Question

1a. How is biological sample prepared for the insertion into the transmission electron microscope for single particle averaging? b. The single image of a molecule in cryo-EM is very noisy. What is done to improve the clarity of the image? What does that mean in terms of statistics?

Answer 1

1a. Sample preparation in TEM (transmission electron microscope) can be a complex procedure. TEM specimens should be less than 100 nanometers thick for a conventional TEM. Unlike neutron or X-Ray radiation the electrons in the beam interact readily with the sample, an effect that increases roughly with atomic number squared (Z²). High quality samples will have a thickness that is comparable to the mean free path of the electrons that travel through the samples, which may be only a few tens of nanometers. Preparation of TEM specimens is specific to the material under analysis and the type of information to be obtained from the specimen.

Materials that have dimensions small enough to be electron transparent, such as powdered substances, small organisms, viruses, or nanotubes, can be quickly prepared by the deposition of a dilute sample containing the specimen onto films on support grids. Biological specimens may be embedded in resin to withstand the high vacuum in the sample chamber and to enable cutting tissue into electron transparent thin sections. The biological sample can be stained using either a negative staining material such as uranyl acetatefor bacteria and viruses, or, in the case of embedded sections, the specimen may be stained with heavy metals, including osmium tetroxide. Alternately samples may be held at liquid nitrogen temperatures after embedding in vitreous ice. In material science and metallurgy the specimens can usually withstand the high vacuum, but still must be prepared as a thin foil, or etched so some portion of the specimen is thin enough for the beam to penetrate. Constraints on the thickness of the material may be limited by the scattering cross-section of the atoms from which the material is comprised.

Tissue sectioning

Biological tissue is often embedded in a resin block then thinned to less than 100nm on an ultramicrotome. The resin block is fractured as it passes over a glass or diamond knife edge. This method is used to obtain thin, minimally deformed samples that allow for the observation of tissue ultrastructure. Inorganic samples, such as aluminium, may also be embedded in resins and ultrathin sectioned in this way, using either coated glass, sapphire or larger angle diamond knives. To prevent charge build-up at the sample surface when viewing in the TEM, tissue samples need to be coated with a thin layer of conducting material, such as carbon.

Sample staining

TEM samples of biological tissues need high atomic number stains to enhance contrast. The stain absorbs the beam electrons or scatters part of the electron beam which otherwise is projected onto the imaging system. Compounds of heavy metals such as osmium, lead, uranium or gold (in immunogold labelling) may be used prior to TEM observation to selectively deposit electron dense atoms in or on the sample in desired cellular or protein region. This process requires an understanding of how heavy metals bind to specific biological tissues and cellular structures.

Mechanical milling

Mechanical polishing is also used to prepare samples for imaging on the TEM. Polishing needs to be done to a high quality, to ensure constant sample thickness across the region of interest. A diamond, or cubic boron nitride polishing compound may be used in the final stages of polishing to remove any scratches that may cause contrast fluctuations due to varying sample thickness. Even after careful mechanical milling, additional fine methods such as ion etching may be required to perform final stage thinning.

Chemical etching

Certain samples may be prepared by chemical etching, particularly metallic specimens. These samples are thinned using a chemical etchant, such as an acid, to prepare the sample for TEM observation. Devices to control the thinning process may allow the operator to control either the voltage or current passing through the specimen, and may include systems to detect when the sample has been thinned to a sufficient level of optical transparency.

Ion etching

Ion etching is a sputtering process that can remove very fine quantities of material. This is used to perform a finishing polish of specimens polished by other means. Ion etching uses an inert gas passed through an electric field to generate a plasma stream that is directed to the sample surface. Acceleration energies for gases such as argon are typically a few kilovolts. The sample may be rotated to promote even polishing of the sample surface. The sputtering rate of such methods is on the order of tens of micrometers per hour, limiting the method to only extremely fine polishing.

Ion etching by argon gas has been recently shown to be able to file down MTJ stack structures to a specific layer which has then been atomically resolved. The TEM images taken in plan view rather than cross-section reveal that the MgO layer within MTJs contains a large number of grain boundaries that may be diminishing the properties of devices.

Ion milling

More recently focused ion beam methods have been used to prepare samples. FIB is a relatively new technique to prepare thin samples for TEM examination from larger specimens. Because FIB can be used to micro-machine samples very precisely, it is possible to mill very thin membranes from a specific area of interest in a sample, such as a semiconductor or metal. Unlike inert gas ion sputtering, FIB makes use of significantly more energetic gallium ions and may alter the composition or structure of the material through gallium implantation.

Replication

Samples may also be replicated using cellulose acetate film, the film subsequently coated with a heavy metal such as platinum, the original film dissolved away, and the replica imaged on the TEM. Variations of the replica technique are used for both materials and biological samples. In materials science a common use is for examining the fresh fracture surface of metal alloys.

b. In single-particle cryo-EM, a solution of biologic molecules is frozen and imaged with a low electron dose (5–15 electrons/Å2). This reduces radiation damage, preserves the atomic structures, but compromises the signal-to-noise ratio (SNR) of the individual two-dimensional (2D) projection images. Production of a good initial three-dimensional (3D) model ab initio from noisy images is difficult, and therefore, many high-quality cryo-EM maps are generated by alignment to initial 3D models obtained by crystallography or small-angle X-ray scattering.

New feasible orientations are accepted with a probability determined by the uniform distribution over all orientations that improve the correlation. If no orientations improve the correlation, the r highest scoring orientations are included in the weighting scheme. The indeterminism of the correlation function defines a probabilistic rule for moving from the current alignment to the new neighbor. Alignments that are more robust toward uncertainties in the reconstruction are found by modeling this uncertainty in the set of “acceptable” alignments. Probabilistic orientation assignment accounts for the large errors in the goal function landscape introduced by noise.