SEM Sample Preparation

Fixation

Same as for TEM, but less concerned for the details of interior of cells


Samples with hard exteriors may be air dried


Dehydration

For SEM, the alcohol must be removed without causing drying artifactsWhen water evaporates, the energy required to break water-water bonds will cause extensive cell damage. (Grapes, when air dried, look like raisins)

To minimize this damage,
---air dry from liquids with lower vapor pressure

1)Hexamethyldisilazane
has low vapor pressure so has reduced evaporation characteristics
---dry from a solid directly to a gas without the liquid interface
2a) Peldri is a solid at room temp
warm to 40C to liquify, then infiltrate sample.  When the sample is cooled, the Peldri becomes a solid. Under vacuum, the Peldri  sublimes (converts directly from a solid to a gas), leaving a dry sample.  It is hard to completely sublime so it can contaminate the column.
2b) freeze drying--  a sample is frozen, then maintained frozen under vacuum.  The ice sublimes as above, leaving a dry sample without the effects of drying from a liquid interface.  Get less drying artifact, but  it requires rapid freezing with its inherent problems, then requires transfer of sample while frozen, and finally requires a much longer drying time, dependent on size
---dry from a liquid without having a liquid interface
            critical point drying
Critical Point Dryer very simplified diagram and operation (Fig 7.6)
 
rely on the phase diagram showing the interfaces between solid, gas and liquid at various temperatures and pressures
--at the critical point, a liquid and a gas are in equilibrium and there is no change in phase between liquid and gas

critical point of water is 374 C and 3184 psi

carbon dioxide 31 C and 1072 psi

            freon 19-28 and 432-700 psi  (but destroys the ozone layer)


Conductive coating

    The SEM illuminates the sample with a stream of electrons. Since the beam of electrons is highly negative, it will cause the sample to become highly negatively charged and lead to image distortions. This charging will deflect the beam and disrupt image quality. To avoid this, the sample must be conductive or made conductive during sample preparation. There are some fixations that can make the sample conductive (e.g. multiple rounds of osmium fixation to increase the conductive metal in cells).  Alternatively, the accelerating voltage can be reduced (less than 2 kV), which reduces the surface charging and usually allows a sample to be viewed without being conductive.

Otherwise, the sample can be coated with a thin layer of metal that will conduct the electrical charge to ground. This takes place in a evaporator or sputter coater.

purpose of coating

1. reduce charging

2. reduce thermal damage by improved heat transfer

3. enhance SE reflection by providing homogenous source of SE

4. reduce mass loss due to beam damage

The evaporator was previously discussed as part of shadowing and freeze fracture replica production (Chapter 6).


Sputter coater diagram and operation (Fig. 7.3)
 

evacuate to remove water vapor and oxygen--these plasmas can cause surface damage

apply voltage to form a plasma of argon+ and electrons-

e- are trapped by anode ring and magnetic fields causing e to spiral out away from sample

argon+ hits target with high energy, kicking out metal in all directions