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Biomedical subjects

P Echlin

Publications and source records attributed to P Echlin.

At least 19 recordsLinked to original sources
PubMed1999

Structural and dynamic properties of crystalline and amorphous phases in raffinose-water mixtures.

PURPOSE: To obtain an improved characterisation of the raffinose-water solid-solid and solid-liquid state diagram, and to study the thermophysical behaviour of the solid amorphous phase. This information is expected to shed light on the potential of raffinose as a pharmaceutical excipient, for stabilising labile preparations at high temperatures. METHODS: X-ray diffraction, scanning electron microscopy, polarised-light microscopy, differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) were applied to study raffinose pentahydrate and its behaviour during progressive dehydration. RESULTS: Isothermal dehydration of raffinose pentahydrate led to its gradual amorphisation, but also to minor changes in the diffractograms, suggesting the probability of lower stable hydrates. Their existence was confirmed by DSC. Anhydrous raffinose was found to be completely amorphous, and this was supported by the gradual disappearance of birefringence during dehydration. In contrast, electron micrographs, taken during the dehydration process, exhibited no changes in the original ultrastructural crystal morphology. The widths of the glass-to-fluid transitions and the absolute specific heats of crystalline and amorphous phases in the vitreous and fluid states were used to estimate some structural and relaxation characteristics of amorphous raffinose-water mixtures. CONCLUSIONS: Raffinose forms the most "fragile" glass of those pharmaceutical excipients for which data are available. In its thermomechanical properties, it is superior to trehalose and should therefore be effective as a long-term stabiliser for dried biopharmaceutical preparations at temperatures up to 65 degrees C.

Calorimetry, Differential Scanning↗
PubMed1998

Low-Voltage Energy-Dispersive X-ray Microanalysis of Bulk Biological Materials.

: Low-voltage energy-dispersive X-ray microanalysis has a number of distinct advantages for measuring the concentration of light elements (Z = 11-20) in a largely organic matrix. Between 10 and 4 kV, there is a 50-fold decrease in the size of the incident beam-specimen interaction volume which enables discrete subcellular compartments to be analyzed. Experiments with mineral and organic samples of known chemical composition show that two of the most widely used analytical algorithms are capable of providing quantitative data at accelerating voltages as low as 5 kV. This technique is used at 5 kV to analyze frozen hydrated fracture faces of leaves of the tea plant which are known to contain aluminium. Higher levels of aluminium are found in the cell walls and are associated with increased levels of silicon and magnesium. The advantages and disadvantages of the experimental approach are discussed in relation to analysis carried out at higher voltages.

Journal Article↗
PubMed1991

Ice crystal damage and radiation effects in relation to microscopy and analysis at low temperatures.

There are several limitations to the low-temperature techniques which are currently being used for the preparation, examination and analysis of biological and organic samples by means of high-energy beam instrumentation. The low thermal conductivity of samples and the inadequacy of rapid cooling techniques means that, with the exception of thin-film suspensions and the surface of impact-cooled bulk specimens which may be vitrified, ice crystals of varying sizes will be present in nearly all samples which are quench cooled. Data are presented which indicate the depth to which adequate cryo-fixation may be achieved for both morphological and analytical studies. Although dynamic processes may be time resolved in the outer parts of quench-cooled samples, the decreased freezing rate below the surface makes resolution of these processes much less certain. The quality of information which may be obtained from quench-cooled samples is limited by radiation damage. Low-dose microscopy of vitrified thin-film suspensions of macromolecules continues to provide valid structural information at the molecular level. The increased doses needed for X-ray microanalysis present serious problems with the high spatial resolution analysis of thin frozen-hydrated sections although much less damage is observed in dried samples. A case is presented for using the outer fracture faces of frozen-hydrated bulk samples for low-resolution analysis of cells and tissues.

Electron Probe Microanalysis↗
PubMed1981

Application of scanning electron microscopy to x-ray analysis of frozen-hydrated sections. I. Specimen handling techniques.

X-ray microanalysis of frozen-hydrated tissue sections permits direct quantitative analysis of diffusible elements in defined cellular compartments. Because the sections are hydrated, elemental concentrations can be defined as wet-weight mass fractions. Use of these techniques should also permit determination of water fraction in cellular compartments. Reliable preparative techniques provide flat, smooth, 0.5 micrometers-thick sections with little elemental and morphological disruption. The specimen support and transfer system described permits hydrated sections to be transferred to the scanning electron microscope cold stage for examination and analysis without contamination or water loss and without introduction of extraneous x-ray radiation.

Animals↗
PubMed1978

Low temperature scanning electron microscopy: a review.

Low temperature scanning electron microscopy is useful for morphological and analytical studies both in situations where low temperature techniques are used during specimen preparation and where low temperature stages are used for specimen examination and analysis. Examples are given of different low temperature specimen preparation techniques and how they may be applied to different types of specimen. There are still a number of problems associated with morphological identification in fully frozen-hydrated samples and it is important to carry out parallel studies using more conventional transmission electron microscopy and light microscopy preparation techniques. A number of criteria are presented, some or all of which may be used to establish the existence of the frozen-hydrated state.

Animals↗
PubMed1977

Polymer cryoprotectants in the preservation of biological ultrastructure. I. Low temperature states of aqueous solutions of hydrophilic polymers.

The solid states formed by vitrified and frozen aqueous solutions of some hydrophilic polymers, able to act as biological cryoprotectants, have been studied by differential scanning calorimetry and freeze fracture electron microscopy. Glass transitions, devitrification, recrystallization and melting behaviour of aqueous solutions of polyvinylpyrrolidone, hydroxyethyl starch and dextran have been established. The vitrified polymer solutions exhibit a characteristic microspheral morphology which is not induced by the quench cooling process but is an inherent feature of the solutions themselves.

Cryoprotective Agents↗
PubMed1977

Polymeric cryoprotectants in the preservation of biological ultrastructure. II. Physiological effects.

A study has been made of the physiological effects of three non-penetrating polymeric cryoprotective agents on sixteen different plant and animal cells and tissues. The cryoprotectants, when used at concentrations at which they are effective in preventing ice-crystal formation, generally have a lower toxicity to cells and tissue than similar concentrations of glycerol. The relatively low toxicity of these substances suggests that they would be more suitable as cryoprotectants for morphological and analytical studies than the commonly used low molecular weight compounds.

Cell Survival↗
PubMed1977

Polymeric cryoprotectants in the preservation of biological ultrastructure. III. Morphological aspects.

Two high molecular weight polymers, polyvinylpyrrolidone (PVP) and hydroxyethyl starch (HES), have been used as cryoprotectants for preparing specimens to be freeze fractured. Solutions of 25% (w/w) suppress the formation of intracellular ice in single cells and tissue blocks from both plants and animals to the extent that fine structural details of the cell can be elucidates. The mode of action of these cryoprotectants, together with the structures they reveal and the peculiar advantages attached to their use, is discussed.

Cryoprotective Agents↗
PubMed1975

The preparation, examination and analysis of frozen hydrated tissue sections by scanning transmission electron microscopy and x-ray microanalysis.

A method is reported for preparing, examining and analysing frozen hydrated tissue sections using transmission electron microscopy and X-ray microanalysis. Use of this method permits localization and measurement of water soluble or diffusible elements within the hydrated cell matrix. Since any change in total fresh weight of the specimen will affect the concentration of all components, great care has been taken to demonstrate that the mass neither increases nor decreases and to ensure that the tissue remains frozen-hydrated. Criteria for assessing whether or not the tissue remains frozen-hydrated are reported. After quench freezing, 1-2 mum thick sections of mouse liver were cut at 193 degrees K and picked up on a specially designed annular specimen holder covered with an aluminium coated nylon film. Using a transfer device which prevents contamination of the tissue sections while maintaining them at a low temperature (below 143 degrees K), the sections are transferred either to the vacuum evaporator cold stage or the scanning microscope cold stage. The tissue sections may be coated with an aluminium layer to improve electrical and thermal conductivity. The specimens are examined in the scanning transmission imaging mode and analysed using an energy dispersive X-ray analyser. Concentration of intra-nuclear and intra-cytoplasmic K, P, S and Cl are reported for mouse hepatocytes as ratios of the characteristic radiation to the continuum radiation used as a measure of mass. Ratios for all four elements were higher in the nucleus than the cytoplasm. Examples are given of this method as applied to plant and insect tissue.

Animals↗