Ice crystals formed in tissue during cryosurgery. II. Electron microscopy

Tissues frozen by means of a cryosurgical probe have been examined by electron microscopy following techniques designed to preserve the ice crystal spaces.Ice crystals appeared similar whether tissues were quenched or not following cryosurgery and the various techniques of dehydration resulted in similar ice crystal architecture.Ice crystal spaces in the area deep to the freezing probe were intracellular both in epithelium and muscle although in the muscle zone some fibers contained large and others small crystal spaces. It is suggested that this might be due to variations in the local blood supply.At the periphery of the frozen area ice crystals were usually extracellular producing gross distortion of the cells which, however, retained intracellular structural integrity. These results are consistent with the belief of many workers that intracellular ice is lethal while extracellular ice is not, but no evidence of penetration of cell membrane by ice crystals was seen.     

Ice crystals formed in tissues during cryosurgery. I. Light microscopy

The size and distribution of ice crystals formed during cryosurgical procedures in intact animals are not clear. In the present experiment oral mucosa was frozen in situ by means of a surface applied cold probe and was excised and freeze substituted while in the frozen state. It was shown that the form of the frozen tissue was preserved during this procedure and the area frozen was divisible into a zone representing the central part of the lesion and a peripheral zone separating this from normal tissue. Ice crystals within the body of the lesion were intracellular in location but varied somewhat in size. Ice crystals in the boundary zone appeared to be intracellular in the epithelium and both intra- and extracellular in the muscle fibres.It is suggested that the intracellular crystals in the body of the frozen area result in cell death while the extracellular ice in the boundary zone results in a less predictable response.     

Letter: Electron microscopy of pyruvate kinase crystals from Saccharomyces carlsbergensis

Crystals of pyruvate kinase have been analysed by electron microscopy, optical diffraction and filtering, and the following parameters were obtained: $\text{2a = 93}\text{A}\text{?}$, $\text{2b = 126}\text{A}\text{?}$, $\text{l = 35·2}\text{A}\text{?}$. A comparison of these data with the parameters obtained from small-angle X-ray scattering measurements indicates that two molecules of the tetrameric enzyme are arranged in one packing unit.     

Electron microscopy of poly (O-acetyl hydroxy-L-proline) crystals

Single crystals of POAHP II were grown from acetic anhydride, a solvent in which POAHP I is thought to be stable. The single crystals gave an electron diffraction pattern which specified a hexagonal unit cell and the lateral spacing between molecular chains (11.4 Å). There is reasonable indication that molecular chain folding occurs in the POAHP single crystals. Epitaxial crystallization of POAHP II has been observed in the [100] and [320] directions on NaCl, [110] direction on KCl, and [100] direction on KI. Evidence indicates that the molecular axis is normal to the long axis of the POAHP needles and parallel to the substrate surface.     

Electron microscopy of subnanometer surface features on metal-decorated protein crystals

Crystals of heavy riboflavin synthase from Bacillus subtilis were freeze-etched and vacuum-coated at normal incidence with 0.1 to 0.4 nm of gold and silver, respectively. This decoration technique was applied to probe the protein surface for preferential nucleation sites. Image processing of the electron micrographs revealed two particular decoration sites for silver and a different one for gold. According to X-ray crystallography, the riboflavin synthase molecules are spherical and smooth except for a surface corrugation of less than 1 nm, which can not be depicted by heavy-metal shadowing. Thus the decoration sites represent sites of specific physical-chemical interactions between the condensing metal and the protein. The decoration pattern correctly reflects the icosahedral symmetry of the almost spherical protein molecules. Owing to the molecule's symmetry, the position of these topochemical sites with respect to the symmetry axes can be localized within 5A. The packing of the molecules in the crystal can be directly observed on shadowed replicas. Only decoration, however, makes it possible to observe the exact orientation of the molecules within the crystal planes and to derive the true lattice constant along the 6-fold screw axis. This proves decoration to be a technique suitable for studying crystal packing and the molecular symmetry of protein complexes at high resolution. The technique can be applied to crystals that are not large enough or insufficiently ordered for X-ray crystallography.     

Electron microscopy visualization of DNA-protein complexes formed by Ku and DNA ligase IV

The repair of DNA double-stranded breaks (DSBs) is essential for cell viability and genome stability. Aberrant repair of DSBs has been linked with cancer predisposition and aging. During the repair of DSBs by non-homologous end joining (NHEJ), DNA ends are brought together, processed and then joined. In eukaryotes, this repair pathway is initiated by the binding of the ring-shaped Ku heterodimer and completed by DNA ligase IV. The DNA ligase IV complex, DNA ligase IV/XRRC4 in humans and Dnl4/Lif1 in yeast, is recruited to DNA ends in vitro and in vivo by an interaction with Ku and, in yeast, Dnl4/Lif1 stabilizes the binding of yKu to in vivo DSBs. Here we have analyzed the interactions of these functionally conserved eukaryotic NHEJ factors with DNA by electron microscopy. As expected, the ring-shaped Ku complex bound stably and specifically to DNA ends at physiological salt concentrations. At a ratio of 1 Ku molecule per DNA end, the majority of DNA ends were occupied by a single Ku complex with no significant formation of linear DNA multimers or circular loops. Both Dnl4/Lif1 and DNA ligase IV/XRCC4 formed complexes with Ku-bound DNA ends, resulting in intra- and intermolecular DNA end bridging, even with non-ligatable DNA ends. Together, these studies, which provide the first visualization of the conserved complex formed by Ku and DNA ligase IV at juxtaposed DNA ends by electron microscopy, suggest that the DNA ligase IV complex mediates end-bridging by engaging two Ku-bound DNA ends.     

Electron microscopy of the parameres formed by the centromeric heterochromatin of human chromosome 9 at pachytene

The structure and arrangement of the parameres, which are small bodies representing part of the heterochromatin of human chromosome 9 at pachytene, were studied using transmission and scanning electron microscopy. Parameres appear to be denser than other parts of the chromosomes but have a similar fibrous substructure. The most common arrangement is clusters on the axis of the bivalent, consisting of varying numbers of parameres of variable size. The parameres are joined to each other and to the rest of the chromosome by interconnecting fibres. No evidence was obtained for the organisation of parameres into paired lateral loops, as proposed by previous workers using light microscopy. The combination of osmium impregnation of pachytene chromosomes with a backscattered electron detector in the scanning electron microscope produced very clear images of the pattern of chromomeres. This procedure may prove valuable for pachytene mapping of chromosomes because of the greatly improved resolution compared with light microscopy.     

Collagen self-assembly in vitro: electron microscopy of initial aggregates formed during the lag phase

Initial aggregates formed in collagen self-assembly were visualized by electron microscopy, using formaldehyde to fix the state of aggregation at various points in the turbidimetric lag phase. Measurements of the length distributions of monomers and small oligomers show that the first-formed aggregates are dimeric, with the most prevalent dimer having a maximal (approximately equal to 4D; D = 67 nm) stagger between constituent molecules.     

Structural study of the yeast RNA polymerase A. Electron microscopy of lipid-bound molecules and two-dimensional crystals

Two-dimensional crystals of yeast RNA polymerase A (I) were obtained by interaction with positively charged lipid layers. The analysis of single molecular images of lipid-bound RNA polymerases showed that the enzyme was preferentially oriented by the lipid phase, which probably facilitated crystallization. Electron micrographs of the crystals revealed a rectangular unit cell 25.8 nm by 45.6 nm in size containing four RNA polymerase dimers related by P22(1)2(1) symmetry. The projection map showed, at about 2.5 nm resolution, two different views of the enzyme characterized by two bent arms, which appeared to cross at one end. These arms are likely to contain the A190 and A135 subunits and delimit a 3 to 4 nm wide groove. Additional structural features were observed and compared to the Escherichia coli enzyme.     

Electron microscopy of tRNA crystals. II. 4 A resolution diffraction pattern and substantial stability to radiation damage

Electron microscopy was applied to thin crystals of yeast tRNAPhe. The crystals embedded in glucose yield Bragg reflections with a spacing smaller than 4 A. The measurement of radiation damage rate demonstrates that they are 4 to 14 times less susceptible to electron exposures than protein crystals embedded in glucose.     

A novel approach to improve the efficacy of tumour ablation during cryosurgery

Freezing tumours and ablating it using cryosurgery is becoming a popular surgical procedure for treatment of carcinomas. In order to improve the efficiency of the cryosurgical procedure different approaches have been implemented till now, e.g., injecting high thermal conductivity fluid inside the tumour, low latent heat fluids inside the tumour prior to cryosurgery etc. These techniques improve the cryosurgical process to some extent but lack in minimising the damage to the surrounding healthy tissues. In this study, a novel concept is proposed which advocates the use of solutions with specific thermophysical properties around the interface of tumour. Numerical modelling has been done to determine the location of the ice fronts in the presence of this solution around the boundary of the tumour. It is noticed that in the presence of solution layer, owing to its distinct thermophysical properties like low thermal conductivity, not only the cellular destruction is enhanced but also the damage to the surrounding healthy tissue is minimised. Further, results indicate that this strategy leads to a faster ablation rate reducing the surgical time immensely. Also, an optimal offset, the minimum distance between the tip of cryoprobe and the boundary of the tumour, is identified for a given tumour radius with a given active length which gives maximum tumour necrosis in less time. This optimal offset which has been identified for each case will help the surgeons in proper planning of cryosurgery and improving the effectiveness of this technique greatly, making it a better treatment modality than its counterparts in many ways. It is also observed that for a 2 mm increase in activelength of the cryoprobe, the decrease in optimal offset is approximately 1 mm, i.e. optimal offset decreases linearly with an increase in the activelength for a given radius of the tumour. Also, for tumour with different radii, ranging between 10 mm to 15 mm, with same active length, the time taken for complete ablation by the larger tumour is nearly 2.7 times the time taken by the smaller one for every 2.5 mm increase in the tumour radius.     

Effects of ice duration on plankton succession during spring in a shallow polymictic lake

1. Long-term records of air temperature and ice phenology (ice duration), and phyto- and zooplankton time series (1979–1997) were used to study the effects of ice duration on the successional pattern within plankton communities during spring in a shallow polymictic lake. 2. Water temperature in March was significantly lower after cold winters when compared to average or mild winters. Mean water temperature in April was not significantly different after mild, average or cold winters, but showed an overall significant negative correlation with ice duration. 3. Ice duration affected the timing and the magnitude of the peak abundance of diatoms, rotifers and daphnids during spring, but had no direct effects on the timing and maximum of chlorophytes, cryptophytes, cyanobacteria, bosminids and cyclopoid copepods. 4. Plankton groups which appeared first in the seasonal succession (i.e. diatoms, rotifers and daphnids) reached maximum abundance earlier after mild and average winters. The peak abundance of diatoms was negatively correlated with ice duration, whereas that of rotifers and daphnids was independent of the conditions during the preceding winter. 5. Temperature alone was generally a poor predictor of the timing and magnitude of both phyto- and zooplankton maxima. Turbulence may be important in the timing and the magnitude of peaks in diatoms, while total algal biomass was the most important determinant for the timing of the rotifer maximum. The magnitude of the daphnid maxima were significantly influenced by water temperature in March and April, and by rotifer abundance. The magnitude of the bosminid maximum was correlated with food availability and predation, whereas the timing of the maximum was more closely related to water temperature in May. 6. We conclude that, as a result of the low heat storage capacity of shallow lakes, the effects of winter on planktonic communities are short lived, and soon overtaken by the prevailing weather and by biotic interactions.     

Electron microscopy of the marine microalga Dunaliella tertiolecta exposed to triphenyltin

Chemostat-grown cells of the chlorophyte Dunaliella tertiolecta (Butcher) exposed to triphenyltin were examined using transmission electron microscopy. Following a 1-h exposure to 21 and 84μM triphenyltin, mitochondria underwent structural damage and the thylakoid membranes of a small proportion of cells spread from the usual compact arrangement. Prolonging the exposure time resulted in significant cell lysis in cultures exposed to 84μM triphenyltin. Received 05 May 1997/ Accepted in revised form 28 January 1998