• Theory
• Nomenclature
• Microscopes
• X-ray Instrumentation
• Pros and Cons
• Biological Applications
• Problems
• Solutions
• Considerations for Microscope Parameters
• Choice of Microscope Parameters

Theory

Electron Beam/Specimen Interactions

Solid specimens subjected to electron beam excitation in an electron microscope exhibit complex interactions with primary beam electrons. These interactions result in a variety of signals that may be detected in the microscope. To analyze a specimen visually, one might choose to view an image of the specimen by collecting and displaying SE’s, BSE’s or transmitted electrons. To determine information about composition, one might choose to record x-ray or auger electrons.

1. Secondary Electrons

Secondary electrons are specimen electrons ejected by interactions with the beam electrons. They are used for imaging because of their high spatial resolution and topographical sensitivity. They carry very little information about elemental composition. They typically have energies less than 50eV.

2. Backscattered Electrons

Backscattered electrons (BSE’s) are beam electrons that interact with the nucleus of a sample atom and are elastically scattered with little loss of energy. BSE’s come from greater depths within the specimen so they have lower spatial resolution. They provide elemental information because the number of BSE’s produced is directly proportional to the atomic number of the elements within the specimen.

3. X-ray Continuum (Bremsstrahlung)

The beam electron may be scattered inelastically by the coulomb field of an atomic nucleus, thus giving up some of its energy. This energy is emitted in the form of x-ray radiation called bremsstrahlung (German for “braking radiation”). The beam electrons can give up any amount of its energy, so the energy distribution of the emitted x-rays is continuous up to the beam energy. This component of the x-ray signal is called continuum.

4. Characteristic X-rays

Characteristic x-rays are produced by the primary electron beam displacement of an electron in the valence shell of the specimen. As the electron is displaced, an electron from a higher valence shell must fill its orbit. The result is a small amount of energy loss in the form of an x-ray photon. The amount of energy lost is unique to the atom from which it is emitted. X-rays are produced deep within the specimen so they have very poor spatial resolution.

5. Auger Electrons

Sometimes a characteristic x-ray is produced and then reabsorbed within the same atom, ejecting a lower energy electron. This is an Auger electron. The Auger electron possesses an energy exactly equal to the difference between the energy of the original characteristic x-ray and the binding energy of the ejected electron. They carry specific chemical information about the atom from which they originated. They have very low energy (a few eV’s) and therefore carry information about the surface of the specimen (the first few atomic layers).

Nomenclature

The lines are usually named according to the shell in which the initial vacancy occurs and the shell from which an electron drops to fill that vacancy.

Example: If the initial vacancy occurs in the K shell and the vacancy-filling electron drops from the adjacent shell (the L shell), a Ka x-ray is emitted. We will be most concerned with K-, L- and M-series x-rays, so they are referred to as KLM lines.

Microscopes

1. Scanning Electron Microscope

a. the electronics in the SEM make a small probe, but stability is usually poor
b. the SEM is suitable for a variety of attachments
c. sample prep is minimal
d. 1-30 kV accelerating voltages are available
e. the resolution is approximately 20-100Å

2. Microprobe

a. it may or may not have high spatial imaging
b. its column is configured to provide a high intensity, stable probe
c. it is usually equipped with several wavelength spectrometer.
d. it is intended for very accurate quantitative analysis
e. accelerating voltages of 5-30 kV are typical.

3. Scanning Transmission Electron Microscope

a. high resolution is possible
b. phase identification using electron diffraction imaging
c. EDS analysis of very small areas
d. Voltages of 75-300 kV are possible
e. specimen prep is very difficult due to small specimen size
f. the operator has access to all SEM signals as well

X-ray Instrumentation

1. Wavelength Dispersive Spectrometry (WDS)

A small fraction of the x-rays leaving the specimen strike a crystal and are reflected onto the detector. The crystal will reflect only a very narrow range of wavelengths of x-rays so Bragg’s law can be applied to determine elemental content.

Since a particular crystal will diffract only a very narrow wavelength of x-rays, one crystal is useful for detecting only a few atomic numbers. Gypsum crystals will detect atomic numbered elements 11 to 14 and sodium crystals will pick up atomic numbered elements 16 to 37.

The focused x-rays are detected when they pass through a thin plastic window and enter a gas-filled cylinder containing a collector wire kept at a high positive voltage. The x-rays cause an ionization of the argon-methane gas and generate a flow of electrons to the wire. This current is measured and quantified over time. The amount of current is directly related to the energy of the original x-ray, so it is possible to determine the element from which the x-ray was emitted.

2. Energy Dispersive Spectroscopy (EDS)

A. The Detector

In EDS the detector is usually made of a single silicon crystal, 2-3 mm thick. The crystal is chosen for its purity and shape, but even so, conductivity varies greatly and it is necessary to drift lithium into the crystal to fill any vacant lattice spaces.

The atoms in this crystal are covalently bonded. Each x-ray produces a photoelectron in this valence band. A conduction band creating an electron-hole pair dissipates the energy. Each electron-hole pair produces 3.8-3.9 eV. Approximately 1,000 hole pairs are created per x-ray. The current generated by the conducted charge is proportional to the energy of the absorbed x-ray. There can be residual conductivity due to the random excitations of electrons and cryogenic temperatures help to control this.

Because of their extreme sensitivity, detectors must be protected from the interior of the column. Most standard detectors use a beryllium window to achieve this end. Lower energy x-rays (anything lighter than sodium) will not pass through this window.

Next to the crystal is a thin gold layer that aids in conduction. After this comes a space termed the dead layer in which holes appear due to incomplete Li-drifting. Some absorption occurs here, causing a “tail” on spectra peaks.

Many things can happen as the x-ray enters the detector. It may not even reach the crystal because the window, the gold layer or the dead layer may absorb it. It may be too energetic and pass through the crystal with no interaction. Or the interaction between the x-ray and the silicon atoms in the crystal may be such that the silicon atom produces an x-ray of its own which escapes from the detector and is ejected back into the column. This last situation results in the formation of an escape peak. The formation of an escape peak depends upon the angle of incidence into the detector and the energy of the parent peak. The result of the escape is an artifactual small peak 1.47 keV below the parent peak.

B. Preamplifier or Field Effect Transistor

The signal is shuttled here directly after it is collected in the crystal. The bias voltage is subtracted from the pulses. A pulsed optical feedback is used to reduce electronic noise and the result is a “dead time” in which the system cannot take in any data as it processes a signal.

C. Pulse Processor/Amplifier

Pulses from the preamp are shaped and amplified for analog to digital conversion. These pulses must be separated because the height of the pulse carries information about the energy of the x-ray. The pulse pileup rejecter may reject pulses that come into the system together.

D. Analog to Digital Converter and Multichannel Analyzer.

The pulses are converted from analog to digital signals and then sorted into channels according to height. The multichannel analyzer assembles the spectra and lets you control it through the keyboard.

Pros and Cons

WDS Advantages

1. high resolving power, separation of x-ray lines
2. quantification – good for even trace elements
3. good peak to background ratio
4. light elements are easily detectable
5. no liquid nitrogen required

WDS Disadvantages

1. mechanical parts move, prone to error unless properly adjusted and calibrate.
2. only one element analyzed at a time
3. possible interference from high order diffraction lines
4. peak and background must be measured separately

EDS Advantages

1. no x-ray focusing required.
2. high sensitivity – high solid angle
3. no diffraction interference
4. simple mechanical design, no moving parts, easily added to microscope
5. output compatible with computer
6. high count rate delectability, allows smaller probe diameter (less specimen damage
7. whole elemental spectrum displayed, rapid qualitative analysis

EDS Disadvantages

1. constant supply of LN2 (fill 3X weekly)
2. beam must be stable
3. inferior energy resolution compared to crystal spectrometers
4. high background noise due to nondiscriminating x-ray source
5. quantification – poor accuracy at very low concentrations

Biological Applications

X-ray microanalysis can be used to examine biological samples. Applications such as:

1. analyzing cellular electrolytes (diffusible ions)
2. identifications of endogenous and exogenous substances in cells and tissues
3. cell surface labeling
4. examining colorless histochemical reaction products
5. analyzing intermediate stages of histochemical reactions
6. examining ultrastructural histochemistry
7. analyzing microdroplets of body fluids

Biological x-ray analysis is usually performed in a TEM on thin film specimens of 50-100 nm in thickness. There is a tradeoff between morphology and the maintenance and location of ions in their original sites.

Problems

1. K Line Overlap

Osmium, uranium and lead L and M lines can overlap the K lines of the elements of interest. This increases background and decreases the sensitivity of the technique, especially when looking for trace elements.

2. Thickness

Thin sections have a low mass, which will yield a lower count rate.

3. Mass Loss

Low atomic number elements in biological specimens lose mass very easily.

4. Ion Mobility

Normal biological specimen preparation protocols disperse or leach out ions from their original sites. This happens most dramatically in the dehydration steps. Elements most affected are sodium, phosphorus, potassium, sulfur and chlorine.

Solutions

The solutions to the problems of performing x-ray microanalysis on biological tissue lies in the specimen preparation techniques that are used.

1. Freeze Drying

Fresh tissue is placed immediately into a cryogen such as: propane (most common), Freon, butane, ethane or isopentane. It is then transferred to liquid nitrogen and placed in a freeze drying unit for about 2 days at -50°C under a vacuum of 10-3 – 10-5 torr. The vacuum is then slowly lowered and the temperature slowly raised to room temperature. The tissue can then be infiltrated with resin if viewing in the TEM or mounted on a carbon stub for the SEM. The tissue is not processed with chemicals and as a result will have low contrast in the TEM. The tissue can be post stained with alcoholic uranyl acetate if desired. When sectioning, ethylene glycol is used as a boat fluid because the components of interest are water soluble.

2. Freeze Substitution

The fresh tissue is frozen in the same manner as for freeze drying. The tissue is then immersed in anhydrous ether or other intermediate solvents for several days at -90°C. The temperature is slowly raised while at the same time the tissue is infiltrated with resin.

3. Cryoultramicrotomy

A small piece of fresh tissue is placed on the end of a metal pin and quickly frozen in liquid nitrogen. Thin sections are cut dry on a glass knife, mounted on formvar coated grids and freeze dried.

4. Air Drying

This technique is used on small cells such as sperm, bacteria and fungi.

5. Cytochemical Techniques

Chemicals such as potassium pyroantimonate (KSb (OH)6) are combined with an osmium tetroxide fixative and perfused into the tissue. No aldehydes are used. The pyroantimonate reacts with specific cations (most typically calcium and zinc) and antimony precipitates at these sites. The cations are removed in the following processing steps, but the antimony remains. This is not a quantitative technique and the reaction is strongly dependent on pH, PO4 ions in the buffer, osmolarity, type of fixation, washing procedures, staining procedures, etc.

Considerations for Microscope Parameters

1. Mass Loss
Mass loss is the thinning of the specimen by the beam (irradiation damage).

2. Charging
A charge build up will cause ions to migrate from their original sites.

3. Thermal Build Up
Heat builds up in the specimen due to inelastic scattering events.

4. Contamination
The electron beam polymerizes hydrocarbon molecules, which are deposited on the specimen.

Choice of Microscope Parameters

1. Accelerating Voltage
Higher accelerating voltages are better for x-ray analysis. Using a lower accelerating voltage causes more charging, more thermal build-up and more contamination. Higher accelerating voltages provide better resolution even though there will be more mass loss and lower contrast. Imaging near a grid bar can help dissipate charging problems, but will increase x-rays released by the grid material.

2. Tilt
Tilting the specimen will increase contrast and help overcome mass loss by providing a longer path for the electron beam through the specimen.

3. Beam Current
An adequate current density on the specimen is needed to get sufficient count rates but the lowest possible beam current should be used to prevent beam damage to the specimen. Beam current is controlled by adjusting the spot size, brightness, condenser aperature and bias. (The bias adjustment should be a last case scenario).

4. Use of Cold Stage
Controls thermal build-up in the specimen to reduce mass loss and ion migration.