TEM contrast methods (staining)

In order to visualize a specimen in the TEM, the contrasting regions of electron transparency and electron opacity must be present. As in light microscopy, differences in contrast can be accentuated through the use of a stain. The most commonly used stains in electron microscopy are made up of heavy metal salts. This can be explained by mass-thickness contrast. The interaction of electrons with heavy atoms is stronger than with light atoms. If the thickness is homogeneous, areas in which heavy atoms are concentrated appear with darker contrast than such with light atoms (mass contrast). More electrons are scattered in thick than in thin areas; thus, thick areas appear dark (thickness contrast). However, a thick area with light elements might have the same contrast as a thinner area with heavier atoms. Heavy metal salts have atoms of high atomic weight which are especially good at producing differential electron scattering. The samples, in particular those of organic materials, consist of light elements, such as hydrogen, oxygen, phosphorus, sulphur, etc. They have a low electron- scattering power, resulting in the low contrast in an image of untreated material. Staining by soaking in solutions containing heavy metals, such as tungsten, osmium, lead, or uranium increases electron-scattering power, and results in images with more visible specimen details.

Electron staining falls into one of two categories:

• Positive staining in which contrast is imparted to the specimen itself, and is most common method for tissue sections. The heavy metal is absorbed by tissue or particle itself. The stains for positive staining are osmium tetroxide, uranyl acetate, lead citrate and phosphotungstic acid.

  OsO₄: its atomic weight is 190, and it is sufficient to deflect electrons effectively. Because it reacts more readily with lipids than it does with proteins, osmium tetroxide is used as structure specific positive stain.

  Both uranyl acetate (MW= 422) and lead citrate (MW = 1054) are heavy metal salt stains and are both categorized as general or non-specific stains. Uranyl acetate ions are believed to react with phosphate and amino groups (found in nucleic acids and certain proteins) while lead ions are thought to bind to negatively charged molecules such as hydroxyl groups. Because of this ability to stain different cellular components uranyl acetate and lead citrate are often used in conjunction with one another though not simultaneously.

• Negative staining in which the area surrounding the specimen is given increased electron opacity, while the specimen itself remains more translucent. This method is used to stain suspensions of isolated proteins or macromolecules. The stains, commonly used, include phosphotungstate, silicotungstate, uranyl acetate and ammonium molybdate.

The staining techniques are not desirable for most soft matter systems:

- The structure of the sample may be changed during the preparation process, in particular in soft matter.

- Exposing to different chemicals can result in artifacts, which have to be distinguished from genuine structures within the specimen, particularly in biological samples.

SEM Astigmatism

Astigmatism occurs when the electrons sense a non consistent magnetic field as they spiral round the optic axis. As shown in the three-dimensional schematic the two planes at right angles to one another passing through the optical axis are the meridian plane and the sagittal plane, the meridian plane being the one containing the off-axis object point. Rays not in the meridian plane, called skew rays, are focused farther away from the lens than those lying in the plane. In either case the rays do not meet in a point focus but as lines perpendicular to each other. Intermediate between these two positions the images are elliptical in shape. This happened because the soft iron polepieces are not ideal cylindrical and ideal symmetrical. The soft iron may also have micro structural heterogeneities which cause local variations in the magnetic field strength. In addition, if the apertures are not exactly centered on the axis, it can also disturb the field. Moreover, if the apertures are not clean, the contamination charges up and deflects the beam. Astigmatism distorts the image by an amount r_ast, where:

r_ast=βΔf

- β is the maximum semi angle of the collection of the objective lens aperture.

- Δf is the maximum difference in focus induced by the astigmatism.

Astigmatism is simply corrected using stigmators, which are small octupoles that set up a compensating field to balance the heterogeneities causing the astigmatism. There are stigmators in both the illumination system (condenser lens) and the imaging systems (objective lens).

There are several minor defects, such as barrel and pincushion distortion, which also deform the image. They are irregularly seen at very low magnification where electrons traveling close to the bore of the polepiece appear in the image.

Condenser and objective apertures

In electron microscopy the term aperture is commonly used to describe a diaphragm. The condenser aperture associated with each condenser lens. Condenser aperture 1 is usually a fixed aperture located beneath condenser1 that stops electrons traveling at high angles from the gun crossover point. Condenser aperture 2(CA2) is a changeable aperture located beneath condenser lens 2. A small CA2 increases beam coherence. The second condenser lens is equipped with a physical aperture which limits the beam striking the specimen. (Heath J.P., 2005) This is necessary both to protect the specimen from excessive heating and to limit the generation of X-ray from the microscope which might otherwise reach harmful levels.

The size of CA2 determines the maximum semi-angular aperture of illumination, α_c, as viewed from the specimen (Figure 1). This maximum angular aperture is achieved near the point of condenser focus, i.e. when the beam crossover is imaged on the specimen. The larger the aperture angle, the greater the maximum illumination intensity, however the poorer the image quality. If the second condenser lens is defocused, the illumination semi-angular α is defined not by the condenser aperture but by the size of the crossover image and its distance from the specimen. (Alan W. Agar, 1974, p.22-23).

Figure 1: Condenser aperture

The objective lens is equipped with two important controls: the objective aperture and the astigmatism corrector. Figure 2 show how electron are scattered through different angels by the specimen, and how the objective aperture, placed at the back focal plane of the lens, stops a number of these scattered electrons. By exchanging the aperture for one of a different size, the effective aperture of the objective lens can be varied, thus varying the proportion of electrons from any given object point stopped by the aperture. The semi-angular aperture also affects the resolution. Typically, an objective aperture is 50mm in diameter. For a focal length of 2.5 mm, the defined semi-angular aperture is 10^-2 radian. (Alan W. Agar, 1974, p.26-27)

Figure 2: Objective aperture

Schottky and cold field-emission guns comparing in electron microscopes.

 

A field emission gun is based on a release of electrons from surface of sharply pointed tungsten tip as a result of application of a strong electric field(>10⁷ Vm^-1). The field emission is caused by applying a voltage between the tip and an aperture metal anode.

Higher brightness is achieved from the gun that consists of two electrodes: the first electrode has the extraction voltage and the second one has the final accelerating voltage, required for the electron gun. There are two types of field emission gun differ mainly by their tips:

• CFE - cold field emitter is the base type. Application of the very strong electric field causes the surface energy barrier to deform to such an extent that it becomes physically very narrow. There becomes a finite probability of electrons from the Fermi level penetrating the barrier even at ambient temperature. Usually made of single crystal tungsten sharpened tip with radius of about 100 nm, the needle is so sharp that electrons are extracted directly from the tip.
• SE – Schottky emitter. The Schottky emitter combines the high brightness and low energy spread of the cold field emitter with the high stability and low beam noise of thermal emitters. A commercial Schottky emitting cathode is less sharp than the others; there a monatomic layer of ZrO is formed on the tungsten surface. This coating reduces the work function of the tungsten from 4.54 to 2.8 eV. At an operating temperature of about 1800 K, this emitter may not be as bright as the cold field emitter but it delivers stable high currents and is less demanding in operation.

The follow table summarizes the properties of common sources:

Source type	Brightness[A/cm2/sr]	Source size [nm]	Energy spread (eV)	Vacuum requirement (Torr)
SE	~108	20	0.9	10-9
CFE	~109	5	0.22	10-10