Lecture 2: Electron microscopy

Magnification of electron microscopy

x 1k → 200k

How does electron microscopy work

Electrons emitted from filament and accelerated in an electric field, which is focused by a condenser lens

What then happens in transmission electron microscopy

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

What then happens in scanning electron microscopy

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

What is the microscope column maintained at

A very high vacuum

What is used to preserve biological fine structures during preparation

Fixation

What are the molecules used in fixation

Glutaraldehyde (protein amino groups)

Osmium tetroxide (proteins and lipids)

Potassium permanganate (membranes)

What does glutaraldehyde do

Covalently cross links proteins to neighbours

What does Osmium tetroxide do

Stabilises lipid belayers and proteins

What has to be done to the tissues before analysis

Dehydrate, embed, cut then rapidly freeze (cryopreservation)

How to increase contrast/ image clarity in a specimen

Have a range of contrasting electron densities by impregnating with metal

What is used in EM to detect proteins

Antibodies and Protein A-gold

What image does a TEM create

2D black and white

And an SEM

3D black and white