electron microscopy

units and wavelengths

• Electron 

  • Wavelengths (0.004 nm) 

  • Resolution (0.2 nm) 

• Light 

  • Wavelengths (400 – 710 nm) 

  • Resolution (200 nm) 

  • Super resolution microscopy (20 nm) 

electron microscopy

• X 1k – 200 k m 

• Electrons emitted from a filament and accelerated in an electric field 

• Condenser lens focuses the electron beam 

• Column is maintained at a very high vacuum (thus no living specimens) 

• No colour images 

transmission electron microscope

Microscope 

• Electrons scatter or hit a fluorescent screen at the bottom of the microscope 

Techniques (fixation) 

• Biological fine structure needs to be preserved during sample preparation (are chemically fixed) 

• Glutaraldehyde (covalently cross links proteins to neighbours) 

• Osmium tetroxide (stabilises lipid bilayers and proteins) 

• Potassium permanganate (membranes) 

• Small pieces of sample (0.5 mm^3) used to allow rapid infiltration of fixative 

Techniques (sectioning) 

Images  

• 2D images 

scanning electron microscope

Microscope 

• Electrons are focused on a metal coated specimen, electrons from the metal are collected by a detector 

• Electrons are scattered, detected and a 3D image of the surface is created 

• Specimen is dehydrated, and coated with a thin layer of heavy metal 

Images 

• 3D images 

image clarity

• Depends upon having a range of contrasting electron densities in the specimen 

• Tissues often impregnated with heavy metals (osmium, uranium, lead) to provide contrast 

localisation of cell components

• Antibody linked to colloidal gold. Different sizes can differentiate two different compounds simultaneously 

• Enzyme localisation by linking product to heavy metal (e.g. lipases with Pb) 

• Enzyme localisation if products are electron dense (e.g. peroxidase) 

• Cannot use fluorescence (as no colour)