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Stack #217457
Electron microscopy
| Question | Answer |
|---|---|
| Able to resolve structures 0.0004 microns apart | Electron microscope |
| Magnification range of x1000 to x500,000 | Electron microscope |
| Obtain extra resolving power by replacing the light source of a light microscope w/an electronic gun | Electron microscope |
| an electrified tungsten filament that emits electrons | Electric gun |
| the electric gun,electron beam and specimen are all maintained under the | vacuum |
| aimed at the specimen and focused by varying the strength of electromagnetic fields | Electron beam |
| is visualized by projection onto a fluorescent screen | resulting image |
| specimen either transmits electrons producing clear areas in the image electron-lucent | transmission electron microscope |
| deflects electrons producing dark areas in the image-electron-defencse | transmission electron microscope |
| a two-dimensional image is seen | transmission electron microscope |
| used to dx kidney diseases and tumor identification | transmission electron microscope |
| an electronic beam sweeps across the surface of a specimen resulting in a three dimensional image | scanning electron microscope |
| its hightes effective magnification is much less than the transmission electron microscope but it has a greaterd depth of focus | scanning electron microscope |
| used to study cell surfaces | scanning electron microscope |
| used primarily in research | scanning electron microscope |
| provides excellent preservation of cytologic detail | advantages of primary osmium tetroxide fixation |
| renders lipids insoluble, giving excellent memebrane preservation | advantages of primary osmium tetroxide fixation |
| specimens can not be left in fixative for more than 2-4 hours | disadvantages of primary osmium tetroxide fixation |
| penetration is poor so specimens must be minced to 1mm cubes | disadvantages of primary osmium tetroxide fixation |
| hitochemical studies can not be performed | disadvantages of primary osmium tetroxide fixation |
| allows better penetration | advantages of primary aldehyde fixation |
| histochemical studies can be performed | advantages of primary aldehyde fixation |
| EM can be performed on specimens fixed for a long time | advantages of primary aldehyde fixation |
| Formaldehyde and formaldehyde-gluteraldehyde mixtures serve as dual purpose | advantages of primary aldehyde fixation |
| can be used for easy perfusion of tissue | advantages of primary aldehyde fixation |
| when followed by postosmication optimum preservation of cellular details is achieved | advantages of primary aldehyde fixation |
| lipids are not preserved unless secondary osmium tetroxide fixation is employed | disadvantages of primary aldehyde fixation |
| membrane-bound cavities are slightly enlarged beyond a desirable limit | disadvantages of primary aldehyde fixation |
| membranes are electron lucent (clear areas in the image) unless secondary osmium tetroxide fixation is employed | disadvantages of primary aldehyde fixation |
| also known as zamboni/buffered picric acid formaldehyde | PAF |
| specimen can remain at room temperature indefinitely w/o compromising morphology | advantages of primary buffered PAF fixation |
| penetrates tissue rapidly and stabilizes cellular proteins | advantages of primary buffered PAF fixation |
| can be used to fix tissue for light and electron microscope | advantages of primary buffered PAF fixation |
| lipids arent well preserved unless secondary osmium tetroxide fixation is employed | disadvantages of primary aldehyde fixation |
| some cytoplasmic granules and lysosomes may not be preserved | disadvantages of primary aldehyde fixation |
| some background substances may not be preserved | disadvantages of primary aldehyde fixation |
| similar to processing for light microscopy | processing for EM |
| ehthyl alcohol is most commonly used reagent | dehydration |
| acetone, dioxane, 2-ethoxyethanol and dimethyl formamide have been successfully used | dehydration |
| correspond to clearing agents in paraffin embedding. | transitional solvents |
| they are necessary with most epoxy and polyester resin embedments because alcohol will mix very slowly w/epoxy resins and not polyester resin mixture | transitional solvents |
| used with epoxy resins and can be used with polyester resins but styrene is the transitional solvent of choice for polyester resins | propylene oxide |
| was the earliest used embedding media | methylacrylate |
| its advantage was that it would partially volatize in the electron beam, enhancing specimen contrast to such a degree. no stain was necessary for tissue fixed with osmium tetroxide | methylacrylate |
| disadvantage the electron microscope would have to be cleaned weekly instead of every several months | methylacrylate |
| is a polyester resin used for embedding, sections well but must be obtained by foreign sources | vestopal W |
| epon, spurr, and araldite | most commonly used epoxy resins (embedding media) |
| most difficult technique in EM | sectioning |
| have microtome table free of vibration and in a draft free area | sectioning for EM |
| trim the block so that there is a small face to section | sectioning for EM |
| use lens paper to keep the trough liquid of your diamond knife clean when in use | sectioning for EM |
| haste makes waste | do not hurry |
| dont let fingers touch the knife, forceps or any equipment that will be in contrast w/t trough liquied or thin sections | because oil or cellular debris will contaminate the sections |
| oil may be removed by rinsing the blade with acetone and then with water | use oil-free blade |
| Thin sections vary from 50-90 nm | sectioniong for EM |
| how can thickness be accurately determined | from the interference colors shown by the section as they float in the collecting trough |
| Fairly thick sections show bright colors such as purple, blue, green, yellow | Section thickness |
| 50nM thick | Silver sections |
| are too think for use | gray sections |
| give rise to the colors and the color varies very precisely with the distance between the two surfaces | intereference between rays of light reflected from the upper and lower surfaces of the sections |
| used for most thin sectioning | Diamond knives |
| have been used for cutting .5 micron sections. | glass knives |
| What knives are used today | low grade diamond knives |
| avoid touching the edge of the knife with any solid object | use and care of diamond knifes |
| as the sealing material between the knife and the boat may dissolve or destroy the cellular material | avoid using solvents in the trough of the boat |
| do not allow sections to dry on the cutting edge of the knife | section drying |
| remove all unused sections w/hair or eyelash and clean the knife | immediately after picking up the sections |
| use cleaning rod specifically designed for | diamond knife |
| do not use sonication to the cleaning knife | it can loosen the mounting and cement sealing of the sides of the knife |
| it should be soaked overnight in a dilute solution of a nonionic detergent w/a neutral pH. after soaking the knife, rinse w/distilled water and clean with the cleaning rod | if sections have been allowed to dry on the knife edge |
| different types of specimens, require | different types of knives |
| upper and lower edges not parallel | retrim |
| block sides unequal in length | retrim |
| check tightness of specimen block, knife holder and knife | sections are varying rather than uniform thickness |
| knife may be dull | sections are varying rather than unifrom thickness |
| try a faster or slower cutting speed | sections are varying rather than uniform thickness |
| block may be soft heat at 60C for 24 hours | sections are varying rather than uniform thickness |
| check for drafts and air conditioning that may be causing temperature variations | sections are varying rather than uniform thickness |
| check microtome and table for vibrations, keep a steady cutting rhythm | sections are of varying rather than uniform thickness |
| reset microtome advance | sections are skipped or not cut at all |
| knife may be dull | sections are skipped or not cut at all |
| tighten knife and specimen block firmly | sections are skipped or not cut at all |
| block face may be wet-dry with lens paper | sections are skipped or not cut at all |
| block may be soft | sections are skipped or not cut at all |
| check microtome and table for vibrations keep a steady cutting rhythm | sections are skipped or not cut at all |
| reduce cutting speed | chatter or undulations in sections |
| reduce knife clearance angle | chatter or undulations in sections |
| reduce the size of the block face | chatter or undulations in sections |
| check microtome and table for vibrations, do not touch unless manually operated | chatter or undulations in sections |
| raise meniscus level of trough fluid | sections crumble or stick to knife edge |
| clean knife edge | sections crumble or stick to knife edge |
| increase knife clearance angle | sections crumble or stick to knife edge |
| block face may be dirty, clean with lens paper and alcohol | sections crumble or stick to knife edge |
| lower meniscus level of the trough fluid | section lifted by specimen block |
| dry block face with lens paper | section lifted by specimen block |
| increase clearance angle | section lifted by specimen block |
| clean knife edge | section lifted by specimen block |
| block face may be electrified, increase room humidity or touch block face with wet lens paper | section lifted by specimen block |
| check back of knife facet or fluid droplet dry with lens paper | section lifted by specimen block |
| nick in knife cutting edge | split sections or lengthwise lines in sections |
| clean knife edge | split sections or lengthwise lines in sections |
| block may contain glass or dirt,discard block or use and old knife | split sections or lengthwise lines in sections |
| lower meniscus level of trough fluid | face of specimen block gets wet |
| dry the back edge of knife cuttin facet | face of specimen block gets wet |
| increase the room humidity | face of specimen block gets wet |
| staining .5 micron sections for viewing w/the light microspe, staining thin sections 50nm for viewing w/the electron microscope | two types of staining |
| stains .5 micron sections | toluidine blue-basic fuchsin |
| polychromatic | toluidine blue-basic fuchsin |
| nuclei-dark purple | toluidine blue-basic fuchsin |
| cytoplasm-pink to lavender | toluidine blue-basic fuchsin |
| fat - gray/green to gray/blue | toluidine blue-basic fuchsin |
| red blood cells - magenta | toluidine blue-basic fuchsin |
| stains .5 micron sections | toluidine blue |
| nuclei - dark purple | toluidine blue |
| cytoplasm - lavender | toluidine blue |
| fat gray/green to gray/blue | toluidine blue |
| red blood cells - deep blue/purple | toluidine blue |
| stains thin sections | lead citrate solution |
| results: tissue structures will either deflect electrons (electron dense) and will appear as dark structures on the fluorescent screen | lead citrate solution |
| they will transmit electrons (electron lucent) and will be pale to invisible | lead citrate solution |
| the heavy metal stains take up the structures that appear electron dense (membrane) | lead citrate solution |
| View the relationship between cells and the ulatrastructure of the cell itself | transmission electron microscope |
| plamalemma with no breaks, nuclear envolope uniform udilated space between inner and outer membranes, mitochondria no swelling | good fixation shows |
| endoplasmic reticulum regular width and regular arrangement of cisterns or channels | good fixation shows |
| cytoplasm - finely precipitated and not too obvious in most cells | good fixation shows |
| nucleus - appearance varies with fixative, osmium - finely granular, aldehyde aggregated chromatin masses | good fixaton shows |
| pH solutions are buffered between 7.2-7.4. | Factors influencing fixation |
| phosphate, cacodylate,s-collidine and veronal acetate | most common buffers |
| Temperature room temp was previously done at 4c but that caused swellin of perinuclear membrane and destruction of microtubules | Factors influencing fixation |
| Tonicity - should be that of blood plasma around 300 MOsm. fixatives are not ostomotically active dextrose and sucrose are used to adjust tonicity | factors influencing fixation |
| can remain indefinetely in formaldehyde, buffered PAF and formaldehyde-gluteraldehyde | Length of fixation |
| gluteraldehyde 2-4hours then move to a buffer | length of fixation |
| osmium tetroxide 1-2 hours then process | lenght of fixation |
| 90nM thick | gold sections |
Created by:
nperez
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