Phase-plate electron microscopy: a novel imaging tool to reveal close-to-life nano-structures

After slow progress in the efforts to develop phase plates for electron microscopes, functional phase plate using thin carbon film has been reported recently. It permits collecting high-contrast images of close-to-life biological structures with cryo-fixation and without staining. This report reviews the state of the art for phase plates and what is innovated with them in biological electron microscopy. The extension of thin-film phase plates to the material-less type using electrostatic field or magnetic field is also addressed.     

Phase contrast electron microscopy: development of thin-film phase plates and biological applications

Phase contrast transmission electron microscopy (TEM) based on thin-film phase plates has been developed and applied to biological systems. Currently, development is focused on two techniques that employ two different types of phase plates. The first technique uses a Zernike phase plate, which is made of a uniform amorphous carbon film that completely covers the aperture of an objective lens and can retard the phase of electron waves by pi/2, except at the centre where a tiny hole is drilled. The other technique uses a Hilbert phase plate, which is made of an amorphous carbon film that is twice as thick as the Zernike phase plate, covers only half of the aperture and retards the electron wave phase by pi. By combining the power of efficient phase contrast detection with the accurate preservation achieved by a cryotechnique such as vitrification, macromolecular complexes and supermolecular structures inside intact bacterial or eukaryotic cells may be visualized without staining. Phase contrast cryo-TEM has the potential to bridge the gap between cellular and molecular biology in terms of high-resolution visualization. Examples using proteins, viruses, cyanobacteria and somatic cells are provided.     

Retrofit implementation of Zernike phase plate imaging for cryo-TEM

In-focus phase-plate imaging is particularly beneficial for cryo-TEM because it offers a substantial overall increase in image contrast, without an electron dose penalty, and it simplifies image interpretation. We show how phase-plate cryo-TEM can be implemented with an appropriate existing TEM, and provide a basic practical introduction to use of thin-film (carbon) phase plates. We point out potential pitfalls of phase-plate operation, and discuss solutions. We provide information on evaluating a particular TEM for its suitability.     

Isolation of alveolar plates from Coleps hirtus

In the ciliate Coleps hirtus, the alveoli contain rigid alveolar plates that are almost unstudied so far. Neither the exact composition nor the genesis and function are known. A necessary step to study the alveolar plates is to isolate these structures in an adequate amount. Therefore, culture conditions of C. hirtus were optimized to obtain an axenic and dense long-time culture. The protocol we developed to isolate C. hirtus alveolar plates is presented and clean alveolar plates were documented via scanning electron microscopy. The described procedure delivers alveolar plates of very good structure and integrity with preserved filigree details in sufficient amount. They can be analysed via a range of different material and biological characterisations. Since there are indications of a mineral phase within the alveolar plates, the presented results will allow to study C. hirtus alveolar plates also in the context of biomineralisation.     

A fourier tool for the analysis of coherent light scattering by bio-optical nanostructures

The fundamental dichotomy between incoherent (phase independent) and coherent (phase dependent) light scattering provides the best criterion for a classification of biological structural color production mechanisms. Incoherent scattering includes Rayleigh, Tyndall, and Mie scattering. Coherent scattering encompasses interference, reinforcement, thin-film reflection, and diffraction. There are three main classes of coherently scattering nanostructures-laminar, crystal-like, and quasi-ordered. Laminar and crystal-like nanostructures commonly produce iridescence, which is absent or less conspicuous in quasi-ordered nanostructures. Laminar and crystal-like arrays have been analyzed with methods from thin-film optics and Bragg's Law, respectively, but no traditional methods were available for the analysis of color production by quasi-ordered arrays. We have developed a tool using two-dimensional (2D) Fourier analysis of transmission electron micrographs (TEMs) that analyzes the spatial variation in refractive index (available from the authors). This Fourier tool can examine whether light scatterers are spatially independent, and test whether light scattering can be characterized as predominantly incoherent or coherent. The tool also provides a coherent scattering prediction of the back scattering reflectance spectrum of a biological nanostructure. Our applications of the Fourier tool have falsified the century old hypothesis that the non-iridescent structural colors of avian feather barbs and skin are produced by incoherent Rayleigh or Tyndall scattering. 2D Fourier analysis of these quasi-ordered arrays in bird feathers and skin demonstrate that these non-iridescent colors are produced by coherent scattering. No other previous examples of biological structural color production by incoherent scattering have been tested critically with either analysis of scatterer spatial independence or spectrophotometry. The Fourier tool is applied here for the first time to coherent scattering by a laminar array from iridescent bird feather barbules (Nectarinia) to demonstrate the efficacy of the technique on thin films. Unlike previous physical methods, the Fourier tool provides a single method for the analysis of coherent scattering by a diversity of nanostructural classes. This advance will facilitate the study of the evolution of nanostructural classes from one another and the evolution of nanostructure itself. The article concludes with comments on the emerging role of photonics in research on biological structural colors, and the future directions in development of the tool.     

Rat Brain Synaptosomes Prepared by Phase Partition

Synaptosomes from rat forebrain can easily be isolated by combining centrifugation with partition in an aqueous two-phase system composed of dextran T500 and polyethylene glycol 4000 in which synaptosomes have an extreme affinity for the upper phase. The fraction thus obtained has been characterized by electron microscopy and biochemical markers for synaptosomes and some other cell components. The contamination by microsomes, free mitochondria, and myelin was 4.4, 3.2, and 0.1%, respectively. The morphometric analysis of the electron micrographs shows that greater than 60% of the structures are synaptosomes. This preparation of the isolation procedure is remarkably short (less than 1 h), formance as assayed by their respiratory activities and ATP level in the absence and presence of depolarizing agents. Synaptosomes prepared by phase partition release the neurotransmitter glutamate in a Ca2(+)-dependent manner. The duration of the isolation procedure is remarkably short (less than 1 h), no ultracentrifuge is required, and the method can be applied for small- or large-scale preparations.     

聚己内酯静电纺丝纳米纤维支架用于小鼠诱导多能干细胞培养

干细胞联合生物支架材料体外构建功能性组织与器官,成为当前组织再生研究的重要策略,而探求具有良好生物相容性的支架材料是其关键.本研究采用扫描电镜、噻唑蓝（MTT）法、荧光显微染色等方法检测小鼠诱导多能干细胞（murine induced pluripotent stem cells, miPSCs）在聚己内酯（poly ε-caprolactone, PCL）静电纺丝纳米纤维支架上的粘附、增殖等生物学特性,探究聚己内酯纳米纤维支架与miPSCs的生物相容性. 结果显示,miPSC在PCL纳米纤维支架上具有良好粘附性并呈集落样生长,其增殖能力及干性标记物（Oct4-GFP+）的表达均不亚于标准对照组;扫描电镜显示,miPSC在PCL纳米纤维支架材料上呈现出绒毛状突起的表面结构.上述结果表明,PCL纳米纤维支架可促进miPSCs的粘附、自我增殖以及干性维持,两者具有良好的生物相容性,为下一步联合生物支架材料与干细胞构建功能性组织奠定了基础.     

The human neutrophilic promyelocyte

Summary The developmental changes in the neutrophilic promyelocytes from normal human bone marrow have been analyzed by means of phase contrast and electron microscopy. This developmental phase is characterized by the elaboration of primary (azurophillysosomal) granules and the entire intracellular machinery is directed principally toward this goal. The promyelocyte stage has been subdivided into three arbitrary stages based upon morphological, histochemical and functional characteristics which relate to the onset, active production and cessation of primary granulogenesis.Supported by Grant No. AM-HE-12084-12 from the National Institutes of Health, Bethesda, Maryland.Appreciation is expressed to Dr. Arthur Sagone who performed the bone marrow aspirations and to Marjorie Griffith, Anita Topson and Barbara Jordan for their technical assistance.     

Structure of muscle fibres and motor end plates in extraocular muscles of the grass snake, Natrix natrix L.

Six extraocular muscles of the grass snake, Natrix natrix L. together with their motor end plates were examined in the light and electron microscope, and the measurements of the diameter of muscle fibres and the area of their motor end plates were performed. Morphologically, two types of muscle fibres: tonic and red phase ones were distinguished. The former fibres, 2,3 to 14,5 mum in diameter possess single or multiple (up to five on a single fibre) "en grappe" motor end plates, without postsynaptic junctional folds. The latter fibres, 10...40 mum in diameter have single, "en plaque" motor end plates, with numerous postsynaptic junctional infoldings. The morphological features of muscle fibres and motor end plates as well as the correlation between the diameter of muscle fibres and the area of motor end plates are discussed.     

The role of octopamine in locusts and other arthropods

The biogenic amine octopamine and its biological precursor tyramine are thought to be the invertebrate functional homologues of the vertebrate adrenergic transmitters. Octopamine functions as a neuromodulator, neurotransmitter and neurohormone in insect nervous systems and prompts the whole organism to “dynamic action”. A growing number of studies suggest a prominent role for octopamine in modulating multiple physiological and behavioural processes in invertebrates, as for example the phase transition in Schistocerca gregaria. Both octopamine and tyramine exert their effects by binding to specific receptor proteins that belong to the superfamily of G protein-coupled receptors. Since these receptors do not appear to be present in vertebrates, they may present very suitable and specific insecticide and acaricide targets.     

Transient and steady state phase response curves of limit cycle oscillators

The experiment of phase shifts resulting from discrete perturbations of stable biological rhythms has been carried out to study entrainment behavior of oscillators. There are two kinds of phase response curves, which are measured in experiments, according to as one measures the phase shifts immediately or long after the perturbation. The former is the first transient phase response curve and the latter is the steady state phase response curve. We redefine both curves within the framework of dynamical system theory and homotopy theory. Several topological properties of both curves are clarified. Consequently, it is shown that we must compare the shapes of both two phase response curves to investigate the inner structures of biological oscillators. Moreover, we prove that a single limit cycle oscillator involving only two variables cannot simulate transient resetting behavior reported by Pittendrigh and Minis (1964). In other words, the circadian oscillator of Drosophila pseudoobscura does not consist of a single oscillator of two variables. Finally we show that a model which consists of two limit cycle oscillators is able to simulate qualitatively the phase response curves of Drosophila.     

Development of the cell covering in the dinoflagellate Scrippsiella hexapraecingula (Peridiniales, Dinophyceae)

The organization and development of cell coverings in two alternate phases of the life cycle in a marine dinoflagellate, Scrippsiella hexapraecingula Horiguchi et Chihara, were investigated by thin sectioning and freeze‐fracture electron microscopy. In one of these phases, the motile phase, cells have an outermost plasma membrane that is lined with flattened amphiesmal vesicles. Groups of microtubules lie beneath these vesicles. In mature motile cells, thecal plates are completely enclosed in individual amphiesmal vesicles. After settling, the cells enter the second, non‐motile phase. Here, ecdysis occurs, resulting in several steps including formation of the first pellicle layer (PI), fusion of the inner amphiesmal vesicle membranes to form the new plasma membrane, deposition of the second pellicle layer (PM) under PI, and the appearance and fusion of juvenile amphiesmal vesicles to form new territories, which eventually give rise to new thecal plates in the next motile phase. Thus, the pattern in which thecal plates are arranged in motile cells is determined at the time when the amphiesmal vesicles develop into non‐motile cells.     

Perspective: Emerging strategies for determining atomic-resolution structures of macromolecular complexes within cells

In principle, electron cryo-tomography (cryo-ET) of thin portions of cells provides high-resolution images of the three-dimensional spatial arrangement of all members of the proteome. In practice, however, radiation damage creates a tension between recording images at many different tilt angles, but at correspondingly reduced exposure levels, versus limiting the number of tilt angles in order to improve the signal-to-noise ratio (SNR). Either way, it is challenging to read the available information out at the level of atomic structure. Here, we first review work that explores the optimal strategy for data collection, which currently seems to favor the use of a limited angular range for tilting the sample or even the use of a single image to record the high-resolution information. Looking then to the future, we point to the alternative of so-called “deconvolution microscopy”, which may be applied to tilt-series or optically-sectioned, focal series data. Recording data as a focal series has the advantage that little or no translational alignment of frames might be needed, and a three-dimensional reconstruction might require only 2/3 the number of images as does standard tomography. We also point to the unexploited potential of phase plates to increase the contrast, and thus to reduce the electron exposure levels while retaining the ability align and merge the data. In turn, using much lower exposures per image could have the advantage that high-resolution information is retained throughout the full data-set, whether recorded as a tilt series or a focal series of images.     

Phase behavior of freeze-dried phospholipid-cholesterol mixtures stabilized with trehalose

A study is presented of the role of cholesterol content on the gel-to-liquid crystalline phase transition of freeze-dried liposomes stabilized with trehalose, a well known lyoprotectant. The phospholipids considered in this work, DPPC and DPPE, belong to the two predominant phospholipid species found in numerous biological membranes. Cholesterol is found in abundance in mammalian plasma membranes. DSC measurements reveal that cholesterol-containing liposomes exhibit multiple phase transitions upon dehydration. Addition of trehalose to these systems lowers the phase transition temperature and limits the phase separation of the lipidic components upon freeze-drying. This work provides strong evidence for the effectiveness of trehalose in stabilizing cholesterol-containing membranes upon lyophilization.     

Farnesol-DMPC phase behaviour: a H-NMR study

Involved in a number of diverse metabolic and functional contexts, farnesol is a central component of the mevalonate pathway, post-translationally attaches to proteins, and affects a number of other membrane-associated events. Despite farnesol's biological implications, a detailed analysis of how farnesol affects the physical properties and phase behaviour of lipid membranes is lacking. As ²H-NMR spectra are sensitive to molecular motions and acyl chain orientation, they can be used to measure the degree of molecular order present in the system. Also, since the ²H-NMR spectra of fluid and gel phase lipids are very different, they are sensitive probes of membrane phase equilibrium and can be used to determine fluid-gel phase boundaries. In this study, dimyristoyl phosphatidylcholine-d₅₄ (DMPC-d₅₄) bilayers containing varying concentrations of trans-trans farnesol (2.5-20.0 mol%) are investigated over a range of temperatures (8-30 °C). Analysis of these spectra has led to the construction of a farnesol-DMPC-d₅₄ temperature-composition plot. We show that increasing concentrations of farnesol induce a decrease in the fluid-gel phase transition temperature and promote fluid-gel coexistence. Interestingly, farnesol does not seem to affect the quadrupolar splittings (Δv_Q) in the fluid phase, i.e., the organization of farnesol within the bilayer and its interaction with phospholipids does not appreciably influence acyl chain order in the fluid phase.     

Quantification of polyphenolic antioxidants and free radical scavengers in marine algae

While the health benefits of antioxidant compounds from terrestrial plants are widely accepted in Western counties, there is less recognition of the health benefits of marine algal antioxidant compounds. Oceans are an abundant source of biomaterials, with many natural antioxidants derived from marine algae being investigated as potential anti-aging, anti-inflammatory, anti-bacterial, anti-fungal, cytotoxic, anti-malarial, anti-proliferative, and anti-cancer agents. The aim of this work was to quantify and compare polyphenolic content and free radical scavenging activity of algal extracts using normal phase and reverse phase thin layer chromatography. Post-chromatographic derivatization with neutral ferric chloride (FeCl₃) solution and with 2,2-diphenyl-1-picrylhydrazyl (DPPH·) free radical were used to assess total polyphenolic content and free radical scavenging activities in algal samples. Total phenolic content quantified on normal phase plates was correlated to phenolic content established on reverse phase plates. Similarly, free radical scavenging activity established on normal phase and reverse phase plates were in good agreement. However, although free radical scavenging activities determined on normal phase plates were highly correlated with polyphenolic content, this correlation was low for reverse phase plates. Lipophilic reversed phase TLC plates do not effectively separate mixtures of highly polar compounds like flavonoids, phenolic compounds and their glucosides. Thus, although reversed phase plates are recommended for assessment of free radical scavengers, as they do not influence the free radical-antioxidant reaction, they may not provide the best separation of polar phenolic compounds, especially flavonoids, and therefore may not accurately quantify polyphenolic content and free radical scavenging potential.     

Temperature-dependent phase behavior and protein partitioning in giant plasma membrane vesicles

Liquid-ordered (Lo) and liquid-disordered (Ld) phase coexistence has been suggested to partition the plasma membrane of biological cells into lateral compartments, allowing for enrichment or depletion of functionally relevant molecules. This dynamic partitioning might be involved in fine-tuning cellular signaling fidelity through coupling to the plasma membrane protein and lipid composition. In earlier work, giant plasma membrane vesicles, obtained by chemically induced blebbing from cultured cells, were observed to reversibly phase segregate at temperatures significantly below 37 °C. In this contribution, we compare the temperature dependence of fluid phase segregation in HeLa and rat basophilic leukemia (RBL) cells. We find an essentially monotonic temperature dependence of the number of phase-separated vesicles in both cell types. We also observe a strikingly broad distribution of phase transition temperatures in both cell types. The binding of peripheral proteins, such as cholera toxin subunit B (CTB), as well as Annexin V, is observed to modulate phase transition temperatures, indicating that peripheral protein binding may be a regulator for lateral heterogeneity in vivo. The partitioning of numerous signal protein anchors and full length proteins is investigated. We find Lo phase partitioning for several proteins assumed in the literature to be membrane raft associated, but observe deviations from this expectation for other proteins, including caveolin-1.     

Phase organization of circadian oscillators in extended gate and oscillator models

The suprachiasmatic nuclei (SCN) control daily oscillations in physiology and behavior. The gate-oscillator model captures functional heterogeneity in SCN and has been successful in reproducing many features of SCN. This paper investigates the mechanism of phase organization in the gate-oscillator model and finds that only stable fixed points of the phase transition function are essential to phase organization. This obvious finding forms the basis for understanding the complex phase distribution in the gate-oscillator scheme. Extending the model with a dead zone of the phase transition function and the propagation delay of the gate signal which may represent the spatial structure of SCN, the author discusses how some features of experimentally reported phase distribution, such as the existence of anti-phase neurons and fixed phase difference between neurons, could be understood in the framework of the gate-oscillator model. The extended model shows clearly the way in which the interplay between the single-cell property and the property of the network organization influence the phase distribution of SCN neurons.