Standard Procedure

Freeze-dried specimens for mass measurements in the STEM are prepared by the wet film method. 2.3 mm titanium grids are coated with a thick holey carbon film. The open areas are 5-10 µm in diameter which will support the thin (2-3 nm thick) carbon film substrate. The thin carbon film is prepared by ultra-high vacuum evaporation onto a freshly cleaved crystal of rock salt. The thin carbon film is then floated on a dish of clean water. Grids covered with holey film, assembled in rings and caps for handling in the microscope, are placed face down on the floating thin carbon film. The grids are picked up from above one at a time such that the thin carbon film retains a droplet of water. This water is exchanged by washing and wicking, either with water or with injection buffer for the specimen. Two µl of tobacco mosaic virus (TMV) solution, at a concentration of 100 µg/ml, are injected into the drop and allowed to stand for 1 min. The TMV is both a qualitative and a quantitative internal control for all specimens. After four washings, 2 µl of the specimen (its concentration is determined primarily by its size) is injected into the drop and allowed to stand for 1 min. Ten more washings are performed. Half of these can be with injection buffer, but the final few must be with a volatile buffer such as 20 mM ammonium acetate or water. After the final wash, the grid is pinched between two pieces of filter paper (leaving a retained layer of solution less than 1 µm thick) and immediately plunged into liquid nitrogen slush. Six grids are transferred under liquid nitrogen to an ion-pumped freeze dryer with an efficient cold trap, freeze dried overnight by gradually warming to -80°C and transferred under vacuum to the STEM.

Examples

Examples of good STEM specimens are shown in this annotated gallery of earthworm hemoglobin, GroEL, and bacteriophage T4.

Limitations

Although the STEM is an extraodinary machine, there are limitations to the kinds of specimens that it can image with good results. Because it is designed for applications in 'molecular biology" the molecular weight of the specimens should be in the range of 40 kilo daltons to 800 million daltons. Specimen purity, concentration and buffering, may also make a sample unsuitable for use in the STEM. Here, the following limitations are discussed: Size Purity Concentration Buffers and Salts Other Problems

Size

The STEM is capable of 0.25 nm resolution, but freeze-dried biological specimens usually have approximately 2 nm resolution. The STEM can provide valuable information on individual particles in the molecular weight range from 40 kD to several hundred MD. In general, the larger the particle, the more accurate the mass measurement can be. Success in looking at small proteins (less than 100 kD) will depend on their conformation. If parts of them are very extended, they may suffer more mass loss and the mass measurements will be low. Proteins of molecular weight less than 40 kD are likely to be too small to yield good mass measurements unless they are unusually dense and compact. tRNA can be seen in the STEM, but it is very compact and nucleic acids are more radiation resistant than proteins. Double-stranded DNA is also visible in the STEM for the same reasons. At the other extreme, while viruses (and sub-viral particles of large viruses) such as herpes simplex have given excellent results, the STEM is probably not the best choice for looking at very large structures such as bacteria, cells, or organelles. They are essentially opaque, i.e. saturated, in the STEM and so yield little information. For filaments or two- dimensional arrays, the actual size is not a problem. It is only the mass density that is of concern, so they are fine if they are spread out enough and not too dense.

Purity

The physical purity of the specimens is very important for STEM preparations. Essentially everything is visible in the STEM, and impurities may well not wash off the grid. Most biologists have some assay for the biological activity of their specimens. However, many preparations contain biologically inert but physically visible material that may make it difficult to obtain good STEM data. Particulates from columns or gels are a problem. Additives for activity such as BSA, PEG, or trypsin are a problem. Precipitations can concentrate the specimen, but also enrich contaminants. If a multi- subunit enzyme has 90% activity, but the remaining 10% has fallen apart, that can be very visible in the STEM. Frequently, a final purification over an appropriate sizing column (such as an A5M) where the intact specimen comes off in the void volume gives adequate purity. At times, the particles of interest can be distinguished from contaminants. However, it is clearly preferable to eliminate contaminants when possible.

Concen- tration

The necessary concentration of a given specimen to give a good distribution of particles on a grid is somewhat empirical. However, for data taking it is very important. If the particles are too close together, they cannot be measured individually, but if they are too sparse, it is very difficult to obtain both particles and the control TMV in an image. Also, searching for rare events in the STEM is slow. As a guide, large particles such as viruses need to be applied to the grid at a relatively high concentration such as 200-300 µg/ml. For smaller proteins, a concentration of 50 µg/ml might be adequate. For any given specimen, it is not known how well it will adhere to the carbon grid. For a new specimen, a series of dilutions is usually made. A highly concentrated specimen can always be diluted, but there is little that can be done with a specimen that is too dilute.

Buffers and Salts

The ideal specimen for the STEM is one that can be applied to the grid in water or 20 mM ammonium acetate which volatilizes during freeze-drying. However, many biological specimens have additional requirements. Most salts and buffers can be tolerated in the injection buffer for the specimen. The assumption is that if the specimen is absorbed to the carbon grid in a few washes of its own buffer, it will be able to tolerate some final washes with a volatile solution such as water or ammonium acetate. For example, low levels (1-2 mM) of Mg++, Ca++, ATP (or non-hydrolyzable analogues) are often needed for biological activity and usually will wash away. Low concentrations of glycerol or sucrose also usually wash off, as will DTT or mercaptoethanol. NaCl at levels as high as 100 mM usually washes off with ammonium acetate, but KCl at more than a few mM occasionally causes problems. Phosphate buffers should be avoided if possible since they usually leave a background high in "spots", which is a real problem for interpreting site-specific cluster labeling experiments. Tris buffers often, but not always, have a bad background, whereas Hepes, Mops, and Pipes are usually better. For a new specimen in an unusual buffer, a grid of TMV by itself washed with the buffer is made as a control. Some specimens, such as membrane proteins, have to be solubilized in a detergent and these are always difficult to work with although some successful results have been obtained.

Other Problems

Some specimens which have met all the above criteria simply do not work out. Harm can come to the specimen during its attachment to the carbon film (a part can attach firmly while the rest of the molecule flails around), during the freeze-drying process, and during the data-taking process (from radiation damage in the electron beam). An occasional specimen will not attach to the carbon film. Double-stranded DNA, for example, does not attach reliably even at high concentrations. However, with polylysine pre-treatment of the carbon grid, DNA will bind at concentrations of less than 1 µg/ml. Polylysine pre-treatment will also help some other types of specimens. All proteins denature to some extent at the air-water interface of the drop on the grid. Usually, washing and wicking removes this denatured film. However, some proteins continue to denature at the interface until there is nothing left on the grid. Sometimes washing with low concentrations of organic solvents such as ethanol or acetonitrile will help. Some specimens which fall apart on the carbon film benefit from a brief fixation with glutaraldehyde just prior to injection. If this does help, the increase in mass is less than 10%. Occasionally specimens will be all right on some parts of the grid but not in other areas. The STEM staff is continually working on specimen preparation, especially with "difficult" specimens, to try to solve these problems.

Updated 25 Oct 2000	Security Notice   Webteam   Site Map   STEM   Biology   BNL