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