Cell techniques

• What is the optical resolution limit?

  Minimum distance for recognizing object details
• How does wave length affect resolution?

  Smaller wave length equals better resolution
• What is the resolution limit of light microscopy?

  200 nm
• What type of cells can be observed with light microscopy?

  Cells that are alive
• What is the resolution limit of electron microscopy?

  0.05 nm
• What does electron microscopy reveal about cells?

  Ultrastructure of the cell
• What can electron microscopy visualize?

  • proteins
  • single molecule analysis
  • structural information from average omage reconstruction
• What does freeze fracture electron microscopy analyze?

  Membrane topology and ultrastructural information
• What is a technical limitation of electron microscopy?

  Cells must be dehydrated and dead
• What does fluorescence microscopy provide that electron microscopy does not?

  Information on cellular dynamics
• What is the wavelength range of visible light?

  390-700 nm
• What is the Stokes shift?

  Difference between excitation and emission light
• What is the absorption and emission spectrum of Rhodamine used for?

  To stain mitochondria
• What is fluorescence?

  Emission of light by absorbed light
• How does the fluorescence microscope work?

  • Uses optical filters for excitation and emission
  • different fluorescent dyes or fluorescent proteins have different absorption and emission spectra, specific filters allow to visualise them in the same sample

  
• What is the principle of immunocytochemistry?

  Visualizing proteins using antibodies and fluorescence
• What are antibodies?

  Protective protein complexes made by the immune system
• How are antibodies produced?

  Made by B-cells, a type of white blood cell
• What do antibodies bind to?

  Epitopes on antigens
• What is a technical limitation of immunocytochemistry?

  Cells are not alive and not motile
• What is GFP and where does it come from?

  Green fluorescent protein from Aequorea victoria

  
• What was the 2008 Nobel Prize in Chemistry awarded for?

  Development of fluorescent proteins in biology
• How is GFP used in biological applications?

  As a reporter to analyze proteins in living cells
• What happens when the GFP gene is fused to other genes?

  GFP fusion protein behaves like the original protein
• What does FRAP stand for?

  Fluorescence recovery after photobleaching

  
• What does photoactivation do in fluorescent proteins?

  • Makes proteins visible after laser radiation
  • fluorescent protein is invisible and needs activation at ~400nm before it can be detected at 488nm
  • ~400nm laser light induces a chemical reaction
• How does photoactivation affect fluorescence intensity?

  Induces about 100-fold increase in fluorescence
• What are the strengths and weaknesses of fluorescence microscopy?

  Strengths:

  • Provides information on cellular dynamics
  • Can visualize multiple proteins simultaneously

  
  

  Weaknesses:

  • Requires specific filters
  • Cells must be fixed or permeabilized
• What are the steps in primary immunocytochemistry?

  1. Cell is chemically fixed
  2. Plasma membrane is removed
  3. Primary antibodies with fluorophore are added
  4. Antibodies bind to specific antigens
• What are the steps in secondary immunocytochemistry?

  1. Cell is chemically fixed
  2. Primary antibodies bind to target antigens
  3. Secondary antibody with fluorophore binds to primary antibodies
• What are the applications of fluorescent proteins in biological research?

  • Tagging proteins in living cells
  • Analyzing subcellular structures
  • Studying cellular dynamics
• What was the nobel prize in chemistry 2008 for

  developing and expanding the use of fluorescent proteins in biological applications
• What did Martin Chalfie discover in 1993

  GFP can be used to tag proteins 
• How does GFP work

  1. transgenic organism made with GFP-coding sequence
  2. GFP fusion protein behaves in same way as the original protein
  3. directly reveals its location and activites due to fluorescence
• What did Roger Tsien win a nobel prize for in 2008

  development of a palette of fluorescent proteins
• What does FRAP do

  • reveals differences in membrane fluidity and protein mobility
  • nucleus doesn’t recover
  • nuclear protein not mobile
• what does the GFP-Rab5a fusion protein analyse

  intracellular motility in a fungal cell