopy


  Study of molecules by recording the EM radiation with the intrinsic nuclear spin of molecules placed in a strong magnetic field

  
• Rabi


  Method for measuring the magnetic properties of atomic nuclei, known as atomic beam magnetic resonance

  
• NMR Spectroscopy Concepts


  • Nuclear spin
  • Energy levels
  • Resonance condition, excitation, and relaxation
  • Chemical shift
  • Spin-spin coupling

  
• 1H NMR spectroscopy


  Molecule utilized is hydrogen (so only molecules with hydrogen are analyzed)
• Nuclear spin


  Nuclei with odd number of protons and/or neutrons possess nuclear spin (angular momentum, quantum mechanics)
• Energy levels


  Two states with corresponding energy levels according to its interaction with an external magnetic field; alpha state (aligned with external, low energy) and beta state (against the direction of external, high energy); strength of magnetic field and characteristics of nuclei influence spin and behavior; the presence of electrons affect the resonance frequency (lower energy is required)

  
• Resonance condition, excitation, and relaxation


  Resonance (energy difference of two spin states matches energy of external magnetic field), uses RF pulses to excite the nuclear spins from equilibrium to higher energy state; relaxation (return to their equilibrium state), free induction decay FID emitted during the relaxation

  
• Relaxation types


  T1 longitudinal (return to normal alignment with external field); T2 transverse (desynchronization from other resonated atoms)
• Chemical shift


  Electron distribution (electrons around nuclei generate their own magnetic field); deshielded (electrons are removed from near the nucleus, higher ppm count); factors (electronegativity, proximity to functional groups, hybridization, ring currents)

  
• Spin-spin coupling

  Hydrogen nuclei sensing each other (n+1 rule)
• NMR Applications


  • Chemical laboratories
  • Materials science
  • Drug discovery and development
  • Agriculture and food
  • MRI
  • Cancer diagnosis
• NMR


  Molecular structure, dynamics; depends on the orientation of the nuclei with respect to an external magnetic field; used to determine the structure of a compound
• NMR Spectroscopy Principles


  All nuclei are electrically charged and have spins, transfer of energy is possible if it coincides with the frequency (spin flip) occurs

  
• Free induction decay (induction vs. time chart) converted to intensity to frequency chart
• Principle of magnetic resonance: the magnetic moments of the nuclei align with the field resulting in an excited state which returns to the base state by emitting the energy

  
• 1H spectroscopy


  1. axis is chemical shift, y-axis is intensity of the signal

  
• The larger the mass, the smaller the wavelength
• Electron Microscopy


  • Technique that enables the obtainment of high resolution images
  • Slits diffract light; smaller slits result in a higher diffraction resulting in a blurred image; large objects diffract light, small objects diffract even more
  • Long wavelength has more diffraction (low res), short wavelength has less diffraction (high res); diffraction limitation makes resolution of microscopic images difficult to resolve
  • The diffraction pattern of electrons are similar to photons (wave-matter duality); wavelength of electrons are shorter (h/mv), resulting in higher resolutions

  
• Types of Electron Microscopes


  • Transmission electron microscopy
  • Scanning electron microscopy
• Transmission Electron Microscopy (TEM)


  Passes through the sample before being detected, provides information on the inner structure of the sample, sample is dehydrated; differing energies after passing through the specimen; not visible to the human eye; darker areas in the image are electron dense regions

  
• Scanning Electron Microscopy (SEM)


  Based on emission of secondary and back-scattered electrons and provides information on sample surface; specimens are coated in vapor gold or palladium to allow them to emit electrons; detects backscattered electrons from deeper regions of the sample; secondary electrons show the topography of the surface; study topography of cells

  
• SEM produces 3d images while TEM produces 2d images
• Electron microscopes cannot resolve individual atoms
• Wave-particle duality


  Electrons can exhibit BOTH wave and particle properties; the wavelength of a particle is equal to the ratio of Planck's constant to the momentum of the particle; Broglie-Bohr model shows electrons; Davisson – Germer experiment shows scattering of electrons exhibiting an interference pattern

  
• Resolution


  Ability to show detail in the object being imaged; smallest distance where two neighboring points can be distinguished; intensity pattern depends on diffraction pattern; resolving pattern to separate two points that are near each other; Rayleigh limit is the smallest possible angle between two points to resolve an image; low resolution value of Rayleigh limit shows higher resolution; shorter wavelength of electrons result in higher resolution; higher velocity of the electron also improves resolution by shortening the wavelength

  
• Electron Source


  Thermionic emission, electron field emission (discharge via tunneling in the presence of a strong electric field)

  
• Thermionic Emission


  Heating of cathode before being accelerated by the anode; electrons gain KE due to addition of heat energy (higher than work function energy)
• Electron Field Emission


  A quantum phenomenon occurring when particles move through a barrier that is classically impossible; electron passes through an energy barrier instead of jumping over it; a tungsten tip is subjected to a negative potential

  
• Lorentz Force Law


  Combination of the electric and magnetic forces; used to accelerate the electron beams

  
• Electron Scattering


  The way electrons interact with materials when viewed as particles; scattering is the deflection of electrons; backscattered (elastic), secondary (inelastic, knocked off from the surface); some electrons may produce x-rays or electron-hole pairs or may be absorbed by the sample

  
• Secondary Electrons


  Have low energy and provide topographical information about the surface

  
• Electron microscopes uses EM lenses to focus beam, light microscope uses glass lenses; electron has 1500 times while optical max of 200,00 times; electron resolution is in nanometer, optical is in micro
• Types of Electron Microscopy


  • Transmission electron microscopy (TEM)
  • Scanning electron microscopy (SEM)
  • Scanning transmission electron microscopy (STEM)
  • Environmental scanning electron microscopy

  
• Resolution and Contrast


  Smallest distance at which two neighboring points can be distinguished (light 540 nm, electrons 2.5 pm); signal detection - EM images are monochromatic and essentially intensity maps (false color is added after)

  
• Electron Optics


  EM lenses focus and manipulate the beam using a combination of magnetic and electric fields (Lorentz force) for precise control of beam and trajectory
• Electron Scattering and Detection

  Scattering pattern is used to construct the image; energy dispersive spectroscopy - the x-rays produced by electrons are used to obtain information regarding elements and relative abundance

  
• TEM


  Dark areas indicate absorption of electrons (sparse regions), light areas indicate transmission of electrons (dense regions), dehydrated specimen is embedded in a resin and cut into thin slices, electrons passing through sparse regions will have more energy (less back scattering)

  
• Electron microscopy


  Technique that uses a beam of electrons to image and analyse the structure of materials at the nanoscale