Cutting Sections and Electron Microscopy

Sectioning

Process whereby tissues are cut into uniformly thin slices or "sections" with the aid of a microtome, to facilitate the studies under the microscope

Paraffin Sections

For paraffin embedded tissue blocks which may be cut by rocking and rotary microtome

Celloidin Sections

For celloidin embedded tissues which are usually cut by means of the sliding microtome

Frozen Sections

Which may be cut from tissues that have been fixed and frozen with CO2 or for fresh or fixed tissues frozen with the cryostat

Trimming

• The excess wax is cut off from the block to expose the tissue surface in preparation for actual cutting

• Only thin slices are taken out at a time to prevent the block from cracking

Fine Trimming

• May be done by either setting the thickness adjuster at 15 mm or by advancing the block using the coarse feed mechanism

• The knife is usually tilted at 0-15° angulation on a microtome to allow a clearance angle between the cutting facet and the tissue block

Cold Wax

Provides better support for the harder elements in a specimen allowing thinner sections to be obtained

45-50°C

The sections are then floated out on a water bath set at ___, approximately 6-10°C lower than the melting point of the wax used for embedding the tissue

Floating

• This should expand the section to its original dimensions and ensure that it is completely flat

• The temperature will need to be 5-9°C below the melting point of the wax

Celloidin Embedding

• A slow process, usually taking weeks, and does not produce sections as thin as those produced by paraffin embedding

• Completely avoids the use of heat at any stage

• Shrinkage is absolutely minimal

Electron Microscope

• A type of microscope that uses a beam of electrons to create an image of the specimen

• It is capable of much higher magnifications and has a greater resolving power than a light microscope

Transmission Electron Microscope (TEM)

The source of illumination is a high voltage beam of electrons of very short wavelength, emitted from a tungsten filament at the top of a cylindrical column

1. Electron source

2. Electromagnetic lens system

3. Sample holder

4. Imaging system

Four parts of a TEM

Glutaraldehyde

A di-aldehyde, preserves the tissue’s ultrastructure well but penetrates slower than the monoaldehyde, paraformaldehyde

Paraformaldehyde

A monoaldehyde and penetrates faster than glutaraldehyde, but results in poorer ultrastructure

Polymerization

In this step, tissues embedded in the resin (wrapped in aluminum foil) are allowed to set overnight at room temperature and then placed in an oven at 60°C for 2-3 days

Positive Stains

Deposit electron dense material on the area of interest, so that it stands out as a dark area on a light background

Uranyl acetate and lead citrate

Commonly used stains in positive staining which bind at sites of osmium deposition and lead also binds with and stains nucleic acids and glycogen

Negative Stains

Penetrate and darken the interstices between areas of interest, which then appear light on a dark background. In negative stain microscopy, the electron beam primarily interacts with the stain.

Lead CItrate

The best stain available since it can be used at a high pH and stains a wide variety of cellular components including nuclear components, ribosomes, membranes, microfilaments and glycogen.

Uranyl acetate

May be used during the dehydration process by making the 50% acetone up to 2% with the stain

Phosphotungstic acid

• May be used as a "negative" stain in that it does not bind particularly well to anything but instead causes areas other than cellular (organic) material to appear dark

• It is especially useful for viewing molecules such as proteins (e.g., antibody, DNA) and suspensions of subcellular structures such as membranes (e.g., mitochondria - elementary particles, etc.)

Poor fixation

Can cause autolytic or postmortem changes

Soft blocks

• Where an impression in the resin remains when a fingernail is pressed into it

• This is because poor polymerization and is usually caused by out of-date accelerator

Brittle tissue blocks

This may occur in one block of a batch due to poor infiltration of that solitary block or it may occur in a complete batch of blocks

Hard Tissue

Fragile and break easily when cut due to poor infiltration

Unrelated electron dense material

Throughout specimen when examined under the electron microscope may be caused by lead citrate stain

Scanning Electron Microscope (SEM)

• Scans the surface of coated specimens with an electron beam and by detecting electrons scattered (reflected) by the object, forms an image on a TV like monitor

• Produces images by detecting secondary electrons which are emitted from the surface due to excitation by the primary electron beam

Primary Fixation

Fix specimen with 2.5% glutaraldehyde in 100 mM phosphate buffer at pH 7.0 2-24 hours

Osmium Tetroxide

It is necessary to wash or rinse the specimen following primary aldehyde fixation and before post-fixation with

Scanning Tunneling Electron Microscope (STEM)

A type of electron microscope that shows three-dimensional images of a sample