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Stack #217457

Electron microscopy

QuestionAnswer
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