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Goals of Specimen Preparation Observe specimen near natural state as possible. Preservation of as many features as possible. Avoid artifacts (changes, loss or additional information ). Preparation of Biological Samples. Fixation and washing/rinsing

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slide1

Goals of Specimen Preparation

Observe specimen near natural state as possible.

Preservation of as many features as possible.

Avoid artifacts (changes, loss or additional

information)

slide2

Preparation of Biological Samples

  • Fixation and washing/rinsing
  • Clearing of tissue (light microscopy-optional)
  • Dehydration
  • Embedding
  • Sectioning
  • Mounting
  • Staining
slide3

Fixation

A process which is used to preserve (fix) the structure of freshly killed material in a state that most closely resembles the structure and/or composition of the original living state.

  • Chemical crosslinking - coagulative/noncoagulative
  • Coagulative:
  • original killing agents (alcohols, Farmer’s, FAA, Bouins)
  • Low pH Unbuffered
  • Coagulates cellular components - like frying an egg.
  • Non Coagulative: Formaldehyde, Glutaraldehyde, Osmium Tetroxide
slide4

Formaldehyde

Usually in form of paraformaldeyde powder or 37% to 16% aqueous solution

  • Low MW makes it one of the best penetrating of all the fixatives, thus it is widely used in fixation of resistant materials, such as seeds, spores, plant material, etc., usually in conjunction w/ another aldehyde.
  • Formalin contains many impurities, so formaldehyde for use in EM is normally prepared from the dissolution, heating, and alkalination of powdered paraformaldehyde. Since this solution contains no inhibitors, it has a shelf life of only a few weeks.
slide5

Glutaraldehyde

  • Glutaric acid dialdehyde, a 5 Carbon dialdehyde, is the most widely applied fixative in both scanning and transmission electron microscopy.
  • Most highly cross-linking of all the aldehydes. GTA fixation is irreversible.
  • In TEM, buffered GTA has the reputation of providing the best ultrastructural preservation in the widest variety of tissue types of any known chemical fixative.
slide6

Osmium Tetroxide (OsO4)

  • A non-polar tetrahedral molecule with a
  • molecular weight of 254 and solubility water
  • and a variety of organic compounds.
  • Its principle utility is its ability to stabilize and stain lipids- preferentially unsaturated fatty acids
  • Commercially available as a coarse yellow crystalline material packaged in glass ampoules sealed under inert gas. Similarly packaged aqueous solutions are also available.
  • An additive, non-coagulative type of fixative, but lacks the ability to crosslink many proteins.
  • Very poor rate of penetration
slide7

Basic factors affecting chemical fixation

pH (Isoelectric point)

Total ionic strength of reagents

Osmolarity

Temperature

Length of fixation

Method of application of fixative

slide8

Buffers

Solution containing a weak acid and its salt.

Serves to hold pH steady during the fixation process.

  • Chemical fixation is a complex set of oxidative and reductive reactions, thus [H+] is constantly changing.
  • All fixatives have an optimal pH at which rate of crosslinking is greatest.
  • At a specific pH, all proteins have a point, the isoelectric point (IEP) where the numbers of + and - charges are equal. Fixation is most effective at the IEP.
slide9

Tonicity

    • Osmolality of fixatives, buffers, and tissue fluids can be measured with an OSMOMETER
    • Effect of tonicity:
      • 1.Isotonicity
      • Environment and
      • Sample similar
      • 2.Hypertonicity
      • Environment higher osmolarity
      • Water moves out of sample
      • 3.Hypotonicity
  • Environment lower osmolarity
  • Water enters sample

5 mOsm

5

3

8

slide10

Dehydration

  • Reasons for dehydration:
    • Water in incompatible with conditions inside an electron column.
    • Most of the materials used to infiltrate and embed specimens prior to ultrathin sectioning are hydrophobic.
  • Methods of Dehydration:
    • Organic solvent Series
      • Tissue is transferred through a series of organic solvents in increasing concentration.
      • Ethanol and acetone are the most commonly used.
      • Water content is slowly reduced to the point that the tissue is in 100% solvent. and is thus completely dehydrated.
slide11

Embedding and Sectioning

  • Requirements for cutting any material into thin slices:
    • Support - biologicals tend to be soft. Inducing hardness in them gives them the mechanical support needed for sectioning.
      • Accomplished by lowering temperature (freezing) or infiltration with some material that can be hardened.
    • Plasticity - resiliency as opposed to brittleness.
slide12

Embedding and Sectioning

  • Cryosectioning
    • Commonly done for light microscopy.
      • ie hospital operating room biopsies.
      • Rapid.
      • Preservation is usually sufficient for a rapid diagnosis.
      • Overall resolution is low.
    • Ultrathin cryosectioning
      • Technically demanding
      • Requires expensive specialized equipment
      • Ultrastructural preservation often poor due to freezing artifact.
      • Usually done only when tissue cannot be exposed to chemical fixatives...as in some immunolabeling, analytical work.
slide14

Embedding and Sectioning

  • Embedment
    • Light microscopy
      • Tissue infiltrated with molten paraffin wax - which is allowed to cool and harden.
      • Requires dehydration and infiltration with a paraffin solvent - aromatic hydrocarbon (xylene, toluene, benzene).
      • Provides sufficient support to section to about 3 micrometers minimum with a steel knife.
      • Paraffin can infiltrate deeply into tissue, allowing large blocks and ultimately large sections to be obtained.
slide15

Embedding and Sectioning

Paraffin Sectioning for Light Microscopy

slide17

TEM Embedment

    • Tissue infiltrated with a resin which is polymerized by heat, chemicals, or U.V.
    • Provides support to section infiltrated tissue to about 40 nm minimum.
    • Infiltration is limited...specimens can be no more than a few mm thick.
    • The required thinness of the sample and the friction during cutting limits the section size to about 1 mm2 maximum.
slide18

Types of Resins

    • Acrylics - ie methyl, butyl methacrylates (plexiglass) - "Open-structured" - allows for better stain penetration and Antibody rxn
    • Epoxies - epon, araldite, Quetol, Spurr - for most general work
    • Polycarbonates - vestopal - fiberglass resin
  • Infiltration
    • In resin/solvent mixture in increasing concentration
    • Ethanol/resin or acetone resin often used
    • Propylene oxide/resin is most effective
  • Polymerization
    • Thermal - 50-70 C, depending on resin mix
    • U.V. - usually done to avoid heat
    • of polymerization. Often done at low temp.
slide19

Ultramicrotomy

  • Ultramicrotome Knives:
    • Diamond - 1.5 - 6mm cutting edge
    • Latta-Hartmann (glass) - 6mm cutting edge (~1mm useable)
    • Both use water to support and lubricate the section as it is cut (decreases friction)
slide20

Making a glass knife:

  • Use of a glass knifemaker to score a 1" glass square
slide21

A scored 1" glass square (top) and the resultant glass knife:

Making the water trough

Tape or plastic

a) Cutting edge

b) Knife angle (45o)

c) Corner

d) Shelf

slide22

Evaluating a glass knife - factors to consider:

    • Age - degrade rapidly due to edge flaking
    • Quality of cutting edge - flat, concave, convex
    • Amount of cutting edge - judged by the stress line. A "spur" is normal.
    • Contamination - on edge or sides.
slide23

Setting up the Microtome

Block

face

Sample

Block

Knife

edge

Glass

Knife

slide24

Syringe - adjusting water in trough

Loop - assist picking up sections

Eyelash tools - assist with section manipulations

slide25

Standard Preparation

Tissue

TEM

SEM

Chem.

Fixation

Cryo

Fixation

Chem.

Fixation

Cryo

Fixation

Rinse/store

Substitution

Rinse/store

En bloc

staining

Cryo-

sectioning

Dehydration

Dehydration

Dehydration

Drying

Resin

infiltration

Mounting

Sectioning

Coating

Post staining

slide26

Support Films

Formvar, Carbon, Collodion

-Used when sections or samples are smaller than support of grid.

-100 mesh or less, slot grids

-Fragile or very thin sections

Avoid when possible because:

Usually has holes or uneven thickness

Added thickness affects clarity and contrast

slide28

Formvar coated grids

Holey formvar

Formvar and carbon

slide29

Negative Staining

Positive staining - forms a complex with specimen

Negative - stain and specimen do not interact and specimen remains electron transparent

Advantages:

1) Improved resolution

2) Speed

3) Unique information

4) Simplicity

slide30

Disadvantages:

1) Repeatability

2) Limited surface topography

3) Toxicity

slide31

Choice of stain:

1) High density to provide high contrast

2) High solubility and minimal reaction to sample

3) High melting and boiling point (beam stable)

4) Precipitant formed is extremely fined grained

Stains commonly used:

Phosphotungstate, sodium tungstate, uranyl acetate and uranyl nitrate

slide32

Brief procedure:

Small grid and support film (formvar, paraloidin. Sometimes carbon added.

Thin suspension of sample and excess removed.

Dry then add negative stain and remove

Factors affecting staining:

concentration of stain

pH of stain

time

- Dry and view.

slide37

Negative stain of purified RhMV virus labelled with anti-RhMV and detected with anti- rabbit conjugated to 10 nm gold. Bar = 100 nm.

Photograph provided by Fred Gildow Lab, Department of Plant Pathology, Penn State.