Electron microscopy Flashcards
what 2 factors does resolution depend on?
wavelength
numerical aperture of lens
how does electron microscopy work?
electrons are emitted from a filament and accelerated in an electric field. condenser lens focusses the electron beam. high vacuum
TEM
electrons either scatter or hit a fluorescent screen at the bottom of the microscope
ideal for viewing the internal structure of cells
SEM
electrons focussed on a metal coated specimen and electrons from the metal are collected by a detector
two common fixatives used in electron microscopy
glutaraldehyde
osmium tetroxide
fixation
preserving samples during preparation
fixatives cross link molecules
glutaraldehyde
covalently cross links proteins to neighbours
osmium tetroxide
stabilises lipid bilayers and proteins
why do fixed tissues need to be cut into extremely thin sections
electrons have limiting penetrating capability
how are thin sections achieved
dehydrate specimen
permeate it with resin that polymerises
cut thin sections from resin
rapid freezing
ice crystals do not form which can damage the specimen
electron density contrast
The clarity of images in electron microscopy heavily depends on having a range of contrasting electron densities in the specimen. Electron density refers to how many electrons are present per unit volume in a material. Higher electron density materials interact more with the electron beam, appearing darker on the micrograph. Lower electron density materials allow more electrons to pass through, appearing lighter.
contrast agents
To enhance the contrast in biological specimens, tissues are often impregnated with heavy metals. This process, known as staining, makes different structures stand out more clearly. Heavy metals like osmium, uranium, and lead are commonly used because they have high electron densities, which provides the necessary contrast.
tomography
serial sections can be used to reconstruct 3D images
