Fast, robust, and accurate determination of transmission electron microscopy contrast transfer function

Transmission electron microscopy, as most imaging devices, introduces optical aberrations that in the case of thin specimens are usually modeled in Fourier space by the so-called contrast transfer function (CTF). Accurate determination of the CTF is crucial for its posterior correction. Furthermore, the CTF estimation must be fast and robust if high-throughput three-dimensional electron microscopy (3DEM) studies are to be carried out. In this paper we present a robust algorithm that fits a theoretical CTF model to the power spectrum density (PSD) measured on a specific micrograph or micrograph area. Our algorithm is capable of estimating the envelope of the CTF which is absolutely needed for the correction of the CTF amplitude changes.     

Automated determination of parameters describing power spectra of micrograph images in electron microscopy

The current theory of image formation in electron microscopy has been semi-quantitatively successful in describing data. The theory involves parameters due to the transfer function of the microscope (defocus, spherical aberration constant, and amplitude constant ratio) as well as parameters used to describe the background and attenuation of the signal. We present empirical evidence that at least one of the features of this model has not been well characterized. Namely the spectrum of the noise background is not accurately described by a Gaussian and associated "B-factor;" this becomes apparent when one studies high-quality far-from focus data. In order to have both our analysis and conclusions free from any innate bias, we have approached the questions by developing an automated fitting algorithm. The most important features of this routine, not currently found in the literature, are (i). a process for determining the cutoff for those frequencies below which observations and the currently adopted model are not in accord, (ii). a method for determining the resolution at which no more signal is expected to exist, and (iii). a parameter-with units of spatial frequency-that characterizes which frequencies mainly contribute to the signal. Whereas no general relation is seen to exist between either of these two quantities and the defocus, a simple empirical relationship approximately relates all three.     

Ingestion of latex beads by filopodia of adherent mouse peritoneal macrophages. A scanning electron microscopical and reflection contrast microscopical study

Scanning electron microscopy (SEM) of mouse peritoneal macrophages attached to glass shows that these cells have filopodia, i.e., cord-like extensions arising from the cell surface. To confirm that these extensions are not the result of the preparative procedure required for SEM or cell-surface material left behind by cells moving on the substrate surface, the cells were studied with a reflection contrast microscope prior to the preparative procedure. The results indicate that reflection-contrast microscopy and SEM both show the same filopodia for a given cell. The filopodia appear to be functional components of the cytoplasm, as shown by their ability to ingest latex beads.     

Modes of coenzyme Q function in electron transfer

Summary In the mitochondrial respiratory chain, coenzyme Q acts in different ways. A diffusable coenzyme Q pool as a common substrate-like intermediate links the low-potential complexes with complex III. Its diffusion in the lipids is not rate-limiting for electron transfer, but its content is not saturating for maximal rate of NADH oxidation. Protein-bound coenzyme Q is involved in energy conservation, and may be part of enzyme supercomplexes, as in succinate cytochromec reductase. The reason for lack of kinetic saturation of the respiratory chain by quinone concentration is in the low extent of solubility of monomeric coenzyme Q in the membrane lipids. Assays of respiratory enzymes are performed using water soluble coenzyme Q homologs and analogs; several problems exist in using oxidized quinones as acceptors of coenzyme Q reductases. In particular, for complex I no acceptor appears to favorably substitute the endogenous quinone. In addition, quinone reduction sites in complex III compete with the sites in the dehydrogenases, particularly when using duroquinone. The different extent by which these sites operate when different donor substrates (NADH, succinate, glycerol-3-phosphate) are used is best explained by different exposure of the quinone acceptor sites in the dehydrogenases.     

Interpretation of the electron microscopical appearance of collagen fibrils from corneal stroma

The appearance in the electron microscope of mechanically-dispersed corneal collagen has been observed after positive staining with phosphotungstic acid and/or uranyl acetate and after negative staining with phosphotungstate ions. The distributions of positive stains (both cationic and anionic) were similar to those observed in other type I collagens (e.g. skin, tendon). A high correlation was found between charge density in the fibril and the distribution of charged amino acids predicted from the sequence of calf skin collagen. This correlation could be improved by including type III sequence data, suggesting the presence of 20% type III collagen within each fibril. Negative staining showed the usual collagen D-periodicity but without a clear gap/overlap structure. Detailed analysis revealed at least six sites where stain penetration was inhibited. Specific staining of glycosides using N,N,N′,N′-tetramethylethylenediamine(TEMED)-osmate suggested that these sites identify the covalent attachment of disaccharides to the collagen. Using synchroton X-ray diffraction from TEMED-osmate stained corneas we have determined the locations of the stain ions (and hence the glycosides) in the moist tissue. The results demonstrate that even though the detailed charge distribution and axial molecular packing in corneal collagen are similar to other type I collalgens, carbohydrate material, probably disaccharide, is attached at fairly regular intervals. This does not occur in other type I collagens. In particular, the presence of glycoside in the overlap region may play a role in producing the narrow uniform fibrils which are essential for the transparency of the cornea.     

Antisense DNA parameters derived from next-nearest-neighbor analysis of experimental data

Background  

The enumeration of tetrameric and other sequence motifs that are positively or negatively correlated with in vivo antisense DNA effects has been a useful addition to the arsenal of information needed to predict effective targets for antisense DNA control of gene expression. Such retrospective information derived from in vivo cellular experiments characterizes aspects of the sequence dependence of antisense inhibition that are not predicted by nearest-neighbor (NN) thermodynamic parameters derived from in vitro experiments. However, quantitation of the antisense contributions of motifs is problematic, since individual motifs are not isolated from the effects of neighboring nucleotides, and motifs may be overlapping. These problems are circumvented by a next-nearest-neighbor (NNN) analysis of antisense DNA effects in which the overlapping nature of nearest-neighbors is taken into account.     

Effects of kinetic coupling on experimentally determined electron transfer parameters

Coupled electron transfer (ET) occurs when a relatively slow nonadiabatic ET reaction is preceded by a rapid but unfavorable adiabatic reaction that is required to activate the system for ET. As a consequence of this, the observed ET rate constant (k(ET)) is an apparent value equal to the product of the true k(ET) and the equilibrium constant for the preceding reaction step. Analysis of such reactions by ET theory may yield erroneous values for the reorganizational energy (lambda), electronic coupling (H(AB)), and ET distance that are associated with the true k(ET). If the DeltaG degrees dependence of the rate of a coupled ET reaction is analyzed, an accurate value of lambda will be obtained but the experimentally determined H(AB) will be less than the true H(AB) and the ET distance will be greater than the true distance. If the temperature dependence of the rate of a coupled ET reaction is analyzed, the experimentally determined value of lambda will be greater than the true lambda. The magnitude of this apparent lambda will depend on the magnitude of DeltaH degrees for the unfavorable reaction step that precedes ET. The experimentally determined values of H(AB) and distance will be accurate if DeltaS degrees for the preceding reaction is zero. If DeltaS degrees is positive, then H(AB) will be greater than the true value and the distance will be less than the true value. If DeltaS degrees is negative, then H(AB) will be less than the true value and the distance will be greater than the true value. Data sets for coupled ET reactions have been simulated and analyzed by ET theory to illustrate these points.     

High voltage electron microscopical analysis of chromosomal number in the slime mold Echinostelium minutum de bary

Utilizing serial thick sectioning techniques in combination with high voltage electron microscopy and stereopair analysis a count of 124 chromosomes was determined for a metaphasic plasmodial nucleus of the slime mold E. minutum.     

Influence of the experimental setup on the determination of enzyme kinetic parameters

For the design of bioconversion processes parallel experimentation in microtiter plates is commonly applied to reduce the experimental load, although data accuracy and reproducibility are often reduced. In an effort to quantify the impact of different microscale experimental systems on the estimation of enzyme kinetic parameters from progress curves, we comprehensively evaluated the enzymatic reduction of acetophenone in both open and closed polystyrene and quartz microtiter plates as well as quartz cuvettes. Differences in conversion of up to 50% over time were observed increasing from polystyrene MTPs to quartz MTPs to quartz cuvettes. Initial reaction velocities increased systematically from polystyrene to quartz MTPs and cuvettes. The experimental errors decreased in the same order showing highest experimental error of about 20% in polystyrene. We further evaluated reasons causing the deviations within one system as well as between the systems. The choice of reaction vessel material, temperature effects and substrate cross contaminations in MTPs were shown to be of importance in the experimental results. Although the experimental data differed between the reaction vessels, no distinct trends in estimated kinetic parameters were found. While the microkinetic parameters vary up to an order of magnitude between different systems, the corresponding macrokinetic parameters lie in the same range for all systems varying by 29–118%. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:87–95, 2017     

Spectroelectrochemical determination of the heterogeneous electron transfer kinetics of soluble spinach ferredoxin

Heterogeneous electron transfer rate parameters for soluble spinach ferredoxin are reported using a recently developed single potential step spectroelectrochemical technique. The reductive kinetics were measured by monitoring the $\text{decrease}$ in absorbance as a function of time for several overpotential steps at methyl viologen modified optically transparent gold minigrid electrodes. These measurements yielded an average formal heterogeneous electron transfer rate constant (k_f,h^o′ = 6.5 (±1.3) × 10^?5 cm/s) and electrochemical transfer coefficient (α = 0.60 ± (0.16)) at pH 7.5. These results are the first heterogeneous electron transfer rate parameters reported for this species.     

X-ray structure determination of the cytochrome c2: reaction center electron transfer complex from Rhodobacter sphaeroides

In the photosynthetic bacterium Rhodobacter sphaeroides, a water soluble cytochrome c2 (cyt c2) is the electron donor to the reaction center (RC), the membrane-bound pigment-protein complex that is the site of the primary light-induced electron transfer. To determine the interactions important for docking and electron transfer within the transiently bound complex of the two proteins, RC and cyt c2 were co-crystallized in two monoclinic crystal forms. Cyt c2 reduces the photo-oxidized RC donor (D+), a bacteriochlorophyll dimer, in the co-crystals in approximately 0.9 micros, which is the same time as measured in solution. This provides strong evidence that the structure of the complex in the region of electron transfer is the same in the crystal and in solution. X-ray diffraction data were collected from co-crystals to a maximum resolution of 2.40 A and refined to an R-factor of 22% (R(free)=26%). The structure shows the cyt c2 to be positioned at the center of the periplasmic surface of the RC, with the heme edge located above the bacteriochlorophyll dimer. The distance between the closest atoms of the two cofactors is 8.4 A. The side-chain of Tyr L162 makes van der Waals contacts with both cofactors along the shortest intermolecular electron transfer pathway. The binding interface can be divided into two domains: (i) A short-range interaction domain that includes Tyr L162, and groups exhibiting non-polar interactions, hydrogen bonding, and a cation-pi interaction. This domain contributes to the strength and specificity of cyt c2 binding. (ii) A long-range, electrostatic interaction domain that contains solvated complementary charges on the RC and cyt c2. This domain, in addition to contributing to the binding, may help steer the unbound proteins toward the right conformation.     

New experimental approach to the estimation of rate of electron transfer from the primary to secondary acceptors in the photosynthetic electron transport chain of purple bacteria

A method for calculating the rate constant ($\text{K}{}_{\mathrm{<mtext>A</mtext><mtext>1</mtext><mtext>A</mtext><mtext>2</mtext>}}$) for the oxidation of the primary electron acceptor (A₁) by the secondary one (A₂) in the photosynthetic electron transport chain of purple bacteria is proposed.The method is based on the analysis of the dark recovery kinetics of reaction centre bacteriochlorophyll (P) following its oxidation by a short single laser pulse at a high oxidation-reduction potential of the medium. It is shown that in Ectothiorhodospira shaposhnikovii there is little difference in the value of $\text{K}{}_{\mathrm{<mtext>A</mtext><mtext>1</mtext><mtext>A</mtext><mtext>2</mtext>}}$ obtained by this method from that measured by the method of Parson ((1969) Biochim. Biophys. Acta 189, 384–396), namely: $\text{(4.5±1.4) · 10}{}^3\text{s}{}^{\mathrm{?1}}$ and $\text{(6.9±1.2) · 10}{}^3\text{s}{}^{\mathrm{?1}}$, respectively.The proposed method has also been used for the estimation of the $\text{K}{}_{\mathrm{<mtext>A</mtext><mtext>1</mtext><mtext>A</mtext><mtext>2</mtext>}}$ value in chromatophores of Rhodospirillum rubrum deprived of constitutive electron donors which are capable of reducing P⁺ at a rate exceeding this for the transfer of electron from A₁ to A₂. The method of Parson cannot be used in this case. The value of $\text{K}{}_{\mathrm{<mtext>A</mtext><mtext>1</mtext><mtext>A</mtext><mtext>2</mtext>}}$ has been found to be $\text{(2.7±0.8) · 10}{}^3\text{s}{}^{\mathrm{?1}}$.The activation energies for the A₁ to A₂ electron transfer have also been determined. They are 12.4 kcal/mol and 9.9 kcal/mol for E. shaposhnikovii and R. rubrum, respectively.     

Immunocytochemical localization of GABAergic neurones at the electron microscopical level

Summary Antibodies prepared to purified brain glutamic acid decarboxylase (GAD), the synthesizing enzyme for the neurotrasmitter, -aminobutyric, acid (GABA), have been utilized with an unlabelled antibody method to localize GABAergic neurones in both light and electron microscopic preparations. A modification of Sternberger's peroxidase-antiperoxidase (PAP) complex is used to localize the site of anti-GAD binding, and the PAP complex is visualized with diaminobenzidine and H₂O₂. The reaction product is visible in both the light and electron microscopes. The ability to localize and identify labelled profiles in the electron microscope provides more functional information than light microscopical preparations. For example, the GAD-positive reaction product occurs mostly in association with synaptic vesicles within axon terminats, and this localization indicates the importance of GAD for the packaging and storage of GABA. The somata and dendrites of neurones giving rise to these terminals are visualized in colchicine-injected material. The GABAergic neurones form axo-somatic, axo-dendritic, axo-axonal and dendro-dendritic synapses in various regions of the rat central nervous system. Pretreatments of animals with anterograde degeneration have shown the significance of some of the GABAergic terminals that form axo-axonal synapses in the spinal cord.An many brain regions, such as the cerebral cortex, hippocampus and olfactory bulb, virtually all of the GABAergic synapses are derived from local circuit neurones. In other regions such as the cerebellum and neostriatum, the GABAergic terminals are derived from both local circuit neurones and the local axon collaterals of projection neurones that have their somata within these regions. A third type of configuration of GABAergic terminals occurs in the globus pallidus and substantia nigra where these terminals are derived from distant brain regions, axon collaterals of projection neurones and from local circuit neurones. Together, these results indicate the complex organization of the GABAergic system of the brain that has been vividly revealed with electron in croscopical immunocytochemistry.     

Objective characterization of cells in terms of microscopical parameters: An example from muscle histochemistry

Summary A quantitative histochemical analysis of almost 200 mouse triceps surae muscle fibres is presented, together with some data from similar surveys of rabbit skeletal muscles. In the main work, ten parameters are considered: mean diameter, and reaction intensities (estimated as apparent absorbances) of three markers for oxidative-lipolytic metabolism, three for metabolism associated with glycolysis and three for the type of myosin. Cytoarchitecture of one deposit (succinate dehydrogenase) is also noted.Frequency histograms for each parameter and correlation analyses for all possible pairings demonstrate that markers within the same metabolic system are not truly equivalent. Therefore, fibre typing in terms of just one marker from each system cannot be independent of the markers used. Selecting the most sharply discriminatory pair-myosin ATPase (following formalin and alkaline pretreatment) and glycogen phosphorylasea — one isolates four more-or-less discrete clusters of points. Taking succinate dehydrogenase also into account, to indicate characteristic oxidative levels within clusters, one can label the indicated fibre types (following a well-established muscle terminology) as fast, essentially glycolytic (FG), fast, oxidative and glycolytic (FOG), fast, essentially-oxidative (FO) and slow, essentially oxidative (SO). However, with either -glycerophosphate dehydrogenase or the periodic acid-Schiff reaction (PAS) as glycolytic marker, the FO-group is not separated from the FOG; and with oxidative level as a primary clustering criterion FG and FOG groups are continuous. An entirely different basis for classification-cytoarchitecture-also suggests four fibre types but the divisions most comparable to the FG/FOG and FOG/FO boundaries are best located somewhat differently again.Some of the techniques of formal cluster analysis are next introduced. These are ways of searching for similarities (defined in terms of various objective criteria) in large volumes of essentially multivariate data.Within a sample, considered representative of acceptably artefact-free fibres, virtually the same four groups as before are identified. The only difference is that what were earlier grouped as the most oxidative FG fibres are now classed FOG; the acid-pretreated myosin ATPase reaction (previously little considered) contributes to this reclassification. At the five cluster level, a strong tendency exists for this small group, designated FG(O), to appear separately. At the two-cluster level classical distinctions in terms of high/low oxidative capacity, glycolytic capacity and myosin ATPase activity are each favoured by different similarity criteria.Surveying the whole sample of fibres, only criteria which favour non-rambling clusters produce similar results to those above. However, an artefact in one reaction, for which there is strong internal evidence, is able to explain almost all other effects. These results do not prove the biological rightness of a 4–5 cluster pattern, but they do demonstrate its mathematical strength and the reactions upon which it depends.The suggestion is made that cluster analysis and related multivariate statistical methods could profitably be applied to a wide range of further problems in cell taxonomy.     

New sensitive light microscopical detection of colloidal gold on ultrathin sections by reflection contrast microscopy. Combination of reflection contrast and electron microscopy in post-embedding immunogold histochemistry.

One simple post-embedding method for combined light- and electron microscopy is presented. Different types of antigens in normal rat and mouse kidneys as well as in tissues from cases of experimental induced nephritis were stained after Lowicryl K4M embedding by an immunogold (silver) method. The (silver-enhanced) gold particles were visualized by light microscopy, e.g. bright-field (BFM)- and reflection contrast (RCM) microscopy, as well as by electron microscopy. The potentials of RCM visualization in this field were investigated, resulting in the successful detection of colloidal gold (15 nm) particles, or silver enhanced gold particles, on ultrathin sections. Furthermore, an increased detection sensitivity of RCM compared with BFM together with an increase in the sensitivity of the immunostaining by RCM visualization was found. The different ways to use RCM, alone or in combination with bright-field- or phase contrast microscopy for visualization of plastic sections varying in thickness, type of plastic and staining, are discussed.     

Fluorescent analysis of excess electron transfer through DNA

The DNA base stack provides unique features for the efficient long-range charge transfer. For the purpose of investigating excess electron transfer process through DNA, we developed a new method for fluorescence analysis of excess electron transfer based on reductive cleavage of a disulfide bond and a thiol-specific fluorescent probe. Excess electron transfer was detected by monitoring the fluorescence of emissive pyrene monomer generated by the reaction of pyrene maleimides with the cleaved disulfide bond (thiols). Mechanism of reductive cleavage of disulfides through excess electron transfer and subsequent reaction with the fluorescent probes were discussed. This facile and sensitive detection by fluorescence method can be applied for mechanistic study of excess electron transfer.     

An electron microscopical study of developing sympathetic neurons in man

Summary An electron microscopic study has been made of the sympathetic ganglia of a 15 and a 17 week old male human fetus. The fetal sympathetic neurons were densely packed in a scanty connective tissue matrix which also contained blood vessels. The fetal sympathetic neurons had a large, electron-light nucleus with one or two nucleoli, and was of a somewhat mottled appearance due to irregularly dispersed aggregates of fine and coarse granules. The perikaryon usually formed a thin envelope around the nucleus and contained, except for large pigment granules, all intracytoplasmic structures which were also found in mature sympathetic neurons. Adjacent sympathetic cells were either in immediate contact, or slightly separated by a wedge of electron-light satellite expansions, or lined by primitive axons. The satellite cells were in the early state of development. Electron-dense axons either stood side by side with, or were slightly engulfed by light Schwann cell expansions and formed distinct bundles surrounded by a common basement membrane. There was practically no trace of myelin formation or Schwann cell wrapping characteristic for unmyelinated fibers as seen in the adult.This investigation was supported (in whole) by United States Public Health Service Grant NB-01879-05, Institute for Nervous Diseases and Blindness.Grateful acknowledgment is made to Professor Dr. John Lind who madea vailable the fetal material through the Laboratory of Prenatal Growth and Development, Karolinska Institutet, Stockholm, Sweden.The authors wish to thank Docent Dr. Gunnar Bloom who provided the facilities necessary to prepare the fetal material for electron microscopical examination, in his laboratory for Cell Research, Karolinska Institutet, Stockholm, Sweden.