Cryo-EM--the first thirty years.

Thirty years ago, in December 1981, The Journal of Microscopy published a very short paper entitled 'Vitrification of pure water for electron microscopy'. It turned out to be important for the development of cryo-electron microscopy and it contributed to reverse, from foe to friend, the status of water in electron microscopists' minds. This change has brought obvious gains. The future will tell how many more are still to come.

Comparison of different methods for thin section EM analysis of Mycobacterium smegmatis.

Bacteria are generally difficult specimens to prepare for conventional resin section electron microscopy and mycobacteria, with their thick and complex cell envelope layers being especially prone to artefacts. Here we made a systematic comparison of different methods for preparing Mycobacterium smegmatis for thin section electron microscopy analysis. These methods were: (1) conventional preparation by fixatives and epoxy resins at ambient temperature. (2) Tokuyasu cryo-section of chemically fixed bacteria. (3) rapid freezing followed by freeze substitution and embedding in epoxy resin at room temperature or (4) combined with Lowicryl HM20 embedding and ultraviolet (UV) polymerization at low temperature and (5) CEMOVIS, or cryo electron microscopy of vitreous sections. The best preservation of bacteria was obtained with the cryo electron microscopy of vitreous sections method, as expected, especially with respect to the preservation of the cell envelope and lipid bodies. By comparison with cryo electron microscopy of vitreous sections both the conventional and Tokuyasu methods produced different, undesirable artefacts. The two different types of freeze-substitution protocols showed variable preservation of the cell envelope but gave acceptable preservation of the cytoplasm, but not lipid bodies, and bacterial DNA. In conclusion although cryo electron microscopy of vitreous sections must be considered the 'gold standard' among sectioning methods for electron microscopy, because it avoids solvents and stains, the use of optimally prepared freeze substitution also offers some advantages for ultrastructural analysis of bacteria.

Twisting in a crowd.

Given our current understanding of nuclear structure, it is difficult to imagine how the nucleus performs its varied functions and controls the traffic of its many components. For example, how can densely packed chromatin be transcribed without the helical nature of the DNA resulting in entangled DNA and RNA and a stalled RNA polymerase? Here, Jacques Dubochet discusses a model of transcription in which DNA rotates around its axis, rather than RNA polymerase rotating around the DNA. Furthermore, he suggests that a view of chromatin as 'liquid' may help in understanding a wide range of nuclear functions.

Chromatin conformation and salt-induced compaction: three-dimensional structural information from cryoelectron microscopy.

Cryoelectron microscopy has been used to examine the three-dimensional (3-D) conformation of small oligonucleosomes from chicken erythrocyte nuclei after vitrification in solutions of differing ionic strength. From tilt pairs of micrographs, the 3-D location and orientation of the nucleosomal disks, and the paths of segments of exposed linker can be obtained. In "low-salt" conditions (5 mM NaCl, 1 mM EDTA, pH 7.5), the average trinucleosome assumes the shape of an equilateral triangle, with nucleosomes at the vertices, and a length of exposed linker DNA between consecutive nucleosomes equivalent to approximately 46 bp. The two linker DNA segments converge at the central nucleosome. Removal of histones H1 and H5 results in a much more variable trinucleosome morphology, and the two linker DNA segments usually join the central nucleosome at different locations. Trinucleosomes vitrified in 20 mM NaCl, 1 mM EDTA, (the salt concentration producing the maximal increase in sedimentation), reveal that compaction occurs by a reduction in the included angle made by the linker DNA segments at the central nucleosome, and does not involve a reduction in the distance between consecutive nucleosomes. Frequently, there is also a change in morphology at the linker entry-exit site. At 40 mM NaCl, there is no further change in trinucleosome morphology, but polynucleosomes are appreciably more compact. Nevertheless, the 3-D zig-zag conformation observed in polynucleosomes at low salt is retained at 40 mM NaCl, and individual nucleosome disks remain separated from each other. There is no evidence for the formation of solenoidal arrangements within polynucleosomes. Comparison of the solution conformation of individual oligonucleosomes with data from physical measurements on bulk chromatin samples suggests that the latter should be reinterpreted. The new data support the concept of an irregular zig-zag chromatin conformation in solution over a range of ionic strengths, in agreement with other in situ (McDowall, A.W., J.M. Smith, and J. Dubochet. 1986, EMBO (Eur. Mol. Biol. Organ.) J.5: 1395-1402; Horowitz, R.A., D.A. Agard, J.W. Sedat, and C.L. Woodcock, 1994. J. Cell Biol. 125:1-10), and in vitro conclusions (van Holde, K., and J. Zlatanova. 1995. J. Biol. Chem. 270:8373-8376). Cryoelectron microscopy also provides a way to determine the 3-D conformation of naturally occurring chromatins in which precise nucleosome positioning plays a role in transcriptional regulation.

Nucleosome arcs and helices.

Crystals and other regular arrangements of nucleosome cores have been obtained and analyzed in the electron microscope. Two types of regular structures have been studied in detail, the nucleosome arcs and cylinders. The latter are composed of concentric cylindrical layers of intertwined right-handed helices of nucleosome cores. These studies lead to the following conclusions and concepts. The overall structure of the nucleosome core is a short, wedge-shaped cylinder measuring about 110 by 110 by 60 angstroms. Nucleosome cores interact primarily between top and bottom planes. Nucleosome cores exhibit large conformational variability. A pivot allowing two degrees of rotational freedom is postulated in the region of the 70th base pair to account for this property of the nucleosome.

Compression and crevasses in vitreous sections under different cutting conditions.

Compression and crevasses are common cutting artefacts in cryo-ultramicrotomy of vitreous sections. They can be reduced or suppressed under optimal cutting conditions. In the present study, compression and thickness were measured for different cutting speeds and knife angles. It was found that compression decreased with feed and that crevasses appeared only above a certain thickness. The optimal feed for vitreous sections was between 50 and 80 nm. The thickness, calculated by two independent methods, was quantitatively related to feed and compression.

Are the light-harvesting I complexes from Rhodospirillum rubrum arranged around the reaction centre in a square geometry?

The basic photosynthetic unit containing the reaction centre and the light-harvesting I complex (RC-LHI) of the purple non-sulphur bacterium Rhodospirillum rubrum was purified and reconstituted into two-dimensional (2D) membrane crystals. Transmission electron microscopy using conventional techniques and cryoelectron microscopy of the purified single particles and of 2D crystals yielded a projection of the RC-LHI complex at a resolution of at least 1.6 nm. In this projection the LHI ring appears to have a square symmetry and packs in a square crystal lattice. The square geometry of the LHI ring was observed also in images of single isolated particles of the RC-LHI complex. However, although the LHI units are packed identically within the crystal lattice, a new rotational analysis developed here showed that the reaction centres take up one of four possible orientations within the ring. This fourfold disorder supports our interpretation of a square ring symmetry and suggests that a hitherto undetected component may be present within the photosynthetic unit.

Cryo-electron microscopy of vitrified insect flight muscle.

Frozen hydrated sections of vitrified unstained insect flight muscle in the rigor and relaxed states were studied by cryo-electron microscopy. High resolution images with good contrast were obtained. As judged from direct images and from the comparison of optical diffractograms with X-ray diffraction patterns obtained from glycerinated muscle fibres, the vitrified specimen is well preserved. Preparation artifacts encountered in conventional plastic sections are avoided. The preservation of the relaxed structure also seems better than in freeze-etched replicas as the 14.5 nm repeat of crossbridges on thick filaments is easily visible.

The twist, writhe and overall shape of supercoiled DNA change during counterion-induced transition from a loosely to a tightly interwound superhelix. Possible implications for DNA structure in vivo.

A cryo-electron microscopy study of supercoiled DNA molecules freely suspended in cryo-vitrified buffer was combined with Monte Carlo simulations and gel electrophoretic analysis to investigate the role of intersegmental electrostatic repulsion in determining the shape of supercoiled DNA molecules. It is demonstrated here that a decrease of DNA-DNA repulsion by increasing concentrations of counterions causes a higher fraction of the linking number deficit to be partitioned into writhe. When counterions reach concentrations likely to be present under in vivo conditions, naturally supercoiled plasmids adopt a tightly interwound conformation. In these tightly supercoiled DNA molecules the opposing segments of interwound superhelix seem to directly contact each other. This form of supercoiling, where two DNA helices interact laterally, may represent an important functional state of DNA. In the particular case of supercoiled minicircles (178 bp) the delta Lk = -2 topoisomers undergo a sharp structural transition from almost planar circles in low salt buffers to strongly writhed "figure-eight" conformations in buffers containing neutralizing concentrations of counterions. Possible implications of this observed structural transition in DNA are discussed.

Opposite effect of counterions on the persistence length of nicked and non-nicked DNA.

Using cryo-electron microscopy we reconstructed the three-dimensional trajectories adopted in cryovitrified solutions by double-stranded DNA molecules in which the backbone of one strand lacked a phosphate at regular intervals of 20 nucleotides. The shape of such nicked DNA molecules was compared with that of DNA molecules with exactly the same sequence but without any single-stranded scissions. Upon changing the salt concentration we observed opposite effects of charge neutralization on nicked and non-nicked DNA. In low salt solutions (10 mM Tris-HCl, 10 mM NaCl) the applied dense nicking caused ca 3.5-fold reduction of the DNA persistence length as compared with non-nicked DNA. Upon increasing the salt concentration (to 150 mM NaCl and 10 mM MgCl2) the persistence length of non-nicked DNA appreciably decreased while that of nicked DNA molecules increased by a factor of 2.

Determination of DNA persistence length by cryo-electron microscopy. Separation of the static and dynamic contributions to the apparent persistence length of DNA.

Axial deflection of DNA molecules in solution results from thermal motion and intrinsic curvature related to the DNA sequence. In order to measure directly the contribution of thermal motion we constructed intrinsically straight DNA molecules and measured their persistence length by cryo-electron microscopy. The persistence length of such intrinsically straight DNA molecules suspended in thin layers of cryo-vitrified solutions is about 80 nm. In order to test our experimental approach, we measured the apparent persistence length of DNA molecules with natural "random" sequences. The result of about 45 nm is consistent with the generally accepted value of the apparent persistence length of natural DNA sequences. By comparing the apparent persistence length to intrinsically straight DNA with that of natural DNA, it is possible to determine both the dynamic and the static contributions to the apparent persistence length.

Sedimentation and electrophoretic migration of DNA knots and catenanes.

Various site-specific recombination enzymes produce different types of knots or catenanes while acting on circular DNA in vitro and in vivo. By analysing the types of knots or links produced, it is possible to reconstruct the order of events during the reaction and to deduce the molecular "architecture" of the complexes that different enzymes form with DNA. Until recently it was necessary to use laborious electron microscopy methods to identify the types of knots or catenanes that migrate in different bands on the agarose gels used to analyse the products of the reaction. We reported recently that electrophoretic migration of different knots and catenanes formed on the same size DNA molecules is simply related to the average crossing number of the ideal representations of the corresponding knots and catenanes. Here we explain this relation by demonstrating that the expected sedimentation coefficient of randomly fluctuating knotted or catenated DNA molecules in solution shows approximately linear correlation with the average crossing number of ideal configurations of the corresponding knots or catenanes.

Unexpected property of trehalose as observed by cryo-electron microscopy.

Trehalose is an agent useful in maintaining the integrity of many biological systems submitted to various stresses. It is also presumed to improve specimen preparation for electron microscopy and to reduce beam damage. Here we study the effect of trehalose on the preparation and observation by cryo-electron microscopy of thin vitrified films of biological suspensions. We observe that trehalose, as compared to sucrose, can indeed reduce electron beam damage to biological particles, as determined from the dose necessary for the onset of bubbling. Surprisingly, we also find that the contrast of biological particles is higher in a vitrified solution of trehalose than in one of sucrose. This effect can be explained if the water evaporation during the specimen preparation is less in the presence of trehalose than with sucrose, but we do not yet understand the underlying reasons since the evaporation properties of both sugars are similar at a macroscopic level. We conclude that trehalose is truly a remarkable substance and that more investigation is needed in order to fully understand its properties, and that the addition of ca. 3-5% trehalose to biological suspensions is a simple and useful method to reduce commonly arising drying artefacts and water evaporation in the thin film vitrification method.

Making science in a fractal landscape.

There were times when any progress in science was associated with a social benefit. Public support of science was growing continuously without many questions being asked. These times have gone and the public asks now what science is good for and scientists are not yet well prepared to reply. A new social contract between scientists and society should be elaborated based on knowledge, tolerance and transparency.

The cell in absence of aggregation artifacts.

Eduard Kellenberger understood that the conventional resin-embedding, he helped to develop (Ryter, A., Kellenberger, E., 1958. L'inclusion au polyester pour l'ultramicrotomie. J. Ultrastruct. Res. , 2, 200-214), was prone to aggregation artifacts (Kellenberger, E., 1987. The response to biological macromolecules and supramolecular structures to the physics of specimen cryo-preparation. In: Steinbrecht, R.A., Zierold, K. (Eds.), Cryo-techniques in Biological Electron Microscopy, Springer, Berlin, pp. 35-63). He was instrumental in developing various methods to overcome this limitation, for instance, by using low temperature-embedding and partially hydrophilic resins (Carlemalm, E., Garavito, R.M., Villiger, W., 1982. Resin development for electron microscopy and an analysis of embedding at low temperature. J. Microstruct., 126, 123-143; Villiger,W., 1993. Low temperature-embedding with Lowicryl resins. In: Robards, A.W., Wilson, A.J. (Eds.), Procedures in electron microscopy, Wiley, Chichester, UK, pp. 16:7.3-16:7.6). In principle, cryo-electron microscopy of vitreous sections is free of any aggregation artifact since the material remains fully hydrated and is free of chemical fixation or staining. The method is technically difficult still, but recent progress has made it amenable to routine practical applications. We compare here electron microscopical aspects of Zea mays meristem cells prepared by: (1) conventional resin-embedding and sectioning; (2) low temperature-embedding and sectioning of freeze substituted samples; and (3) cryo-sections of vitrified samples. The appearance of the extra-cellular space, the cytoplasm and the nucleoplasm are very different in conditions (1) and (3). They appear as compact, irregular and well delineated structures in conventional resin sections, whereas they are more diffuse and homogeneous in the vitreous sections. In the resin sections, the material seems to form a complex matrix, whereas it looks more like a thick soup in the vitreous sample. Low temperature-embedding (condition 2) shows an intermediate appearance. We suggest that regardless of the difference due to staining and different sectioning conditions, the other image differences are the consequence of aggregation artifacts in the resin-embedded specimens.

Cryo-negative staining.

A procedure is presented for the preparation of thin layers of vitrified biological suspensions in the presence of ammonium molybdate, which we term cryo-negative staining. The direct blotting of sample plus stain solution on holey carbon supports produces thin aqueous films across the holes, which are routinely thinner than the aqueous film produced by conventional negative staining on a continuous carbon layer. Because of this, a higher than usual concentration of negative stain (ca. 16% rather than 2%) is required for cryo-negative staining in order to produce an optimal image contrast. The maintenance of the hydrated state, the absence of adsorption to a carbon film and associated sample flattening, together with reduced stain granularity, generates high contrast cryo-images of superior quality to conventional air-dry negative staining. Image features characteristic of unstained vitrified cryo-electron microscopic specimens are present, but with reverse contrast. Examples of cryo-negative staining of several particulate biological samples are shown, including bacteriophage T2, tobacco mosaic virus (TMV), bovine liver catalase crystals, tomato bushy stunt virus (TBSV), turnip yellow mosaic virus (TYMV), keyhole limpet hemocyanin (KLH) types 1 and 2, the 20S proteasome from moss and the E. coli chaperone GroEL. Densitometric quantitation of the mass-density of cryo-negatively stained bacteriophage T2 specimens before and after freeze-drying within the TEM indicates a water content of 30% in the vitreous specimen. Determination of the image resolution from cryo-negatively stained TMV rods and catalase crystals shows the presence of optical diffraction data to ca. 10 A and 11.5 A, respectively. For cryo-negatively stained vitrified catalase crystals, electron diffraction shows that atomic resolution is preserved (to better than 20 diffraction orders and less than 3 A). The electron diffraction resolution is reduced to ca. 10 A when catalase crystal specimens are prepared without freezing or when they are freeze-dried in the electron microscope. Thin vitrified films of TMV, TBSV and TYMV in the presence of 16% ammonium molybdate show a clear indication of two-dimensional (2-D) order, confirmed by single particle orientational analysis of TBSV and 2-D crystallographic analysis of TYMV. These observations are in accord with earlier claims that ammonium molybdate induces 2-D array and crystal formation from viruses and macromolecules during drying onto mica. Three-dimensional analysis of the TBSV sample using the tools of icosahedral reconstruction revealed that a significant fraction of the particles were distorted. A reconstruction from a subset of undistorted particles produced the characteristic T = 3 dimer clustered structure of TBSV, although the spikes are shortened relative to the structure defined by X-ray crystallography. The 20S proteasome, GroEL, catalase, bacteriophage T2, TMV, TBSV and TYMV all show no indication of sample instability during cryo-negative staining. However, detectable dissociation of the KLH2 oligomers in the presence of the high concentration of ammonium molybdate conforms with existing knowledge on the molybdate-induced dissociation of this molecule. This indicates that the possibility of sample-stain interaction in solution, prior to vitrification, must always be carefully assessed.