A new microbiopsy system enables rapid preparation of tissue for high-pressure freezing

A microbiopsy system was developed to overcome long sampling times for tissues before they are cryo-fixed by high-pressure freezing. A commercially available biopsy gun was adapted to the needs of small-organ excisions, and biopsy needles were modified to allow small samples (0.6 mm x 1.2 mm x 0.3 mm) to be taken. Specimen platelets with a central slot of the same dimensions as the biopsy are used. A self-made transfer device (in the meantime optimized by Leica-Microsystems [Vienna, Austria]) coordinates the transfer of the excised sample from the biopsy needle into the platelet slot and the subsequent loading in a specimen holder, which is then introduced into a high-pressure freezer (Leica EM PACT; Leica Microsystems, Vienna, Austria). Thirty seconds preparation time is needed from excision until high-pressure freezing. Brain, liver, kidney and muscle excisions of anesthetised rats are shown to be well frozen.

A rapid microbiopsy system to improve the preservation of biological samples prior to high-pressure freezing

A microbiopsy system for fast excision and transfer of biological specimens from donor to high-pressure freezer was developed. With a modified, commercially available, Promag 1.2 biopsy gun, tissue samples can be excised with a size small enough (0.6 mm x 1.2 mm x 0.3 mm) to be easily transferred into a newly designed specimen platelet. A self-made transfer unit allows fast transfer of the specimen from the needle into the specimen platelet. The platelet is then fixed in a commercially available specimen holder of a high-pressure freezing machine (EM PACT, Leica Microsystems, Vienna, Austria) and frozen therein. The time required by a well-instructed (but not experienced) person to execute all steps is in the range of half a minute. This period is considered short enough to maintain the excised tissue pieces close to their native state. We show that a range of animal tissues (liver, brain, kidney and muscle) are well preserved. To prove the quality of freezing achieved with the system, we show vitrified ivy leaves high-pressure frozen in the new specimen platelet.

Lamellar body ultrastructure revisited: high-pressure freezing and cryo-electron microscopy of vitreous sections

Lamellar bodies are the storage sites for lung surfactant within type II alveolar epithelial cells. The structure-function models of lamellar bodies are based on microscopic analyses of chemically fixed tissue. Despite available alternative fixation methods that are less prone to artifacts, such as cryofixation by high-pressure freezing, the nature of the lung, being mostly air filled, makes it difficult to take advantage of these improved methods. In this paper, we propose a new approach and show for the first time the ultrastructure of intracellular lamellar bodies based on cryo-electron microscopy of vitreous sections in the range of nanometer resolution. Thus, unspoiled by chemical fixation, dehydration and contrasting agents, a close to native structure is revealed. Our approach uses perfluorocarbon to substitute the air in the alveoli. Lung tissue was subsequently high-pressure frozen, cryosectioned and observed in a cryo-electron microscope. The lamellar bodies clearly show a tight lamellar morphology. The periodicity of these lamellae was 7.3 nm. Lamellar bifurcations were observed in our cryosections. The technical approach described in this paper allows the examination of the native cellular ultrastructure of the surfactant system under near in vivo conditions, and therefore opens up prospectives for scrutinizing various theories of lamellar body biogenesis, exocytosis and recycling.

Neotethys closure history of Anatolia: insights from 40Ar–39Ar geochronology and P–T estimation in high pressure metasedimentary rocks

The multiple high-pressure (HP), low-temperature (LT) metamorphic units of Western and Central Anatolia offer a great opportunity to investigate the subduction- and continental accretion-related evolution of the eastern limb of the long-lived Aegean subduction system. Recent reports of the HP–LT index mineral Fe-Mg-carpholite in three metasedimentary units of the Gondwana-derived Anatolide–Tauride continental block (namely the Afyon Zone, the Ören Unit and the southern Menderes Massif) suggest a more complicated scenario than the single-continental accretion model generally put forward in previous studies. This study presents the first isotopic dates (white mica 40Ar–39Ar geochronology), and where possible are combined with P–T estimates (chlorite thermometry, phengite barometry, multi-equilibrium thermobarometry), on carpholite-bearing rocks from these three HP–LT metasedimentary units. It is shown that, in the Afyon Zone, carpholite-bearing assemblages were retrogressed through greenschist-facies conditions at c. 67–62 Ma. Early retrograde stages in the Ören Unit are dated to 63–59 Ma. In the Kurudere–Nebiler Unit (HP Mesozoic cover of the southern Menderes Massif), HP retrograde stages are dated to c. 45 Ma, and post-collisional cooling to c. 26 Ma. These new results support that the Ören Unit represents the westernmost continuation of the Afyon Zone, whereas the Kurudere–Nebiler Unit correlates with the Cycladic Blueschist Unit of the Aegean Domain. In Western Anatolia, three successive HP–LT metamorphic belts thus formed: the northernmost Tavşanlı Zone (c. 88–82 Ma), the Ören–Afyon Zone (between 70 and 65 Ma), and the Kurudere–Nebiler Unit (c. 52–45 Ma). The southward younging trend of the HP–LT metamorphism from the upper and internal to the deeper and more external structural units, as in the Aegean Domain, points to the persistence of subduction in Western Anatolia between 93–90 and c. 35 Ma. After the accretion of the Menderes–Tauride terrane, in Eocene times, subduction stopped, leading to continental collision and associated Barrovian-type metamorphism. Because, by contrast, the Aegean subduction did remain active due to slab roll-back and trench migration, the eastern limb (below Southwestern Anatolia) of the Hellenic slab was dramatically curved and consequently teared. It therefore is suggested that the possibility for subduction to continue after the accretion of buoyant (e.g. continental) terranes probably depends much on palaeogeography.

Oxygen isotope record of oceanic and high-pressure metasomatism: a P-T-time-fluid path for the Monviso eclogites (Italy)

Fluids are considered a fundamental agent for chemical exchanges between different rock types in the subduction system. Constraints on the sources and pathways of subduction fluids thus provide crucial information to reconstruct subduction processes. The Monviso ophiolitic sequence is composed of mafic, ultramafic and minor sediments that have been subducted to ~80 km depth. In this sequence, both localized fluid flow and channelized fluids along major shear zones have been documented. We investigate the timing and source of the fluids that affected the dominant mafic rocks using microscale U-Pb dating of zircon and oxygen isotope analysis of mineral zones (garnet, zircon and antigorite) in high pressure rocks with variable degree of metasomatic modification. In mafic eclogites, Jurassic zircon cores are the only mineralogical relicts of the protolith gabbros and retain δ18O values of 4.5–6 ‰, typical of mantle melts. Garnet and metamorphic zircon that grew during prograde to peak metamorphism display low δ18O values between 0.2 and 3.8 ‰, which are likely inherited from high-temperature alteration of the protolith on the sea floor. This is corroborated by δ18O values of 3.0 and 3.6 ‰ in antigorite from surrounding serpentinites. In metasomatised eclogites within the Lower Shear Zone, garnet rim formed at the metamorphic peak shows a shift to higher δ18O up to 6‰. The age of zircons in high-pressure veins and metasomatised eclogites constrains the timing of fluid flow at high pressure at around 45–46 Ma. Although the oxygen data do not contradict previous reports of interaction with serpentinite-derived fluids, the shift to isotopically heavier oxygen compositions requires contribution from sediment-derived fluids. The scarcity of metasediments in the Monviso sequence suggests that such fluids were concentrated and fluxed along the Lower Shear Zone in a sufficient amount to modify the oxygen composition of the eclogitic minerals.

Exploring Multi-Component Superconducting Compounds by a High-Pressure Method and Ceramic Combinatorial Chemistry

In this short review, we provide some new insights into the material synthesis and characterization of modern multi-component superconducting oxides. Two different approaches such as the high-pressure, high-temperature method and ceramic combinatorial chemistry will be reported with application to several typical examples. First, we highlight the key role of the extreme conditions in the growth of Fe-based superconductors, where a careful control of the composition-structure relation is vital for understanding the microscopic physics. The availability of high-quality LnFeAsO (Ln = lanthanide) single crystals with substitution of O by F, Sm by Th, Fe by Co, and As by P allowed us to measure intrinsic and anisotropic superconducting properties such as Hc2, Jc. Furthermore, we demonstrate that combinatorial ceramic chemistry is an efficient way to search for new superconducting compounds. A single-sample synthesis concept based on multi-element ceramic mixtures can produce a variety of local products. Such a system needs local probe analyses and separation techniques to identify compounds of interest. We present the results obtained from random mixtures of Ca, Sr, Ba, La, Zr, Pb, Tl, Y, Bi, and Cu oxides reacted at different conditions. By adding Zr but removing Tl, Y, and Bi, the bulk state superconductivity got enhanced up to about 122 K.

Safe high-pressure freezing of infectious micro-organisms

We describe how high-pressure freezing of infectious biological material can safely be accomplished with the help of membrane carriers. The method described is easy to perform; however, careful manipulations are required. Existing safety regulations must still be followed. However, the procedure reduces the risk of dissemination of infectious material.

Structural plasticity of hippocampal mossy fiber synapses as revealed by high-pressure freezing

Despite recent progress in fluorescence microscopy techniques, electron microscopy (EM) is still superior in the simultaneous analysis of all tissue components at high resolution. However, it is unclear to what extent conventional fixation for EM using aldehydes results in tissue alteration. Here we made an attempt to minimize tissue alteration by using rapid high-pressure freezing (HPF) of hippocampal slice cultures. We used this approach to monitor fine-structural changes at hippocampal mossy fiber synapses associated with chemically induced long-term potentiation (LTP). Synaptic plasticity in LTP has been known to involve structural changes at synapses including reorganization of the actin cytoskeleton and de novo formation of spines. While LTP-induced formation and growth of postsynaptic spines have been reported, little is known about associated structural changes in presynaptic boutons. Mossy fiber synapses are assumed to exhibit presynaptic LTP expression and are easily identified by EM. In slice cultures from wildtype mice, we found that chemical LTP increased the length of the presynaptic membrane of mossy fiber boutons, associated with a de novo formation of small spines and an increase in the number of active zones. Of note, these changes were not observed in slice cultures from Munc13-1 knockout mutants exhibiting defective vesicle priming. These findings show that activation of hippocampal mossy fibers induces pre- and postsynaptic structural changes at mossy fiber synapses that can be monitored by EM.