Site author Richard Steane
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The BioTopics website gives access to interactive resource material, developed to support the
learning and teaching of Biology at a variety of levels.
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Transmission Electron Microscope (TEM) | Optical microscope | |
uses | electrons | light [photons] |
principles of operation | electrons pass through / enter (thin) specimen denser parts absorb more electrons so appear darker |
light passes through (transparent sections of) specimen light of different wavelength passes through so parts of image are coloured |
magnification not usually credited in exam answers! |
greater (up to 1,000,000x?) | less (500-1000x) |
resolution & reason |
greater/finer (down to 0.2 nm) electrons have short wavelength |
less (down to 200nm) light has longer wavelength(s) |
detail visible in cell organelles |
greater - e.g. internal detail of membranes in mitochondria and chloroplasts, E.R., Golgi and vesicles- even ribosomes! | less - gross external detail only for nuclei, - maybe mitochondria, chloroplasts (& Golgi ?) if you are lucky |
status of specimens | only dead/dehydrated | live possible |
image colour | no intrinsic colour (shades of grey only) |
own colour of specimen/ stain visible |
specimen preparation | more complex/ difficult / more likely to produce artefacts |
less complex |
section thickness | must be thinner | sectioning not always essential |
focussed using | (electro)magnets | glass lenses |
Stage in process | Reason |
Cut into small (1cm) chunks or squares and just cover with liquid which is: |
(see below) |
... isotonic solution | to prevent damage to mitochondria / chloroplasts /organelles
{not whole cells which will soon be burst!} |
... kept cold using ice bath | to prevent/reduce damage by enzymes |
... buffered (pH between 6.7 and 7.4) | to prevent protein/enzyme denaturation (by acid?) |
Homogenise animal material with blender/ grind plant material with pestle and mortar | to break open cells and release intracellular organelles |
Filter using muslin or coarse filter paper | to remove (large) debris or whole cells |
Centrifuge filtrate (at lower speed/1000 g) | to separate nuclei/cell fragments/ heavy organelles |
Re-centrifuge supernatant/after nuclei/pellet removed at higher speed (3000g) | to get mitochondria/chloroplasts in pellet/at bottom |
Transmission Electron Microscope (TEM) | Scanning Electron Microscope (SEM) | Dissection microscope (stereo microscope) |
Stream of electrons passes through specimen and directly onto screen | Stream of electrons passed from side to side across specimen - secondary electrons reflected/scattered from surface of specimen - require to be collected and integrated to give image |
Light reflected (in all directions) from surface of specimen - may be lit from above or below, or both |
specimen is thin layer (sectioned), on grid in middle of vacuum chamber | specimen is not sectioned (may be left whole), on stud at bottom of vacuum chamber | specimen is not sectioned (may be left whole and alive) and in fresh air! |
parts of specimen contain electron-dense stains | metallic coating on surface | no need for stain |
shows internal detail within cells | shows external view only | shows external view only (but may be used for wide angle view of slides) |
preparation has more stages: fixation, staining, embedding, sectioning | fairly simple preparation (except freeze fracturing/etching) | no processing required |
flat 2-D image | image gives impression of depth - but is not truly stereoscopic | image gives good impression of depth - truly stereoscopic (separate light path and images for left and right eyes) |
controls to move image to left/right and up/down | image can be moved or rotated | specimen can be moved by the thumbs | greater magnification: 1,000,000 x
resolution 0.2 nm |
slightly lower magnification: 100,000 x
resolution 2 nm |
much lower magnification: up to 70 x
resolution irrelevant |