Scanning transmission electron microscopy of biological structures

The design of the scanning transmission electron microscope (STEM) has been conceived to optimize its detection efficiency of the different elastic and inelastic signals resulting from the interaction of the high energy primary electrons with the specimen. Its potential use to visualize and measure biological objects was recognized from the first studies by Crewe and coworkers in the seventies. Later the real applications have not followed the initial hopes. The purpose of the present paper is to describe how the instrument has practically evolved and recently begun to demonstrate all its potentialities for quantitative electron microscopy of a wide range of biological specimens, from freeze-dried isolated macromolecules to unstained cryosections. Emphasis will be put on the mass-mapping, multi-signal and elemental mapping modes which are unique features of the STEM instruments.     

Processing scarce biological samples for light and transmission electron microscopy

Light microscopy (LM) and transmission electron microscopy (TEM) aim at understanding the relationship structure-function. With advances in biology, isolation and purification of scarce populations of cells or subcellular structures may not lead to enough biological material, for processing for LM and TEM. A protocol for preparation of scarce biological samples is presented. It is based on pre-embedding the biological samples, suspensions or pellets, in bovine serum albumin (BSA) and bis-acrylamide (BA), cross-linked and polymerized. This preparation provides a simple and reproducible technique to process biological materials, present in limited quantities that can not be amplified, for light and transmission electron microscopy.     

Imaging of biological structures with the scanning transmission electron microscope

The scanning transmission electron microscope (STEM) is discussed in view of biological applications. Theoretical considerations are given, but the emphasis is directed to practical examples from a range of biological projects. The STEM is most efficiently used in elastic and inelastic dark-field modes providing information on the scattering power of the irradiated sample. Thus, the STEM is an ideal tool for quantitative measurements such as mass-mapping or element-mapping at high resolution. Limitations of such methods due to multiple scattering and quantum noise are briefly reviewed.     

An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy

Scanning confocal microscopes offer improved rejection of out-of-focus noise and greater resolution than conventional imaging. In such a microscope, the imaging and condenser lenses are identical and confocal. These two lenses are replaced by a single lens when epi-illumination is used, making confocal imaging particularly applicable to incident light microscopy. We describe the results we have obtained with a confocal system in which scanning is performed by moving the light beam, rather than the stage. This system is considerably faster than the scanned stage microscope and is easy to use. We have found that confocal imaging gives greatly enhanced images of biological structures viewed with epifluorescence. The improvements are such that it is possible to optically section thick specimens with little degradation in the image quality of interior sections.     

The surface structures and biological properties of Klebsiella strains]

The electron-microscopic method has been used to study the surface structures and biological properties (adhesiveness, antibiotic-resistance, biochemical properties) in 67 strains of Klebsiella isolated from one-year-old children with intestinal infections (56) and from healthy ones (11). It is found that bacterial cells in 58.2% of strains have fimbriae of the 3d type, in 20.9%--of the 1st type. No correlation has been revealed between the type of fimbriae and the studied properties.     

Modification of dimethipin action by light

White light reduced the efficacy of dimethipin in inducing both desiccation and abscission in kidney beans (Phaseolus vulgaris L. cv. Black Valentine). Moreover, light reduced the previously reported inhibitory effect of dimethipin on protein synthesis (Metzger and Keng 1984) in a way that paralleled the reduction in dimethipin-induced morphological changes. Therefore the inhibition of protein synthesis by dimethipin was the parameter measured in experiments designed to characterize the light-induced reduction of dimethipin efficacy. The light effect was directly proportional to both the fluence rate and the duration of the light treatment. A similar effect of light was observed in cultured kidney bean cells devoid of chlorophyll, ruling out the participation of a photosynthetic related process. Moreover, light had no effect on either the metabolism of [2,3]-¹⁴C-dimethipin in kidney bean leaves or uptake of dimethipin into cultured kidney bean cells. No evidence was obtained for photochemical decomposition of dimethipin either. Thus, the light effect is possibly the result of direct modification of the biochemical processes associated with the primary mechanism(s) of dimethipin action, or perhaps promotion of the rate of repair of dimethipin-induced cellular damage.Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.     

Polarized and specular reflectance variation with leaf surface features

The linearly polarized reflectance from a leaf depends on the characteristics of the leaf surface. In the present study the leaf reflectance of a number of plant species with varying surface characteristics was measured at the Brewster angle with a polarization photometer having 5 visible and near-infrared wavelength bands. We found that all leaf surfaces polarized incident light. Differences among species could be explained by variation in surface features. The results support our hypothesis that the polarized light is reflected by the leaf surface, not by its interior. Two mechanisms appeared responsible for the linearly polarized reflectance: (1) specular reflectance and (2) surface particle scattering. In most cases, large values of linearly polarized reflectance could be attributed to specular reflectance from the leaf surface. Attribution required knowledge of the optical dimensions of features on the leaf surface.     

Modification of the poliovirus capsid by ultraviolet light

Ultraviolet (UV) irradiation of type I poliovirus resulted in a modified (M) particle that had lost infectivity, lacked ability to adsorb to HeLa cells, lacked VP4, and reduced in S value. Additional irradiation resulted in the loss of VP2, further reduction in S value, and permeability of the capsid to RNAse, This particle (C) as well as M contain the genome. Acid pH (5.5-65) and sulfhydryl-reducing substances (dithiothreitol. reduced glutathione, and L-cysteine) inhibited UV-induced modification of the capsid. UV irradiation at alkaline pH (7.5-8.5) resulted in more extensive modification of the capsid than irradiation at neutral pH. Ionic compounds were found to inhibit the modifying reaction.     

Visualizing aquatic bacteria by light and transmission electron microscopy

The understanding of the functional role of aquatic bacteria in microbial food webs is largely dependent on methods applied to the direct visualization and enumeration of these organisms. While the ultrastructure of aquatic bacteria is still poorly known, routine observation of aquatic bacteria by light microscopy requires staining with fluorochromes, followed by filtration and direct counting on filter surfaces. Here, we used a new strategy to visualize and enumerate aquatic bacteria by light microscopy. By spinning water samples from varied tropical ecosystems in a cytocentrifuge, we found that bacteria firmly adhere to regular slides, can be stained by fluorochoromes with no background formation and fast enumerated. Significant correlations were found between the cytocentrifugation and filter-based methods. Moreover, preparations through cytocentrifugation were more adequate for bacterial viability evaluation than filter-based preparations. Transmission electron microscopic analyses revealed a morphological diversity of bacteria with different internal and external structures, such as large variation in the cell envelope and capsule thickness, and presence or not of thylakoid membranes. Our results demonstrate that aquatic bacteria represent an ultrastructurally diverse population and open avenues for easy handling/quantification and better visualization of bacteria by light microscopy without the need of filter membranes.     

组蛋白修饰及其生物学效应

组蛋白是染色质的主要成分之一,其氨基端的氨基酸残基可以被共价修饰,进而改变染色质构型,导致转录激活或基因沉默。组蛋白修饰除了简单地调控基因表达,更在于它可以招募蛋白复合体,影响下游蛋白,从而参与细胞分裂、细胞凋亡和记忆形成,甚至影响免疫系统和炎症反应等。不仅如此,最近的研究表明,组蛋白修饰与CTD密码、生物节律、DNA修复之间也存在一定的联系。这些发现证明了组蛋白修饰的重要性。在组蛋白的密码形成与密码破译、修饰级联与招募蛋白质过程中,蛋白复合体的特殊结构域起到的中介作用都是无法替代的。因此,这些特殊结构域将是了解"组蛋白密码"的关键。目前质谱分析等技术的广泛应用,正使得许多新的结构域不断被发现。文章旨在对组蛋白密码的基本内容作一述评,同时对可能的研究热点进行展望。     

Modification of cyclic nucleotide-gated ion channels by ultraviolet light

We irradiated cyclic nucleotide-gated ion channels in situ with ultraviolet light to probe the role of aromatic residues in ion channel function. UV light reduced the current through excised membrane patches from Xenopus oocytes expressing the alpha subunit of bovine retinal cyclic nucleotide-gated channels irreversibly, a result consistent with permanent covalent modification of channel amino acids by UV light. The magnitude of the current reduction depended only on the total photon dose delivered to the patches, and not on the intensity of the exciting light, indicating that the functionally important photochemical modification(s) occurred from an excited state reached by a one-photon absorption process. The wavelength dependence of the channels' UV light sensitivity (the action spectrum) was quantitatively consistent with the absorption spectrum of tryptophan, with a small component at long wavelengths, possibly due to cystine absorption. This spectral analysis suggests that UV light reduced the currents at most wavelengths studied by modifying one or more "target" tryptophans in the channels. Comparison of the channels' action spectrum to the absorption spectrum of tryptophan in various solvents suggests that the UV light targets are in a water-like chemical environment. Experiments on mutant channels indicated that the UV light sensitivity of wild-type channels was not conferred exclusively by any one of the 10 tryptophan residues in a subunit. The similarity in the dose dependences of channel current reduction and tryptophan photolysis in solution suggests that photochemical modification of a small number of tryptophan targets in the channels is sufficient to decrease the currents.     

Cultural transmission and biological markets

Active cultural transmission of fitness-enhancing behavior (sometimes called “teaching”) can be seen as a costly strategy: one for which its evolutionary stability poses a Darwinian puzzle. In this article, we offer a biological market model of cultural transmission that substitutes or complements existing kin selection-based proposals for the evolution of cultural capacities. We demonstrate how a biological market can account for the evolution of teaching when individual learners are the exclusive focus of social learning (such as in a fast-changing environment). We also show how this biological market can affect the dynamics of cumulative culture. The model works best when it is difficult to have access to the observation of the behavior without the help of the actor. However, in contrast to previous non-mathematical hypotheses for the evolution of teaching, we show how teaching evolves, even when innovations are insufficiently opaque and therefore vulnerable to acquisition by emulators via inadvertent transmission. Furthermore, teaching in a biological market is an important precondition for enhancing individual learning abilities.     

Nanoparticle analysis of cancer cells by light transmission spectroscopy

We have measured the optical properties of cancer and normal whole cells and lysates using light transmission spectroscopy (LTS). LTS provides both the optical extinction coefficient in the wavelength range from 220 to 1100 nm and (by spectral inversion using a Mie model) the particle distribution density in the size range from 1 to 3000 nm. Our current work involves whole cells and lysates of cultured human oral cells in liquid suspension. We found systematic differences in the optical extinction between cancer and normal whole cells and lysates, which translate to different particle size distributions (PSDs) for these materials. Specifically, we found that cancer cells have distinctly lower concentrations of nanoparticles with diameters less than 100 nm and have higher concentrations of particles with diameters from 100 to 1000 nm—results that hold for both whole cells and lysates. We also found a power-law dependence of particle density with diameter over several orders of magnitude.     

Rod structures of bound water: a possible role in self-organization of biological systems and nondissipative energy transmission

Cluster–rod structure were designed, which are comprised of tetrahedral atoms with a typical torsion angle of ~38° at interatomic bonds. These structures correspond to a muscle tissue and clathrin lattice by their metrics and topology and can be formed by bound water in these systems. It is shown that the considered rod structures, which are fragments of bound water structures, can also be involved in nondissipative energy transmission as elastic energy storing structures. The estimated length of the bound water rod structure required to absorb the energy of decomposition of an ATP molecule into ADP and a phosphate group is comparable with myosin head sizes and its step along an actin filament. A mechanism of cooperative transition of the rod structure to a fragment of the ice Ih structure was demonstrated. This transition is accompanied by nondissipative release of stored energy.     

Modification by temperature of conduction and ganglionic transmission in the gastropod nervous system

The pedal ganglia of the terrestrial gastropod Ariolimax contain junctions between nerve fibers which are shown to be preferential points of fatigue and which exhibit facilitation (summation) of preganglionic impulses to produce a postganglionic spike. These characteristics in conjunction with others previously reported (reversible susceptibility to nicotine, convergence of preganglionic impulses, and inhibition of transmission through setting up a refractory state in the postganglionic fiber) are considered sufficient to indicate synaptic transmission in the pedal ganglia. The mean conduction velocity of the fastest fibers in the pedal nerves is 0.52 meter per second for preganglionic and 0.50 meter per second for postganglionic fibers at 7.56°C. The conduction rates at 21.76°C. are respectively 0.80 meter per second and 0.83 meter per second. The mean ganglionic delay is 0.033 second at 7.56°C. and 0.019 second at 21.76°C. The mean Q₁₀'s for conduction velocity are thus 1.37 for preganglionic and 1.42 for postganglionic fibers. The mean Q₁₀ for ganglionic delay is 1.49. If the assumption is made that the Q₁₀ for ganglionic delay is that of a limiting reaction, this figure then represents a value below which the Q₁₀ for synaptic delay is statistically improbable.     

Seasonal changes in light transmission by bud scales of spruce and pine

A bundle of optical fibres and a photometer were used to measure light transmission through bud scales of Norway spruce [Picea abies (L.) Karst.] and Scots pine (Pinus silvestris L.). Percentage transmission increases with wavelength from none below 480 nm to 3% the far-red. Transmission of all wavebands measured was lowest in late summer and winter and highest in the autumn and spring. For most of the year in pine, and only from July to September in spruce, there is a depression of red light (660–670 nm) transmission. This affects the ratio of 660/730 nm light penetrating the apical domes. This ratio is lowest in July and August, at the time of initiation of female strobili. The seasonal variation in transmission of light by bud scales is discussed in relation to the possible consequences for the control of flowering.     

Modification by surface association of antimicrobial susceptibility of bacterial populations

In the majority of natural situations in which bacteria are found, they are associated with and attached to surfaces. In the presence of moisture and nutrients, they grow to form extensive bacterial films which are often enveloped within copius exopolymeric matrices. Biofilms are ubiquitous to many different situations in industry, the environment and medicine. Their presence can be either beneficial or more commonly detrimental to such systems. In this respect, biofilm populations possess physiological properties distinct from those of unattached, planktonic bacteria. Moreover, it is generally accepted that bacteria growing within a biofilm are more resistant to antimicrobial agents than their planktonic counterparts. However, although the consequences of attachment to antimicrobial resistance have been known for many years, the mechanistic bases for such effects have still to be fully elucidated. In this article the nature of different resistance mechanisms, including those of the exopolymeric matrix, environmental modulation, attachment-specific physiologies and quorum sensing are reviewed.