Bhaskar Ganguly Ph.D. Scholar (Vety. Biochemistry) Animal Biotechnology Center Deptt. Of Vety. Physiol. & Biochem. C.V.A.Sc.

Pantnagar. INDIA

SECTIONING
Electron microscopy with accelerating voltage of 100 kV requires sections ranging in thickness from 10 nm to 100 nm. The section must be thin and yet able to bear high vacuum and electron bombardment. Section must be of a uniform, known thickness and free from chatter, wrinkles, breaks and folds. Factors that determine the quality of a section are:

Ultramicrotome Cutting edge Knife angle Cutting speed Embedding material Fluid in collection trough

Knives
Glass knives: First introduced by Latta & Hartmann (1950) Easy availability, safer handling and cheaper than diamond knives Require frequent replacement, and quality of section is slightly inferior; Section thickness lower than 30 nm is difficult to achieve Two main types of defects:
Striations/ Ridges/ Hackle Marks - due possibly to

vibrations during making knives Knife defects due to dust particles, hard inclusions in the specimen, etc.
Should be prepared just prior to sectioning Coating with tungsten improves sectioning quality

Knives (contd)
Diamond knives: Superior to glass knives Offer clearer and sharper cytological features in the sections; less labor intensive, long life, yields extremely thin sections (1-2 nm) Require more care, difficult wetting of surface and costly Sapphire knives: Superior toughness at low cost
Poor wetting

Thin Sectioning
Section Thickness: Actual thickness - determined by interference color Nominal thickness determined by ultramicrotome setting Description:
Thin section 8-100 nm (0.1 m) Semi-thin section 0.1-0.2 m Thick section 2.5-10 m

High

thickness - structural overlap, more chromatic errors, resolution is limited to about 1/10th of section thickness; low thickness - poor contrast

Thin Sectioning (contd)

Actual Thickness: Determined by Interference color Observed by visualization of light reflected by sections floating at the surface of liquid in the trough; visualization angle should not exceed 45
Dark Grey Grey Silver Gold Purple Blue

20 nm 25-30 nm 50-65 nm 150 nm 190 nm 240 nm

Thin Sectioning (contd)

Sectioning angle:

Direction of block advance

Kn ife

Direction of block descent

: Clearance angle : Knife angle : Shear/ Rake angle

Thin Sectioning (contd)

Trimming & Preparation of Block Face:

Thin Sectioning (contd)

Sectioning:

Grids & Support Films

Electron microscope grids are analogous to the glass slides

used for specimen support in light microscopy. These grids come in a variety of sizes and shapes of their perforations. Many have a regular coordinate grid pattern, e.g., 200 mesh, which have 200 holes per inch and about 60-70% open space. Grids with fewer holes have more open space. When samples are smaller than the hole, some electron transparent material (film) is required to maintain the specimen in position on the grid. Suitable electron transparent support films can be constructed of carbon evaporated on a freshly cleaved sheet of mica or a more easily prepared plastic film. Three plastics are commonly used for support films: Collodion, Parlodian and Formvar (polyvinyl formal). All can be produced by the same technique and with reasonable ease.

Preparation of support films:

STAINING
Biological material largely consist of low atomic

weight elements and show poor contrast under electron microscopy. Staining is aimed at improving contrast of the specimen. Two types:
Positive Staining: Electron dense material is used

to selectively stain the site of interest Negative Staining: Used to study surface morphology, electron dense material is coated around the specimen; Surface of specimen appears as the interface between light specimen and dark stain

Uranyl Acetate and Lead Citrate Staining

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