Microscopes

• Parts of flagellum

  head, rod, motor, hook
• Ultrastructure: fine structure, especially within a cell, that can be seen only with the high magnification obtainable with an electron microscope.
• Types of microscopy:

  • light
  • fluorescence
  • confocal
  • electron
• Resolution of light microscope = 0.2 micrometers
• Magnification: the ratio of an object's image size to its real size
• Resolution: the measure of the minimum distance of two distinguishable points
• Contrast: visible difference in brightness or colour between parts of the sample
• Types of light microscope:

  • dissecting
  • compound brightfield
• Dissecting light microscope

  A) eyepiece

  B) focus knob

  C) top lighting

  D) lighting controls

  E) stage clips

  F) stage plate

  G) bottom lighting

  H) stereo head

  I) diopter

  
• Compound light microscope

  A) eyepiece lens

  B) barrel

  C) arm

  D) stage clips

  E) objective lens

  F) stage

  G) diaphragm

  H) lamp

  I) focus knobs

  J) base

  
• Improve resolution of a light microscope

  use shorter wavelength of radiation
• disadvantages of light microscopy:

  specimen must be partially transparent

  limited resolution
• advantage for light microscopy:

  can use living cells
• Staining: enhances contrast
• fluorescence: shows location of specific molecules
• Phase-contrast microscopy: Uses light to create a contrast between the phase of the unstain and the background.
• differential-interference-contrast microscopy (DIC) is a light microscopy uses optical modifications to exaggerate difference in density, stained samples must be used
• How to prepare a sample for brightfield microscopy: Use a microtome arm to slice the specimen embedded in wax or resin into thin sections
• confocal: uses laser ans special potics for optical sectioning.

  Fluorescently stained samples

  Narrow depth of focus
• Advanced light microscopy:

  Permits observation of transparent living cells

  Light phase shifts induced by specimen are used to generate contrast

  Phase contrast (refracted and unrefracted light)

  Differential interference contrast (two light beams)
• First discovered fluorescent protein GFP, green fluorescent protein, in aequorea Victoria
• Fluorescence microscopy

  A) detector

  B) excitation filter

  C) emissions filter

  D) dichronic mirror

  
• confocal microsopy

  A) detector

  B) emission pinhole

  C) out of focus light

  D) dichronic mirror

  E) objective lens

  F) excitstion light

  G) focal planes

  H) laser

  I) excitation aperature

  J) sample

  K) in focus light

  
• Confocal Scanning Microscopy

  Generates 3D images of living cells

  Removes out-of-focus images optical sectioningy

  Can look inside thick specimens (eggs, embryos, tissues)
• Sample preparation for a light microscope
• sample preparation for a light microscope:

  whole mount: small transparent species can be mounted directly onto slide

  Tissue sections

  Fixation: prevents autolysis and preserves the structure of the tissue

  Dehydration and clearing: removes water for wax impregnation

  Embedding: infiltration with molten wax and transferred to a mold

  Section: 5microns thick are cut on a microtome

  Staining:
• Transmission electron microscopes

  1. Electron beam passes through the specimen.
  2. Image is focused and magnified by magnetic objective and projector lenses
  3. Electron image is converted to a visible image by a fluorescent screen
  4. Photographed
  5. kept within a vaccum
• Resolution of electron microscope:

  0.08nm
• Transmission electron microscope

  A) viewing screening or photographic film

  B) projector lens

  C) electromagnetic objective lens

  D) specimen

  E) condensor

  F) beam of electrons

  G) anode

  H) tungsten filament (cathode)

  
• Transmission electron microscope sample preparation:

  • whole mounts
  • Fixation: Glutaraldehyde (protein crosslinking) followed by second fixation step in osmium tetroxide (lipid crosslinking)
  • dehydration: in ethanol series
  • embeddeing: in resin
  • sectioning: 50nm thick cut using an ultramicrotome
  • staining: heavy metals
• Scanning electron microscope:

  Electron beam is scanned across the surface of the specimen

  Electrons are reflected from the surface of the specimen

  Electronic detector converts into an electronic signal which is displayed on a screen
• SEM represents the topology and the depth of focus give image a 3D appearance
• SEM sample preparation: Fixed and dired, viewed under vaccum

  Fixation: same as TEM

  Dehydration: water is replace with ethanol

  Crititcal point drying: technique to remove all ethanol in a way that minimises shrinkage

  Coating specimens: coated in gold to prevent damage from electron beam damage
• Cell fractionation is the process of separating a cell into its different organelles.

  1. cell homogenised to release organelles
  2. differential centrifugation
• Differential centrifugation: Separates a mixture of substances based on their relative densities and size by using increasing durations and g forces