TEM SPECIMEN PREPARATION

Dehydration

Samples need to be transferred from water or buffer to solvent compatible with the following steps. Use either alcohol or acetone, in a series of increasing concentration through 100%

At the completion of this step, samples are ready for embedding

Embedding

Function of embedding medium-- provide a rigid matrix to allow for slicing

When don't you need an embedding medium?

very small samples, such as viruses, proteins, DNA, or fragments of cell organelles

cryosections

shadowing--replicas

 to support these samples, need grids coated with a thin layer of plastic formvar

carbon

 Characteristics: 1) Maintain contents

2) Maintain antigenicity

3) Readily soluble

4) Maintain volume

5) Beam stable

6) Beam transparent

7) Cutable

8) Stainable

9) Evenly polymerizable

10) Low viscosity

 History and types of materials

1802 Lee gelatin glycerin

1879 nitrocellulose

1881 paraffin

1950s acrylics (Lowicryl, LR White)

1962 Luft Epoxy resins (Epon 812) (EMBed, PolyBed)

Epon (long carbon chain with epoxide groups at each end) (pours like honey)
 
 
 
 
 
 
 
 
 
 

Spurr (6 carbon ring with 2 epoxide groups attached) (pours like water)
 
 
 
 
 
 
 
 

Embedding medium is a mix of components--the resin by itself does not exhibit many of the aforementioned characteristics. For this reason, it is usually mixed with other components. The ratio of these other components determined the characteristics of the final plastic Resin can have Flexiblizers (DER 736) and Hardeners added (DDSA, NSA, and NMA).

To accelerate the conversion of liquid form to plastic form, a Catalyst (DMP-30, BDMA) is added to chemically polymerize the mixture at elevated temperature. To avoid baking the plastic, some mixtures can be polymerized using UV light at low temperature
 
 
 
 

Reversible embedding

Purpose: to provide a rigid matrix for tissue so it can be sliced, then removed to allow access of reagents to the tissue without the presence of embedding media to inhibit the access

polymethyl methacrylate (remove with acetone)

diethylene glycol distearate (remove with n-butyl alcohol)

Trimming

After the embedding medium has been polymerized (hardened), the excess plastic must be trimmed away with razor blades to expose the tissue itself

Slicing

Once trimmed, embedded samples must be sliced using a microtome for light microscope slices, called sections, and an ultramicrotome for thinner EM sections.  For the light microscope, section thickness various depending on the embedding medium (5-10 um for paraffin, 1 micron for expoxy resin.  For the TEM, slices are about 70-100nm thick, or 10,000 slices to the mm.

Slices are cut using knives made of glass (cheaper) or diamonds (long-lasting)

Slices are collected onto screens, called grids, from the surface of water.