MCB 11 - 2ND MODULE

• Microscopy

  • Robert Hooke (cork cells, fungi, compound)

  • Anton van Leeuwenhoek (compound microscope, bacteria)

  • Galileo Galilei (compound microscope)

  • Joseph Jackson Lister (achromatic lens)

  • 1940 – Electron Microscope
• What makes good microscope?

  -adequate magnifying power (enlarge specimen)

  -high resolving power (distinguish 2 points or objects from each other)

  -provide good contrast (morphology, structure, between specimen and background)

  -serve your purpose
• LIGHT MICROSCOPES

  • use light waves and mirrors
• Simple

  • short focal length
  • only 1 lens
  • magnification: 300x
• Compound or Complex

  -2 sets of lenses

  -magnification: 1000x
• parts of light microscope

  A) revolving nosepiece

  B) eyepiece

  C) objective lens

  D) mechanical stage

  E) coarse focus knob

  F) stage

  G) fine focus knob

  H) diaphragm

  I) condenser

  J) illuminator

  K) rheostat

  L) x-y mechanical stage knobs

  
• ELECTRON MICROSCOPES

  • use electron beams as energy source

  • has higher magnification and resolving power

  • for objects smaller than 0.2 mm in diameter

  • in vacuum
• parts of electron microscope

  A) electron source

  B) sample port

  C) viewing screen

  D) evacuated chamber

  
• Types of Light Microscopes

  1. bright field
  2. dark field
  3. phase contrast
  4. differential interference contrast
  5. fluorescence
  6. confocal
  7. two-photon
• BRIGHT FIELD

  • objects under study are darker

  • microscopic field is brightly lit

  • gross morphology
• Bacillus anthracis viewed in bright field

  
• Treponema pallidum viewed in dark field

  
• DARK FIELD

  • microscopic field is dark

  • objects under study are luminous

  • for specimens that are:

  • invisible in the ordinary light microscope

  • cannot be stained by standard methods

  • distorted by staining

  • capsule
• PHASE CONTRAST

  • principle is based on variations in the refractive indices (measure of bending or refracting of a beam of light on entering a denser medium)

  • not necessary to fix or stain cells

  • detailed examination of internal structure
• Unstained Epithelial Cells in phase contrast

  
• DIFFERENTIAL INTERFERENCE CONTRAST

  -principle is based on variations in the refractive indices

  • advantage: no diffraction halo associated with phase contrast

  • disadvantage: the three-dimensional appearance may not represent reality
• Fonsecaea pedrosoi viewed in differential interference contrast

  
• FLUORESCENCE

  • makes use of fluorochromes

  • visualizes specimens that fluoresce

  • detection of immunological reactions
• Neisseria gonorrhoeae viewed in fluorescence

  
• Schistosoma mansoni viewed in fluorescence

  
• CONFOCAL

  -useful for thick specimens like biofilms

  -used to visualize structures
• Roof-dwelling Cyanobacterium Biofilm viewed in confocal

  
• TWO-PHOTON

  • useful for examining living cells within intact tissues

  • currently limited to advanced clinical and research laboratories
• Mouse Intestine Cells Stained With Fluorescent Dyes viewed in two-photon

  
• Types of Electron Microscopes

  1. TRANSMISSION ELECTRON MICROSCOPE (TEM)
  2. SCANNING ELECTRON MICROSCOPE (SEM)
• TRANSMISSION ELECTRON MICROSCOPE (TEM)

  • ultrastructure in thin section of the cells

  • can project images in a much higher resolution—up to the atomic level of thinner objects

  • examine viruses
• Cells in Biofilm viewed in Transmission Electron microscope

  
• SCANNING ELECTRON MICROSCOPE (SEM)

  • surface features of viruses and cells

  • reveals a 3-D image
• Staphylococcus aureus viewed in Scanning Electron Miscroscope (SEM)

  
• Scanning Probe Microscope

  -does not use light or electrons, but rather very sharp probes that are passed over the surface of the specimen and interact with it directly
• Types of Scanning Probe Miscroscope:

  1. scanning tunneling microscope (STM)
  2. atomic force microscope (AFM)
• scanning tunneling miscroscope

  -uses a probe that is passed just above the specimen as a constant voltage bias creates the potential for an electric current between the probe and the specimen
• atomic force microscope

  -establishes a constant current and measures variations in the height of the probe tip as it passes over the specimen.
• Pure Gold Surface viewed under scanning tunneling microscope

  
• Nanocellulose viewed under atomic force microscope

  
• Living or Natural State 


  Advantages:

  • observation of unaltered/undistorted characteristics

  • cellular processes can be studied

  • motility can be observed

  • simple to prepare

  Disadvantage:

  • refractive index of cells almost similar to that of water
• Stained Preparations

  Advantages:

  • provides contrast

  • slides can be preserved

  • specimens are killed

  Disadvantages:

  • more complicated and tedious to prepare

  • expensive
• Three basic steps in staining microorganisms:

  1. Smear Preparation
  2. Fixation
  3. Staining
• Smear – a thin, dry film of microorganisms
• Fixation Purpose:

  • kills the cells

  • makes the cells sticky

  • increases apparent diameter of cells