visualizing cells and molecules

• Visible to the naked eye


  • Visible using light microscopy (uses visible light to detect small objects)
  • Visible using electron microscopy (uses a beam of electrons to create an image of a specimen)
• Different microscopes


  • Can see different things
• Limit of resolution in light microscopy


  ~200 nm, depends on the wavelength of light
• Superresolution microscopy


  Can increase the resolution of conventional light microscopy (down to 20 nm)
• How a light microscope works

  1. Lenses in the condenser focus light on the specimen

  2. Lenses in the objective, tube lens, and eyepiece work together to focus the image of the illuminated specimen in the eye
• Dark-field microscopy


  You see a dark field, with a bright object. Works because oblique rays of light that do not enter the specimen are not directed to the objective; only light that is scattered ('redirected') by parts of the cell will enter the objective. Regions that scatter more light into the objective will appear brighter.
• Phase-contrast microscopy


  Built on the principle that the "phase" of light will change as it passes through a cell (due to a cell's refractive index). Light waves can become "out of sync" after passing through parts of a cell with different refractive indeces (for example, thick or dense parts, like a nucleus, will slow the light considerably).
• Phase-contrast image


  • Dark-field image
• Fluorescence


  Can be used to visualize different parts of cells, or molecules. These parts of the cell are not "naturally colored" in these ways.
• Fluorophore


  Fluorescent chemical compound that can re-emit light upon light excitation. Electrons will be "excited" upon absorbing a photon. Fluorescence occurs as the excited electron returns to its ground state and emits a photon of light at a longer wavelength.
• Fluorescence microscopes possess filters


  1. Filter set #1: Allows light of only a certain wavelength through (selects for excitation light)

  2. Filter set #2: Allows light of only a certain wavelength through (selects for emission light)
• Different fluorescent probes require different combinations of filters
• Fluorescently-labeled antibodies


  Can be used to detect molecules. A fluorophore could in theory just be conjugated to a primary antibody (which recognizes the antigen of choice directly), but a stronger signal can be achieved by conjugating the fluorophore to a secondary antibody (because multiple will bind to one primary antibody). One big drawback: often requires fixation (which kills cells in the process).
• Fluorescent proteins


  Organisms and cell lines can be engineered to make their own fluorescent proteins and labels… without the introduction of foreign materials!
• Aequorea victoria


  Is naturally bioluminescent: Aequorin emits blue light when bound by calcium. Blue light excites Green Fluorescent Protein, ultimately allowing it to emit green light.
• Harnessing GFP in other systems


  1. Step 1: molecularly clone the GFP-coding sequence from A. victoria into a bacterial expression vector

  2. Step 2: transform bacteria with this GFP-expression vector (or a control vector)

  3. Step 3: put these bacteria under blue light. The result: bacteria glow green!
• Harnessing GFP in animals

  1. Step 1: molecularly clone the GFP-coding sequence from A. victoria into a plasmid for C. elegans expression (use the mec-7 promoter, which is active only in neurons)

  2. Step 2: inject worms with this plasmid. The result: neurons of the worm glow green!
• GFP


  • Is not toxic to other cells; organisms can survive, develop and function normally
  • Fluorophore function is not species-specific, and only requires blue light
• Nobel prize in Chemistry 2008
• Useful applications of GFP and derivatives in cell biology


  • Track the expression of a gene
  • Track organelles
  • Track proteins
  • Measure protein mobility
  • Monitor protein-protein interactions
  • Photoactivation
• Resolution limit of light microscopy


  Individual points of light cannot actually appear as points, but instead appear as airy discs. This is due to scattering/diffraction of light as it passes through a specimen and the microscope. Thus, two sites of fluorescence that are close together (<200nm) cannot be resolved by standard light microscopy.
• PALM & STORM


  Two related types of superresolution microscopy. How can individual points be resolved? Photoactivate (and then bleach) individual points within a field at any one time. Repeat many times until all of the points are detected.
• Building complete superresolution images by plotting single molecules
• Cellular example of super-res

  • Microtubules are better resolved, and more representative of actual size (25 nm)
• Electron microscopy


  Wavelength of electrons is much smaller that visible light (0.004 nm for 100,000 V of acceleration). So, the limit of resolution is also smaller.
• Immunogold labeling


  Can be used to get high-res localization data with EM. Before imaging: incubate specimen with primary antibodies (to proteins), and then secondary antibodies attached to collodial gold particles (very electron dense, so will show as dark dots).
• CryoEM


  Involves the rapid freezing of molecules (to about -160˚C). Macromolecular complexes can be visualized without staining, drying, or fixation. In order to extract useful structural information, many individual molecules need to be averaged, creating a single-particle reconstruction.
• Transmission Electron Microscopy (TEM) is used to observe thin sections of tissue or whole cells that are stained with heavy metals.
• Electron microscopes have higher resolution than optical microscopes, allowing them to see smaller structures within the cell.