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Resolution in transmission electron microscopy (TEM) now is limited by the properties of specimens, rather than by those of instrumentation. The long-standing difficulties in obtaining truly high-resolution structure from biological macromolecules with TEM demand the development, testing, and application of new ideas and unconventional approaches. This review concisely describes some new concepts and innovative methodologies for TEM that deal with unsolved problems in the preparation and preservation of macromolecular specimens. The selected topics include use of better support films, a more protective multi-component matrix surrounding specimens for cryo-TEM and negative staining, and, several quite different changes in microscopy and micrography that should decrease the effects of electron radiation damage; all these practical approaches are non-traditional, but have promise to advance resolution for specimens of biological macromolecules beyond its present level of 3-10 Å (0.3-1.0 nm). The result of achieving truly high resolution will be a fulfillment of the still unrealized potential of transmission electron microscopy for directly revealing the structure of biological macromolecules down to the atomic level.

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Irradiation of an amorphous layer of dried sodium phosphate buffer (pH = 7.0) by transmission electron microscopy (100-120 kV) causes rapid formation of numerous small spherical bubbles [10-100 A (= 1-10 nm)] containing an unknown gas. Bubbling is detected even with the first low-dose exposure. In a thin layer (ca. 100-150 A), bubbling typically goes through nucleation, growth, possible fusion, and end-state, after which further changes are not apparent; co-irradiated adjacent areas having a slightly smaller thickness never develop bubbles. In moderately thicker regions (ca. over 200 A), there is no end-state. Instead, a complex sequence of microstructural changes is elicited during continued intermittent high-dose irradiation: nucleation, growth, early simple fusions, a second round of extensive multiple fusions, general reduction of matrix thickness (producing flattening and expansion of larger bubbles, occasional bubble fission, and formation of very large irregularly-shaped bubbles by a third round of compound fusion events), and slow shrinkage of all bubbles. The ongoing lighter appearance of bubble lumens, maintenance of their rounded shape, and extensive changes in size and form indicate that gas content continues throughout their surprisingly long lifetime; the thin dense boundary layer surrounding all bubbles is proposed to be the main mechanism for their long lifetime.

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All common negative stains are salts of heavy metals. To remedy several technical defects inherent in the use of heavy metal compounds, this study investigates whether salts of the light metals sodium, magnesium, and aluminum can function as negative stains. Screening criteria require aqueous solubility at pH 7.0, formation of a smooth amorphous layer upon drying, and transmission electron microscope imaging of the 87-A (8.7-nm) lattice periodicity in thin catalase crystals. Six of 23 salts evaluated pass all three screens; detection of the protein shell in ferritin macromolecules indicates that light metal salts also provide negative staining of single particle specimens. Appositional contrast is less than that given by heavy metal negative stains; image density can be raised by increasing electron phase contrast and by selecting salts with phosphate or sulfate anions, thereby adding strong scattering from P or S atoms. Low-dose electron diffraction of catalase crystals negatively stained with 200 mM magnesium sulfate shows Bragg spots extending out to 4.4 A. Future experimental use of sodium phosphate buffer and magnesium sulfate for negative staining is anticipated, particularly in designing new cocktail (multicomponent) negative stains able to support and protect protein structure to higher resolution levels than are currently achieved.

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Recent research progress using X-ray cryo-crystallography with the photon beams from third-generation synchrotron sources has resulted in recognition that this intense radiation commonly damages protein samples even when they are held at 100 K. Other structural biologists examining thin protein crystals or single particle specimens encounter similar radiation damage problems during electron diffraction and imaging, but have developed some effective countermeasures. The aim of this concise review is to examine whether analogous approaches can be utilized to alleviate the X-ray radiation damage problem in synchrotron macromolecular crystallography. The critical discussion of this question is preceded by presentation of background material on modern technical procedures with electron beam instruments using 300-400 kV accelerating voltage, low-dose exposures for data recording, and protection of protein specimens by cryogenic cooling; these practical approaches to dealing with electron radiation damage currently permit best resolution levels of 6 A (0.6 nm) for single particle specimens, and of 1.9 A for two-dimensional membrane protein crystals. Final determination of the potential effectiveness and practical value of using such new or unconventional ideas will necessitate showing, by experimental testing, that these produce significantly improved protection of three-dimensional protein crystals during synchrotron X-ray diffraction.

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We tested the hypothesis that chronically ischemic (IS) myocardium induces autophagy, a cellular degradation process responsible for the turnover of unnecessary or dysfunctional organelles and cytoplasmic proteins, which could protect against the consequences of further ischemia. Chronically instrumented pigs were studied with repetitive myocardial ischemia produced by one, three, or six episodes of 90 min of coronary stenosis (30% reduction in baseline coronary flow followed by reperfusion every 12 h) with the non-IS region as control. In this model, wall thickening in the IS region was chronically depressed by approximately 37%. Using a nonbiased proteomic approach combining 2D gel electrophoresis with in-gel proteolysis, peptide mapping by MS, and sequence database searches for protein identification, we demonstrated increased expression of cathepsin D, a protein known to mediate autophagy. Additional autophagic proteins, cathepsin B, heat shock cognate protein Hsc73 (a key protein marker for chaperone-mediated autophagy), beclin 1 (a mammalian autophagy gene), and the processed form of microtubule-associated protein 1 light chain 3 (a marker for autophagosomes), were also increased. These changes, not evident after one episode, began to appear after two or three episodes and were most marked after six episodes of ischemia, when EM demonstrated autophagic vacuoles in chronically IS myocytes. Conversely, apoptosis, which was most marked after three episodes, decreased strikingly after six episodes, when autophagy had increased. Immunohistochemistry staining for cathepsin B was more intense in areas where apoptosis was absent. Thus, autophagy, triggered by ischemia, could be a homeostatic mechanism, by which apoptosis is inhibited and the deleterious effects of chronic ischemia are limited.

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A new assay using low-dose electron diffraction to measure the protection of protein structure against damage from drying is described. When thin single crystals of catalase are dried within water alone, low-dose electron diffraction yields no Bragg spots. Drying within an experimental aqueous solution that permits detection of diffraction spots thereby indicates a positive result, and the extent of these Bragg reflections into the high angle range gives a quantitative measure of the degree of protection. Bragg spots out to 3.73.9 are recorded for drying within 100 mM solutions of the known structure-preserving sugars, sucrose, tannin, and trehalose. The ability of trehalose to maintain native protein structure during drying starts between 10 and 25 mM, and changes only slightly at concentrations above this threshold; with drying in 150-mM trehalose, catalase crystals yield diffraction spots out to 3.7. Drying within the organic nonsugar polymer polyvinylpyrrolidone gives Bragg spots to 4.0. This new assay should be useful to measure the unexamined structure-preserving capabilities of modified sugars, other nonsugars, and mixtures to identify which protective matrix maintains native protein structure to the greatest extent during drying; electron crystallography using that optimal matrix should yield protein structure at improved levels of high resolution.

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