Cryogenic temperature electron microscopy types

Cryogenic-temperature transmission electron microscopy (cryo-TEM), namely TEM imaging of liquids ultrafast cooled to cryogenic temperatures has become an indispensable tool to acquire high-resolution direct-images of self-aggregating liquids, containing features on the nanoscopic scale. A wide range of systems of low- and high-molecular weight solutes, synthetic and biological, has been studied by the technique. While most cryo-TEM work has been done on aqueous systems, more recently the technique has been extended to non-aqueous solvents as well.
The term ‘cryo-TEM’ actually refers to two techniques: direct-imaging cryo-TEM, by which a thin vitrified sample is examined by the TEM at cryogenic temperatures, and freeze-fracture-replication cryo-TEM (FFR), by which a carbon-metal replica of the fractured fast-cooled specimen is examined at room temperature by the TEM.

Direct-imaging cryo-TEM allows in situ imaging of delicate structures of soft matter, including liquid systems, providing unique information not obtainable by other methods. Cryo-TEM can provide high-resolution images of complex fluids in a near in situ state. Samples embedded in a thin layer of vitrified solvent do not exhibit artifacts that would normally occur when using chemical fixation or staining-and-drying techniques. Cryo- TEM has been useful in imaging biological molecules in aqueous solutions.

Cryo-TEM technique involves production of frozen hydrated specimens prepared, maintained and observed within a layer of vitreous ice. Below 273 K, ice may exist as hexagonal or cubic crystals, or as the vitreous form, which is essentially a supercooled liquid. Vitrification occurs when cooling is very rapid (~10⁵ degrees). Vitrification of a thin specimen is usually achieved by rapid plunging it into liquid hydrocarbon, mostly ethane (an efficient cryogen), held at the liquid nitrogen temperature. This helps to avoid formation of ice crystals and thus the damage they cause, allowing the investigation of a specimen in the fully hydrated, close to physiological form. The vitreous state persists if the temperatures are maintained below the devitrification level (~130 K). Specimens can effectively be observed in the absence of the background noise contributed by normal support films.

Freeze-fracture-replication (FFR) is an indirect way to get an image from a specimen, meaning the image is obtained from a replication of the sample and not from the sample itself. This method includes few steps of preparation (see figure):

1. Freezing – the sample is cooled (in rapid cooling) to a very low temperature

2. Fracturing – the top part of the sample is cracked open

3. Etching – by sublimation of the ice that on the sample (not necessary step)

4. Replication – the surface of the sample is coevered with heavy metal (e.g. Platinum) by deposition at an angle of 45º or less to the horizon, to enhance contrast (“shadowing”). Above it, a carbon layer is added for mechanical stabilization of the replica.

5. Dissolving the specimen – the replica is floated onto the surface of a powerful solvent to dissolve away the specimen.

6. Washing – the replica is washed and picked up on a cooper grid for examination in the cryo TEM.

Scheme of the different stages preparation of the replica

This technique provide image of the topography of the specimen, fine imaged and fine particles can be imaged. System with high viscosity, or that containing large particles that cannot be accommodated in the thin specimen of direct imaging cryo-TEM, cannot be examined in direct-imaging cryo-TEM, but can be examined in the FFR technique.

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

Electron microscope lens defects

We are know about the next lens defects: spherical aberration, chromatic aberration, astigmatism.

Spherical aberration is an inability of a lens to focus all incident beams from a point source to a point. The outer zones of the lens have a greater strength, and light rays or electrons originating from a point are not imaged at a point. The one point the envelope of the imaged rays has a minimum diameter known as the circle or disc of least confusion. This limiting disc has a diameter d_s given by: d_s =1/2*C_s*α³ =>C_s- spherical aberration coefficient, α - semi–angular aperture of the lens.

Chromatic aberration, the other major distortion, is caused by the faster (shorter wavelength) electrons focusing at a different position from the slower ones, a problem that only a perfect electron beam could solve completely. The chromatic aberration coefficient C_s is the parameter which expresses this quality , and the resolution d_c as limited by chromatic effects is given by

Where α is the objective semi-angular aperture.

Astigmatism is a defect of magnetic field asymmetry resulting in differing lens strengths in two directions at right angles. The amount of the astigmatism of the lens is defined as the distance between the focal lines. The astigmatism of an objective lens in a good microscope will be typically 1 μm or less.

Characteristics parameters of an electron microscope lens

Electromagnetic lens contain soft-iron pole-pieces and copper coils. The soft-iron pole-pieces sit in the hole down the middle of the lens are surrounded by copper coils through which the current runs to magnetize the pole pieces. As the result creates a electromagnetic field (or electrostatic), depending on current and number of copper coils: B=mNI/L

The resolution of the electron lens is the minimum resolvable distance in the object. The resolution of electromagnetic lens is customarily defined in terms of the Rayleigh criterion. The finite size of the lens results in diffraction of the rays at the outermost collection angle of the lens, usually defined by a limiting aperture. This diffraction results in a point being imaged as a disk (called the Airy disk). The distance apart of the two incoherent point sources is defined as the theoretical resolution of the lens r_th and is given by the radius of the Airy disk: r_th =0.61*(λ/α).

The focal length of a lens is a measure of its strength, and it is defined as the distance between the lens and the beam cross-over (which is also known as the focal point) when all the beams coming into the lens are parallel to one another. The higher the field region of a condenser-objective lens the shorter its focal length.

The Depth of field, D_ob is a measure of how much of the object that we are looking at remains in focus at the same time; the term depth of focus refers to the distance over which the image can move relative to the object and still remain in focus. D_ob=d_ob/a_ob . When d=1nm, α=5*10^-3radians, =>D_ob=200nm

The Depth of focus, D_im is the extent of the region around the image plane in which the image will appear to be sharp. This depends on magnification, M_T. D_im=(d_ob/a_ob)M_T²

Both depth of field and depth of focus are strongly dependent on changes in aperture (hence the semi angle α) and working distance (d_ob).