305 electron microscopy

transmission electron microscopy

investigate structures and functional relationships at high resolution, correlate ultrastructure with light microscopy data, identify presence of microorganisms, localise biologically important molecules and elements, build 3D models of complex cell structures, confirm or refute results from other techniques

most common TEM measurement unit

nanometre - 10^-9 m

EM disadvantages

expensive, difficult to operate and maintain, specimen prep often complicated expensive and time consuming, images can be confusing and difficult to interpret, few EM pathologists

stereo light microscope

looks at surface features, light is shone onto specimen and reflected back

compound light microscope

projected image through specimen or section of specimen, light comes up from bottom and passes through specimen to lens

scanning electron microscope

looks at surface features, beam is against surface and look at electrons bouncing off

transmission electron microscope

looks through the specimen, pass electrons through sample to form image that is a projection through the specimen

TEM structure

electron gun at top emits electrons, condenser lens system converges electron beam, specimen, objective lens image formed and focused, projector lens system enlarges image, digital or 35mm film camera, fluorescent screen

tungsten filament

cheaper, short life, lower resolution, heated, electrons ripped off by energy 100kV or 200kV

lanthanum hexaboride crystal

mid price, mid life, mid resolution, tip smaller than tungsten, smaller point source gives higher resolution

field emission

expensive ($10,000+), long life (10,000+ hrs), high resolution, very tiny tip gives concentrated beam of electrons

the electron gun

accelerating voltage TEMs tend to use either 100kV 200kV or 300kV, higher kV gives lower contrast but higher resolution and the electrons also have more energy so thicker specimens can be imaged, most TEMs used for routine pathology will be 100kV

EM lenses

usually electromagnets, coils of wire around a steel pole-piece that forms the shape of the lens, prone to chromatic and spherical aberrations, prone to hysteresis (residual magnetism), also easily changed in strength by changing current, calibration important using internal standard

the specimen rod

the rod holds the 3 mm diameter specimen grid in the electron beam, all specimens must fit on this standard grid, the stages moves the grid in X Y and Z

high vacuum system

inside the column there is a vacuum which runs 24/7, require for passage of the electron beam down the column and insulating the high voltage electron source

image formation

when electrons hit the specimen, many different interactions occur at the atomic level, each type of interaction generates signal that carries information about the specimen, analysis of these signals gives us different information about our sample, in ultrastructural pathology we are mostly interested in the transmitted electrons, these are the electrons that form a bright field image of our sample

contrast in the TEM

black and grey areas highly scattering, grey and white areas have little scattering, high atomic number elements scatter the electrons and give us contrast, for biological specimens we normally have to add these elements but sometimes they are already present.

preparing biological specimens for investigation in the TEM

the sample comes from a living hydrated and dynamic biological system, we want to examine it as close to living state as possible, specimen has to cut thin enough for electrons to pass through, durable enough to survive the hostile environment inside the TEM column and have sufficient contrast to create an electron image

specimen requirements

must be stable in a high vacuum, must be thin 50nm to 300 nm thick, should be conductive (electrons have negative charge), variable electron density (to give contrast)