Low-Temperature Microscopy and Analysis

Echlin, Patrick

doi:10.1007/978-1-4899-2302-8

Cited by 281 publications

(210 citation statements)

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“…However, the quality of the stained sections is limited because they have to be fractured off tissue samples at very low temperatures (143 K; ref. 29) instead of using cutting procedures at higher temperatures (253 K) or resin-embedding procedures. Such conventional fixation and embedding techniques cannot be applied for our purpose because diffusible substances are likely to be redistributed or even lost during sample preparation.…”

Section: Discussionmentioning

confidence: 99%

Laser postionization secondary neutral mass spectrometry in tissue: a powerful tool for elemental and molecular imaging in the development of targeted drugs

Wittig¹,

Arlinghaus

Kriegeskotte

et al. 2008

Molecular Cancer Therapeutics

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The exact intracellular localization and distribution of molecules and elements becomes increasingly important for the development of targeted therapies and contrast agents. We show that laser postionization secondary neutral mass spectrometry (laser-SNMS) is well suited to localize particular elements and small molecules with subcellular spatial resolution applying the technique exemplary to Boron Neutron Capture Therapy (BNCT). We showed in a murine sarcoma that the drugs used for clinical BNCT, namely L-para-boronophenylalanine (700 mg/kg body weight i.p.) and sodium mercaptoundecahydrocloso-dodecaborate (200 mg/kg body weight i.p.), transport the therapeutic agent 10 B into the cytoplasm and into the nucleus itself, the most sensitive area of the cell. Sodium mercaptoundecahydro-closo-dodecaborate distributes 10 B homogeneously and L-para-boronophenylalanine heterogeneously. When combining laser-SNMS with prompt ;-ray analysis as a screening technique, strategies for BNCT can be elaborated to develop new drugs or to improve the use of existing drugs on scientifically based evidence. The study shows the power of laser-SNMS in the early stages of drug development, also outside BNCT.

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Section: Discussionmentioning

confidence: 99%

Laser postionization secondary neutral mass spectrometry in tissue: a powerful tool for elemental and molecular imaging in the development of targeted drugs

Wittig¹,

Arlinghaus

Kriegeskotte

et al. 2008

Molecular Cancer Therapeutics

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“…Although the qf-fd technique has been available for the last few decades (Eranko, 1954), and despite the widely accepted belief that cell structure is preserved better by physical treatments rather than by chemical fixation (for a review see Echlin, 1992), up until now the potential of this cell preparation procedure has been exploited only to a limited extent. The ultrastructural and immunocytochemical results obtained here seem fully satisfactory.…”

Section: Discussionmentioning

confidence: 99%

“…Although available for many years, and known to offer distinct advantages in terms of both spatial resolution and sensitivity (the estimated detection threshold is three calcium atoms in a 10-nm diameter spot, Shuman and Somlyo, 1987), the technique has not been used extensively in biology. Most of the studies have in fact been carried out on samples first exposed to chemical treatments, with fixatives and/or dehydration agents, that are known to induce, or to be inadequate to prevent, artifactual changes of the cell native state (Echlin, 1992). In our previous work (Grohovaz et al 1996), fibers of a thin frog muscle were first quick frozen and slowly freeze-dried at low temperature [quick freezing-freeze drying (qf-fd)], after which fixation with OSO4 vapors was applied.…”

Section: Introduction Niques Such As High-resolution Immunocytochemistrymentioning

confidence: 99%

High-resolution calcium mapping of the endoplasmic reticulum-Golgi-exocytic membrane system. Electron energy loss imaging analysis of quick frozen-freeze dried PC12 cells.

Pezzati¹,

Bossi²,

Podini³

et al. 1997

MBoC

120

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The calcium pools segregated within the endoplasmic reticulum, Golgi complex, exocytic, and other organelles are believed to participate in the regulation of a variety of cell functions. Until now, however, the precise intracellular distribution of the element had not been established. Here, we report about the first high-resolution calcium mapping obtained in neurosecretory PC12 cells by the imaging mode of the electron energy loss spectroscopy technique. The preparation procedure used included quick freezing of cell monolayers, followed by freeze-drying, fixation with OSO4 vapors, resin embedding, and cutting of very thin sections. Conventional electron microscopy and high-resolution immunocytochemistry revealed a high degree of structural preservation, a condition in which inorganic elements are expected to maintain their native distribution. Within these cells, calcium signals of nucleus, cytosol, and most mitochondria remained below detection, whereas in other organelles specific patterns were identified. In the endoplasmic reticulum, the distribution was heterogeneous with strongly positive cisternae (more often the nuclear envelope and stacks of parallel elements that are frequent in quick frozen preparations) lying in the proximity of or even in direct continuity with other, apparently negative cisternae. The Golgi complexes were labeled strongly and uniformly in all cisternae and part of their vesicles, with no appreciable differences along the cis-trans axis. Weaker or negative signals were recorded from the trans-Golgi network elements and from scattered vesicles, whereas in contrast secretion granules were strongly positive for calcium. These results are discussed in relation to the existing knowledge about the mechanisms of calcium transport in the various organelles, and about the processes and functions regulated by organelle lumenal calcium in eukaryotic cells.

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“…Dimethyl sulfoxide (DMSO) and glycerol are well-known cryoprotectors widely used in cryobiology for the conservation of biological tissues at low temperatures [1]. The main purpose of cryobiology is to find an "optimal" way for cooling biological systems to low temperatures (about the liquid nitrogen temperature), and prevent the crystallisation of water inside biological tissues.…”

Section: Introductionmentioning

confidence: 99%

Ice formation in model biological membranes in the presence of cryoprotectors

Киселев

Lesieur

Kisselev

et al. 2000

Nuclear Instruments and Methods in Physics Research Section A:

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Ice formation in model biological membranes is studied by SAXS and WAXS in the presence of cryoprotectors: dimethyl sulfoxide and glycerol. Three types of phospholipid membranes: DPPC, DMPC, DSPC are chosen for the investigation as well-studied model biological membranes. A special cryostat is used for sample cooling from 14.1 o C to −55.4 o C. The ice formation is only detected by WAXS in binary phospholipid/water and ternary phospholipid/cryoprotector/water systems in the condition of excess solvent. Ice formation in a binary phospholipid/water system creates an abrupt decrease of the membrane repeat distance by ∆d, so-called ice-induced dehydration of intermembrane space. The value of ∆d decreases as the cryoprotector concentration increases. The formation of ice does not influence the membrane structure (∆d = 0) for cryoprotector mole fractions higher than 0.05. IntroductionDimethyl sulfoxide (DMSO) and glycerol are well-known cryoprotectors widely used in cryobiology for the conservation of biological tissues at low temperatures [1]. The main purpose of cryobiology is to find an "optimal" way for cooling biological systems to low temperatures (about the liquid nitrogen temperature), and prevent the crystallisation of water inside biological tissues. One of the problems is a longer time of freeze-induced dehydration (diffusion of water molecules from biological tissues through the membrane to the conserving solvent) than the time of ice formation [2]. The ice-induced dehydration of intermembrane space at the moment of water crystallization is observed by X-ray diffraction [3] and calorimetry [4] for binary phospholipid /water systems. In macromolecular crystallography the portion of X-ray diffraction experiments at cryogenic temperatures is exponentially increasing. In many cases, a dramatic reduction in radiation damage at low temperatures allows complete data sets to be collected from a single crystal. cryoprotectors make it possible to cool samples to low temperature and store them without damage in the crystal [5].In this article ice formation is investigated by small-and wide-angle X-ray scattering (SAXS and WAXS) in ternary phospholipid/cryoprotector/water systems. Three phospholipids are studied: dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylcholine (DSPC). DPPC, DMPC, and DSPC are taken as phospholipids with an equal polar heads and with the difference in the length of hydrocarbon chains. The reason why we use DMSO and glycerol is the fact of their different interactions with the membrane surface. Glycerol penetrates into the region of polar head groups and produces influence on the thickness of the membrane bilayer and the thickness of an intermembrane solvent [6]. DMSO molecules do not penetrate into the region of polar head groups and thus do not influence the membrane thickness [7,8]. DMSO molecules strongly interact with water molecules and form hydrogen bonds. Such interaction is more intensive than the interaction between DMSO a...

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Low-Temperature Microscopy and Analysis

Cited by 281 publications

References 0 publications

Laser postionization secondary neutral mass spectrometry in tissue: a powerful tool for elemental and molecular imaging in the development of targeted drugs

Laser postionization secondary neutral mass spectrometry in tissue: a powerful tool for elemental and molecular imaging in the development of targeted drugs

High-resolution calcium mapping of the endoplasmic reticulum-Golgi-exocytic membrane system. Electron energy loss imaging analysis of quick frozen-freeze dried PC12 cells.

Ice formation in model biological membranes in the presence of cryoprotectors

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