Water is the most abundant component of biological material, but it is systematically excluded from conventional electron microscopy. This is because water evaporates rapidly under the vacuum conditions of an electron microscope. Cryoelectron microscopy has long been seen as a possible avenue to overcome this limitation, but until recently the direct observation of frozen-hydrated specimens was relatively unsuccessful because of a number of serious difficulties. These were, in particular, due to the absence of a good cryospecimen holder, the inherently low contrast of hydrated specimens and the structural damage due to ice crystals formed during freezing. As a consequence, the cryomethods which have flourished in electron microscopy during the last 20 years were not aimed at preserving the hydration of the specimen in the electron microscope. Freezing was only used as an aid to preparation. The objects ultimately observed in the electron microscope were dry and at room temperature. Such cryomethods have recently been reviewed in detail (Robards and Sleytr 1985).

/pdf/cryo-electron-microscopy-of-vitrified-specimens-46t0snibk6.pdf

Cryo-electron microscopy of vitrified specimens.

The autoradiographic demonstration of axonal connections in the central nervous system.

Nanoemulsion: Concepts, development and applications in drug delivery

Water is the most abundant liquid on earth and also the substance with the largest number of anomalies in its properties. It is a prerequisite for life and as such a most important subject of current research in chemical physics and physical chemistry. In spite of its simplicity as a liquid, it has an enormously rich phase diagram where different types of ices, amorphous phases, and anomalies disclose a path that points to unique thermodynamics of its supercooled liquid state that still hides many unraveled secrets. In this review we describe the behavior of water in the regime from ambient conditions to the deeply supercooled region. The review describes simulations and experiments on this anomalous liquid. Several scenarios have been proposed to explain the anomalous properties that become strongly enhanced in the supercooled region. Among those, the second critical-point scenario has been investigated extensively, and at present most experimental evidence point to this scenario. Starting from very low ...

/pdf/water-a-tale-of-two-liquids-hxorql1ud9.pdf

Water: A Tale of Two Liquids

Recently, it has been proposed that organic aerosol particles in the atmosphere can exist in an amorphous semi-solid or solid (i.e. glassy) state. In this perspective, we analyse and discuss the formation and properties of amorphous semi-solids and glasses from organic liquids. Based on a systematic survey of a wide range of organic compounds, we present estimates for the glass forming properties of atmospheric secondary organic aerosol (SOA). In particular we investigate the dependence of the glass transition temperature Tg upon various molecular properties such as the compounds' melting temperature, their molar mass, and their atomic oxygen-to-carbon ratios (O : C ratios). Also the effects of mixing different compounds and the effects of hygroscopic water uptake depending on ambient relative humidity are investigated. In addition to the effects of temperature, we suggest that molar mass and water content are much more important than the O : C ratio for characterizing whether an organic aerosol particle is in a liquid, semi-solid, or glassy state. Moreover, we show how the viscosity in liquid, semi-solid and glassy states affect the diffusivity of those molecules constituting the organic matrix as well as that of guest molecules such as water or oxidants, and we discuss the implications for atmospheric multi-phase processes. Finally, we assess the current state of knowledge and the level of scientific understanding, and we propose avenues for future studies to resolve existing uncertainties.

Glass transition and phase state of organic compounds: dependency on molecular properties and implications for secondary organic aerosols in the atmosphere.

An analysis of grain distributions around a radioactive line source (consisting of polystyrene-3H) showed that the shape of the distribution was independent of the factors that influence resolution, i.e. section and emulsion thickness, silver halide crystal, and developed grain size. These factors did effect the spread of the distribution, however, and thus the distance from the line source within which 50% of the total developed grains fell. We called this distance "half distance" (HD) and determined it for a variety of specimens. When grain distributions were normalized in units of HD, one could plot universal grain distributions for specimens with radioactive sources of various shapes. The use of HD and the universal curves in interpreting radioautograms is discussed.

/pdf/resolution-in-electron-microscope-radioautography-1zn9z6q35x.pdf

Resolution in electron microscope radioautography

Publisher Summary This chapter describes the technique for cryofixation. The goal of ultrastructural fixation methods is to preserve the momentary distribution of all components in a system. For molecular systems, this implies the maintenance of the statistical distribution of all solute components in the solvent and the preservation of the size and shape of the macromolecules in the hydrated state. Cryofixation and freeze-storage of biological materials are important problems in many regards: for the storage of living materials, for the storage and processing of proteins, and for the preservation of structural details. The main problem of cryofixation is that the “fixed” structures are thermodynamically unstable at low temperatures. It is important to have several different cryofixation techniques at hand, if the variety of biological materials and the broad spectrum of secondary techniques are considered, whereby each has its requirements.

Cryofixation : A Tool In Biological Ultrastructural Research

The outer membrane complex of Paramecium was investigated by ultrathin-sectioning techniques and by freeze-etching of unfixed cells without cryoprotectants. Granules were found in the freeze-etched cortical membrane complex in a highly ordered arrangement; morphometric analyses and partial disruption of this membrane complex showed that some of these granular membrane specializations (which we call types a, b, e, f, g ) represent membrane-to-membrane attachment sites. Type a : granules arranged in rings (single or double), 300 nm in diameter, connect plasmalemma and alveolar membranes around trichocysts. Type b : within these a -type rings, a concentric zone (180 nm diameter) of granules connects plasma membrane and trichocyst membrane. Type c : the trichocyst membrane further contains a central ring or patch, 80 nm in diameter, of rather large granules where it is in contact with the crystalline trichocyst matrix. Type d: ‘ciliary necklaces’ are formed by groups of triple rows of 3-6, most frequently 5 granules, with 21-nm periodicity. Rows of granules also connect the alveolar membranes to the apical portion of the trichocyst membrane (type e ) and the alveolar membrane to the plasmalemma around cilia (type f ). Type g : the inner alveolar membrane displays an intense granularity and contains double rows of granules along alveolar septa at attachment sites between 2 alveolar membranes. Upon experimental discharge of the majority of trichocysts only the innermost concentric circles of membrane-bound granules of this region ( b - and c -type) disappeared from the plasmalemma, while the outermost a -type rings of granules persisted for a longer time. Among other possible functions, these regular membrane-to-membrane attachments are likely to maintain the specific cellular shape.

/pdf/membrane-specializations-in-the-form-of-regular-membrane-to-xm5q9q1t89.pdf

Membrane specializations in the form of regular membrane-to membrane attachment sites in Paramecium : a correlated freeze-etching and ultrathin-sectioning analysis

Micrometre-sized water droplets were hyperquenched on a solid substrate held at selected temperatures between 150 and 77 K. These samples were characterized by differential scanning calorimetry (DSC) and X-ray diffraction. 140 K is the upper temperature limit to obtain mainly amorphous samples on deposition within 16–37 min. DSC scans of glassy water prepared at 140 K exhibit on heating an endothermic step assignable to glass → liquid transition, with an onset temperature (Tg) of 136 ± 2 K on heating at 30 K min−1. For Tg of ≈136 K, water relaxes during deposition at 140 K for 16 min, moving towards metastable equilibrium. The apparent increase in heat capacity (ΔCp) depends, for a given rate of heating, on the rate of prior cooling, and a so-called overshoot develops. 140 K deposits cooled at a rate of 5, 2 or 0.2 K min−1 show on subsequent reheating at a rate of 30 K min−1
				ΔCp values of 0.7, 1.1 and 1.7 J K−1 mol−1. This is consistent with liquid-like relaxation at 140 K, and it indicates that different limiting structures are obtained. When these 140 K deposits are in addition annealed at 130 K for 90 min, after slow-cooling at 5, 2 or 0.2 K min−1, their ΔCp values on subsequent reheating are similar to those of hyperquenched glassy water (HGW) deposits made at 77 K and annealed at 130 K. Thus, the previous ΔCp value of 1.6 J K−1 mol−1 obtained with glassy water samples annealed at 130 K (A. Hallbrucker, E. Mayer and G. P. Johari, Philos. Mag. B, 1989, 60, 179) must be an upper-bound limit because it contains a contribution from an overshoot. The Tg value of 140 K deposits, which had relaxed during deposition towards metastable equilibrium, is within experimental error the same as that of 140 K deposits annealed in addition at 130 K. This contradicts Yue and Angell’s (Y. Yue and C. Angell, Nature, 2004, 427, 717) claim for assigning the endothermic step to a sub-Tg peak or a “shadow”
				Tg. Our new data further support the proposed fragile-to-strong transition on cooling liquid water from ambient temperature into the deeply supercooled and glassy state. We also describe in detail experimental aspects to obtain HGW specimens, show the ultrastructure of the deposits using electron microscopy, and discuss the mechanism of our hyperquenching method.

Liquid-like relaxation in hyperquenched water at ≤140 K

Arising from: Y.-Z. Yue & C. A. Angell Nature 427, 717–720 (2004); Yue & Angell reply
 . It has been unclear whether amorphous glassy water heated to around 140–150 K remains glassy until it crystallizes or whether instead it turns into a supercooled and very viscous liquid. Yue and Angell1 compare the behaviour of glassy water under these conditions to that of hyperquenched inorganic glasses, and claim that water stays glassy as it heats up to its crystallization point; they also find a ‘hidden’ glass-to-liquid transition at about 169 K. Here we use differential scanning calorimetry (DSC) heating to show that hyperquenched water deposited at 140 K behaves as an ultraviscous liquid, the limiting structure of which depends on the cooling rate — as predicted by theoretical analysis of the liquid-to-glass transition2. Our findings are consistent with a glass-to-liquid transition-onset temperature (Tg) in the region of 136 K (refs 3,4), and they indicate that measurements of the liquid's properties may clarify the anomalous properties of supercooled water.

/pdf/water-behaviour-glass-transition-in-hyperquenched-water-3a2dlszuf9.pdf

Luis Bachmann

Papers

Resolution in electron microscope radioautography

Cryofixation : A Tool In Biological Ultrastructural Research

Membrane specializations in the form of regular membrane-to membrane attachment sites in Paramecium : a correlated freeze-etching and ultrathin-sectioning analysis

Liquid-like relaxation in hyperquenched water at ≤140 K

Water Behaviour: Glass transition in hyperquenched water?