Cell bio lab final

• The lipid bilayer is composed of _____ with heads pointing outwards.

  Phospholipids
• The diffusion of water in response to a concentration gradient is _____.

  osmosis
• In today's lab exercise, different concentrations of sucrose will be used to observe the tonicity of elodea cytoplasm. 


  sucrose, Elodea's
• _____ important group of lipid molecules in the cell membrane that regulates membrane fluidity.

  Cholesterol
• Safe laboratory practices to be used at all times

  • Use safe laboratory practices
  • Emphasize safe practices during experiments
• Procedures to ensure everyone is familiar with safety procedures and policies

  1. Notify instructor if pregnant, color-blind, or have medical condition

  2. Do not eat, drink, smoke, chew gum or apply cosmetics in the laboratory

  3. No food, drinks, wrappers, or empty containers allowed in the lab

  4. Wash hands before leaving the lab

  5. Wear closed-toe shoes

  6. Wear protective gear like gloves and safety glasses

  7. Use fume hood when instructed

  8. Read labels and ask instructor if unsure

  9. Do not return materials to stock containers

  10. Discard used chemicals and solutions properly

  11. Discard broken glassware in special container

  12. Discard biohazard materials in labeled bags

  13. Assume hot plates are hot

  14. Know location of safety equipment

  15. Report accidents and spills to instructor
• Policies


  • Attendance is mandatory, no make-up labs
  • Clean up bench area and glassware before leaving
  • Record all data in lab notebook
  • Discuss data within group, but individual work must be individual
  • Following safety procedures is part of lab performance
• Microscopy


  The technical field of using microscopes to view samples or specimens

  
• Light microscopes


  • Bright
  • Dark
  • Phase-contrast
  • Fluorescence
• Electron microscopes


  • Transmission Electron
  • Scanning Electron
• Fluorescence Microscope


  • Visible light cannot distinguish between objects closer than 200 nanometers (0.2 μM) to each other
  • Limitation: cannot reveal the detailed structures of tiny functional components in cells called organelles

  
• Electron Microscope


  • Can achieve much higher resolutions
  • Limitation: Require a vacuum and cannot be used on live samples

  
• Magnification


  Enlarges an image
• Magnifications


  • 10x (low power)
  • 40x (high power)
  • 100x (oil immersion)
• Eye pieces


  Magnify between 8x and 12.5x
• Working Distance


  • The distance between the specimen and the objective lens
  • As magnification increases, the distance decreases
  • The greater the specimen is magnified, the greater the chance for the specimen and the lens of the objective to come into contact

  
• Limit of resolution (l.r.)

  The smallest distance by which two neighboring points can be separated and still be observed as separate entities

  
• Limit of Resolution


  1. Calculated using the Abbe Equation: l.r = 0.61λ/N.A.

  2. λ = wavelength of light used to view the object

  3. N.A.= Numerical Aperture, measure of cone angle of light entering the objective lens

  
• As N.A. increases


  The resolving power increases due to the N.A. reducing the diffraction pattern
• High-quality objectives have an N.A. of 1.40
• The lowest possible limit of resolution attainable with a light microscope occurs when a 1.40-N.A. objective is used with a violet filter (λ = 400 nm)
• Histology


  The preparation of samples for microscopic observations

  
• Wright-Giemsa stain


  • Mixture of methylene blue, methylene azure, and the eosinates of both
  • Azures act as bases and stain the basophilic components of the cell blue
  • Eosins act as acids and stain the acidophilic components red
  • The combination of dye and the varying affinity of cell components for each dye will give various parts of the cell a variation of pink, purple, blue and red coloration
• Field of View Distance

  • Diameter of the total circular area you see when looking through the microscope
  • Measured in millimeters (mm) or micrometers (μm)

  
• Estimating Size of a Specimen

  1. Find the image under the scanning objective lens

  2. Move to low power and count the number of cells in one row from left to right (use the middle, longest row) per magnification

  3. Change from the high power, focus with the fine adjustment knob and count the number of cells in the middle, longest row

  4. Repeat the process for oil immersion lens (no need to use oil)

  
• 100x magnification has a Field of View of 1,200 μm
• 400x magnification has a Field of View of 350 μm
• 1000x magnification has a Field of View of 200 μm
• Elodea cells observed under the microscope

  • Pictures of Elodea cells at 100x, 400x, and 1000x magnification with appropriate cell parts labelled

  
• When Werner Kühlbrandt at the Max Planck Institute of Biophysics in Frankfurt, Germany, wrote about the promise of a microscopic technique that could reveal the structure of large biomolecules at near-atomic resolution in 2014, he chose as his headline 'The Resolution Revolution'.
• Electron cryo-microscopy (cryo-EM)


  A technique that fires beams of electrons at proteins frozen in solution to reveal their structures
• We can see whether two proteins are located in the same place, doing the same things, coming together or apart in real time, understand how enzymes work and obtain structures of complexes of proteins in ways we couldn't have dreamed of doing before.
• The 2014 Nobel Prize in Chemistry was awarded to the developers of super-resolution fluorescence microscopy, and the 2017 prize recognized the development of cryo-EM.
• Fluorescence microscopy


  • Allows biologists to observe live samples in real time
  • Visible light cannot distinguish between objects closer than 200 nanometres to each other
• Electron microscopes


  • Can achieve much higher resolutions
  • Require a vacuum and so cannot be used on live samples
• Many advances have been made using these microscopy tools, such as mapping out the relationships between different types of organelle, how fast they were moving and the contacts they made with each other.
• For academic scientists, the ability to determine structures at atomic resolution with electron cryo-microscopy can be very important.
• Advanced imaging techniques


  • Increase the number of processes cell biologists can probe
  • Open up tremendous vistas for the types of questions we can answer
• Electron cryo-microscopy


  • Drug companies are becoming very keen on it as a means to determine the structures of proteins and drug targets
  • Will probably supersede crystallography in the job market
• The proliferation of advanced imaging tools has made microscopy all the more attractive as a focus for cell biologists