Deep learning‐based color holographic microscopy

We report a framework based on a generative adversarial network that performs high‐fidelity color image reconstruction using a single hologram of a sample that is illuminated simultaneously by light at three different wavelengths. The trained network learns to eliminate missing‐phase‐related artifacts, and generates an accurate color transformation for the reconstructed image. Our framework is experimentally demonstrated using lung and prostate tissue sections that are labeled with different histological stains. This framework is envisaged to be applicable to point‐of‐care histopathology and presents a significant improvement in the throughput of coherent microscopy systems given that only a single hologram of the specimen is required for accurate color imaging.     

"Either-or" two-slit interference: stable coherent propagation of individual photons through separate slits

In quantum theory, nothing that is observable, be it physical, chemical, or biological, is separable from the observer. Furthermore, ". all possible knowledge concerning that object is given by its wave function" (Wigner, E. 1967. Symmetries and Reflections. Indiana University Press, Bloomington, IN), which can only describe probabilities of future events. In physical systems, quantum mechanical probabilistic events that are microscopic must, in turn, account for macroscopic events that are associated with a greater degree of certainty. In biological systems, probabilistic statistical mechanical events, such as secretion of microscopic synaptic vesicles, must account for macroscopic postsynaptic potentials; probabilistic single-channel events sum to produce a macroscopic ionic current across a cell membrane; and bleaching of rhodopsin molecules (responsible for quantal potential "bumps") produces a photoreceptor generator potential. Among physical systems, a paradigmatic example of how quantum theory applies to the observation of events concerns the interactions of particles (e.g., photons, electrons) with the two-slit apparatus to generate an interference pattern from a single common light source. For two-slit systems that use two independent laser sources with brief (<1 ms) intervals of mutual coherence (Paul, H. 1986. Rev. Modern Phys. 58:209-231), each photon has been considered to arise from both beams and has a probability amplitude to pass through each of the two slits. Here, a single laser source two-slit interference system was constructed so that each photon has a probability amplitude to pass through only one or the other, but not both slits. Furthermore, all photons passing through one slit could be distinguished from all photons passing through the other slit before their passage. This "either-or" system produced a stable interference pattern indistinguishable from the interference produced when both slits were accessible to each photon. Because this system excludes the interaction of one photon with both slits, phase correlation of photon movements derives from the "entanglement" of all photon wave functions due to their dependence on a common laser source. Because a laser source (as well as Young's original point source) will have stable time-averaged spatial coherence even at low intensities, the "either-or" two-slit interference can result from distinct individual photons passing one at a time through one or the other slit-rather than wave-like behavior of individual photons. In this manner, single, successive photons passing through separate slits will assemble over time in phase-correlated wave distributions that converge in regions of low and high probability.     

Anaerobic Growth of Microorganisms with Chlorate as an Electron Acceptor

The ability of microorganisms to use chlorate (ClO₃^-) as an electron acceptor for respiration under anaerobic conditions was studied in batch and continuous tests. Complex microbial communities were cultivated anaerobically in defined media containing chlorate, all essential minerals, and acetate as the sole energy and carbon source. It was shown that chlorate was reduced to chloride, while acetate was oxidized to carbon dioxide and water and used as the carbon source for synthesis of new biomass. A biomass yield of 1.9 to 3.8 g of volatile suspended solids per equivalent of available electrons was obtained, showing that anaerobic growth with chlorate as an electron acceptor gives a high energy yield. This indicates that microbial reduction of chlorate to chloride in anaerobic systems is coupled with electron transport phosphorylation.     

Electron holography of non-stained bacterial surface layer proteins

We report transmission electron microscopy (TEM) investigations on bacterial surface layers (S-layers) which belong to the simplest biomembranes existing in nature. S-layers are regular 2D protein crystals composed of single protein or glycoprotein species. In their native form, S-layers are weak phase objects giving only poor contrast in conventional TEM. Therefore, they are usually examined negatively stained. However, staining with heavy metal compounds may cause the formation of structural artefacts. In this work, electron microscopy studies of non-stained S-layers of Bacillus sphaericus NCTC 9602 were performed. Compared to other proteins, these S-layers are found relatively stable against radiation damage. Electron holography was applied where information about phase and amplitude of the diffracted electron wave is simultaneously obtained. In spite of small phase shifts observed, the phase image reconstructed from the hologram of the non-stained S-layer is found to be sensitive to rather slight structure and thickness variations. The lateral resolution, obtained so far, is less than that of conventional electron microscopy of negatively stained S-layers. It corresponds to the main lattice planes of 12.4 nm observed in the reconstructed electron phase image. In addition, as a unique feature of electron holography the phase image provides thickness information. Thus, the existence of double layers of the protein crystals could be easily visualized by the height profile of the specimen.     

Three-dimensional structure of lipid vesicles embedded in vitreous ice and investigated by automated electron tomography.

Automated electron tomography is shown to be a suitable means to visualize the shape of phospholipid vesicles embedded in vitrified ice. With a slow-scan charge-coupled device camera as a recording device, the cumulative electron dose needed to record a data set of 60 projections at a magnification of 20,000X can be kept as low as 15 e-/A2 (or 1500 electrons/nm2). The membrane of the three-dimensionally reconstructed vesicles is clearly visible in two-dimensional sections through the three-dimensionally reconstructed volume. Some edges indicating a polygonal shape of the vesicles, frozen from the gel phase, are also clearly recognized. Because of the presently limited tilt angle range (+/- 60 degrees), the upper and lower "caps" of the vesicles (representing about 35% of the surface of the ellipsoidal particles) remain invisible in the three-dimensional reconstruction.     

New locus (ttr) in Escherichia coli K-12 affecting sensitivity to bacteriophage T2 and growth on oleate as the sole carbon source.

The nature of resistance to phage T2 in Escherichia coli K-12 was investigated by analyzing a known phage T2-resistant mutant and by isolating new T2-resistant mutants. It was found that mutational alterations at two loci, ompF (encoding the outer membrane protein OmpF) and ttr (T-two resistance), are needed to give full resistance to phage T2. A ttr::Tn10 mutation was isolated and was mapped between aroC and dsdA, where the fadL gene (required for long-chain fatty acid transport) is located. The receptor affected by ttr was the major receptor used by phage T2 and was located in the outer membrane. Phage T2 was thus able to use two outer membrane proteins as receptors. All strains having a ttr::Tn10 allele and most of the independently isolated phage T2-resistant mutants were unable to grow on oleate as the sole carbon and energy source, i.e., they had the phenotype of fadL mutants. The gene fadL is known to encode an inner membrane protein. The most likely explanation is that fadL and ttr are in an operon and that ttr encodes an outer membrane protein which functions in translocating long-chain fatty acids across the outer membrane and also as a receptor for phage T2.     

A theoretical model for electron transport through chlorophyll-containing bileaflet membranes

Summary A model of electron transport through bileaflet membranes comprised of chlorophylls and carotenoids is proposed and can account for the photoresponses observed in chloroplast-extract membranes. Some features of the model are as follows: In the absence of any specific additive, such as phycocyanin, the energy barrier for electron transfer between chromophores in the membrane and a redox pair in the aqueous solution is significantly high, so that the only realistic mechanism for electron exchange through the interface is by quantum-mechanical tunneling. In an analogy to theories developed for the explanation of semiconductor electrode behavior, the essential requirement of energy overlap between occupied and unoccupied electronic states on either side of the membrane-water interface is explored. The extent of overlap can be controlled by the potential difference developed across the interface. The presence of the carotenoids can reduce the energy barrier for electron movement within she membrane so that excited electrons can pass from the chlorophyll chromophores on one tide of the membrane interface to those on the other side. It is suggested that electron tunneling is the mechanism by which reducing substances belonging to a redox pair of high oxidation-reduction potential deliver their electrons to vacant orbitals in the photosynthetic membranes.     

An electron transfer dependent membrane potential in chromaffin-vesicle ghosts

Adrenal medullary chromaffin-vesicle membranes contain a transmembrane electron carrier that may provide reducing equivalents for intravesicular dopamine beta-hydroxylase in vivo. This electron transfer system can generate a membrane potential (inside positive) across resealed chromaffin-vesicle membranes (ghosts) by passing electrons from an internal electron donor to an external electron acceptor. Both ascorbic acid and isoascorbic acid are suitable electron donors. As an electron acceptor, ferricyanide elicits a transient increase in membrane potential at physiological temperatures. A stable membrane potential can be produced by coupling the chromaffin-vesicle electron-transfer system to cytochrome oxidase by using cytochrome c. The membrane potential is generated by transferring electrons from the internal electron donor to cytochrome c. Cytochrome c is then reoxidized by cytochrome oxidase. In this coupled system, the rate of electron transfer can be measured as the rate of oxygen consumption. The chromaffin-vesicle electron-transfer system reduces cytochrome c relatively slowly, but the rate is greatly accelerated by low concentrations of ferrocyanide. Accordingly, stable electron transfer dependent membrane potentials require cytochrome c, oxygen, and ferrocyanide. They are abolished by the cytochrome oxidase inhibitor cyanide. This membrane potential drives reserpine-sensitive norepinephrine transport, confirming the location of the electron-transfer system in the chromaffin-vesicle membrane. This also demonstrates the potential usefulness of the electron transfer driven membrane potential for studying energy-linked processes in this membrane.     

自由电子激光在生物学中的应用

同步辐射的发展和应用已经极大的推动了自然科学包括生物学的巨大发展,其中结构生物学更是离不开X射线衍射分析,小角散射等。X射线自由电子激光(XFEL)相比同步辐射具有更高强度,完全相干等特点,被称为第四代光源。科学家已经利用XFEL实现了尺度约为1微米的蛋白质晶体的高分辨率结构解析,并且也实现了单颗粒的病毒的低分辨重构。未来,XFEL将会为生物学的发展打开一扇新的大门。     

STRUCTURE OF MEMBRANE HOLES IN OSMOTIC AND SAPONIN HEMOLYSIS

Serial section electron microscopy of hemolysing erythrocytes (fixed at 12 s after the onset of osmotic hemolysis) revealed long slits and holes in the membrane, extending to around 1 µm in length. Many but not all of the slits and holes (about 100–1000 Å wide) were confluent with one another. Ferritin and colloidal gold (added after fixation) only permeated those cells containing membrane defects. No such large holes or slits were seen in saponin-treated erythrocytes, and the membrane was highly invaginated, giving the ghost a scalloped outline. Freeze-etch electron microscopy of saponin-treated membranes revealed 40–50 Å-wide pits in the extracellular surface of the membrane. If these pits represent regions from which cholesterol was extracted, then cholesterol is uniformly distributed over the entire erythrocyte membrane.     

Stochastic heating of electrons by a large-amplitude extraordinary wave in plasma

Stochastic heating of plasma electrons by a large-amplitude electromagnetic wave propagating across a strong external magnetic field is studied theoretically and numerically. An analytic estimate of the threshold wave amplitude at which heating begins is obtained. The dependence of the average electron energy on the magnetic field and plasma density is investigated using particle-in-cell simulations.     

Studies on energy supply for genetic processes. Requirement for membrane potential in Escherichia coli infection by phage T4

In this study the hypothesis considering the requirement for an electrochemical proton gradient in the injection of phage T4 DNA into Escherichia coli cell has been verified experimentally. The phage caused a reversible depolarization of cell membrane, while phage 'ghosts' induced an irreversible depolarization. The phage infection was strictly dependent on E. coli membrane potential value when phage/cell ratio was 5 and higher. When the ratio was close to 1, the decrease in the membrane potential up to -100 mV caused practically no effect on the phage infection. The infection inhibition was observed when the membrane potential was lowered below this 'threshold' value. On the other hand, the decrease in the membrane potential caused no effect on the phage infection under conditions promoting a concomitant increase in the value of the transmembranous pH gradient. The phage DNA transfer through the membrane of ATPase-deficient cells was reversibly inhibited by switching off the respiratory chain - the sole generator of a protonmotive force in these mutant cells. The membrane should be kept in the energized state during the phage DNA entrance into the cell. Adsorption of the phage on E. coli was followed by the reversible release of the respiratory control. Thus the results presented here indicate the requirement of the electrochemical proton gradient across the plasma membrane for injection of phage T4 DNA into E. coli. They support the concept postulating an expenditure of host cell metabolic energy for phage T4 DNA transfer through the membrane.     

Alignment error envelopes for single particle analysis

To determine the structure of a biological particle to high resolution by electron microscopy, image averaging is required to combine information from different views and to increase the signal-to-noise ratio. Starting from the number of noiseless views necessary to resolve features of a given size, four general factors are considered that increase the number of images actually needed: (1) the physics of electron scattering introduces shot noise, (2) thermal motion and particle inhomogeneity cause the scattered electrons to describe a mixture of structures, (3) the microscope system fails to usefully record all the information carried by the scattered electrons, and (4) image misalignment leads to information loss through incoherent averaging. The compound effect of factors 2-4 is approximated by the product of envelope functions. The problem of incoherent image averaging is developed in detail through derivation of five envelope functions that account for small errors in 11 "alignment" parameters describing particle location, orientation, defocus, magnification, and beam tilt. The analysis provides target error tolerances for single particle analysis to near-atomic (3.5 A) resolution, and this prospect is shown to depend critically on image quality, defocus determination, and microscope alignment.     

Redox-linked proton translocation in cytochrome oxidase: the importance of gating electron flow. The effects of slip in a model transducer.

In at least one component of the mitochondrial respiratory chain, cytochrome c oxidase, exothermic electron transfer reactions are used to drive vectorial proton transport against an electrochemical hydrogen ion gradient across the mitochondrial inner membrane. The role of the gating of electrons (the regulation of the rates of electron transfer into and out of the proton transport site) in this coupling between electron transfer and proton pumping has been explored. The approach involves the solution of the steady-state rate equations pertinent to proton pump models which include, to various degrees, the uncoupled (i.e., not linked to proton pumping) electron transfer processes which are likely to occur in any real electron transfer-driven proton pump. This analysis furnishes a quantitative framework for examining the effects of variations in proton binding site pKas and metal center reduction potentials, the relationship between energy conservation efficiency and turnover rate, the conditions for maximum power output or minimum heat production, and required efficiency of the gating of electrons. Some novel conclusions emerge from the analysis, including: An efficient electron transfer-driven proton pump need not exhibit a pH-dependent reduction potential; Very efficient gating of electrons is required for efficient electron transfer driven proton pumping, especially when a reasonable correlation of electron transfer rate and electron transfer exoergonicity is assumed; and A consideration of the importance and possible mechanisms of the gating of electrons suggests that efficient proton pumping by CuA in cytochrome oxidase could, in principle, take place with structural changes confined to the immediate vicinity of the copper ion, while proton pumping by Fea would probably require conformational coupling between the iron and more remote structures in the enzyme. The conclusions are discussed with reference to proton pumping by cytochrome c oxidase, and some possible implications for oxidative phosphorylation are noted.     

Ultrastructure and elemental composition of dormant and germinating Diplodia maydis spores.

The ultrastructure of Diplodia maydis spores was studied in thin sections with a transmission electron microscope. Storage vacuoles were evenly distributed in the two cells. Some of the vacuoles that contained a dense osmiophilic sphere(s) were surrounded by a membrane, and had membranous aggregates around their periphery. The sport wall was composed of an electron-dense layer and an electron-translucent layer. An inner cytoplasmic membrane was present. Dormant and germinating spores were studied with scanning electron microscopy and also with a Si (Li) energy-dispersive X-ray analyzer. The dormant spore was ovate and usually two-celled with a central septum. Germination proceeded via a germ tube from the side of one end of the cell. Of several methods for preparation of specimens for X-ray analysis studied, freeze-dried spores mounted on carbon stubs and then further carbon coated gave the best results. X-ray analyses revealed that spore populations contained large amounts of Si, P, Cl, and K, smaller amounts of S and Ca, and trace amounts of Mg and Al. Analyses of single spores revealed high K and Cl and low P and Mg at one end of the cell with concomitant low K and Cl and high P and Mg in the central portion and other end of the cell. In two-celled germinating spores, high K and Cl occurred in the end of the nongerminating spore cell, whereas the germinating cell contained high P and Mg and low K and Cl. X-ray image maps revealed that K and Cl were located together at one end of the spore.     

Insights from the structure of the yeast cytochrome bc1 complex: crystallization of membrane proteins with antibody fragments

The ubiquinol:cytochrome c oxidoreductase (EC 1.20.2.2, QCR or cytochrome bc1 complex) is a component of respiratory and photosynthetic electron transfer chains in mitochondria and bacteria. The complex transfers electrons from quinol to cytochrome c. Electron transfer is coupled to proton translocation across the lipid bilayer, thereby generating an electrochemical proton gradient, which conserves the free energy of the redox reaction. The yeast complex was crystallized with antibody Fv fragments, a promising technique to obtain well-ordered crystals from membrane proteins. The high-resolution structure of the yeast protein reveals details of the catalytic sites of the complex, which are important for electron and proton transfer.     

Zero-loss energy filtering as improved imaging mode in cryoelectronmicroscopy of frozen-hydrated specimens

One of the remaining problems in attaining higher structural resolution with cryoelectronmicroscopy of frozen-hydrated specimens is the low contrast of micrographs taken close to the electron optical focus. By measuring electron energy loss spectra (EELS) of ice layers we show that a large fraction of incident electrons undergoes an inelastic electron-plasmon scattering process. Thus these electrons do not carry structural information of the protein but increase the background of the electron image and therefore reduce the contrast of the negative. Here we report the improvement in contrast gained by filtering out inelastically scattered electrons using an energy-filtered transmission electron microscope (EFTEM). This gain in contrast permits a dramatic decrease in defocusing values, resulting in improved structural resolution. In addition, the increased signal to noise ratio allows the recording of micrographs at a reduced electron dose. This should result in less damage to vitrified and unstained proteins and other beam-sensitive specimens.     

Nonlinear Electrical Effects in Lipid Bilayer Membranes: II. Integration of the Generalized Nernst-Planck Equations

In this paper the ion transport across a thin lipid membrane is treated using a generalized form of the Nernst-Planck equations. An additional term is introduced into the flux equations to account for the image force acting on the ion. As the membrane thickness is of the same order of magnitude as the range of the image forces, the potential energy of the ion in the membrane is strongly dependent on position. The integration of the flux equations leads to a general expression for the integral membrane conductance lambda as a function of the voltage u. The ratio lambda(u)/lambda(0) (lambda(0) = membrane conductance in the limit u --> 0) depends on the dielectric constant and the thickness of the membrane, but is independent of the ionic radius. When the numerical values of the potential energy function, as calculated by the method of electrical images, are inserted into the expression for lambda(u)/lambda(0), a strongly non-linear current-voltage characteristic is obtained. The theoretical current-voltage curve agrees satisfactorily with the experimental data at a low ionic strength and at low voltages; at higher voltages the observed membrane conductance exceeds the predicted value.     

Role of subunit NuoL for proton translocation by respiratory complex I

The NADH:ubiquinone oxidoreductase, respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with a translocation of protons across the membrane. The complex consists of a peripheral arm catalyzing the electron transfer reaction and a membrane arm involved in proton translocation. The recently published X-ray structures of the complex revealed the presence of a unique 110 ? "horizontal" helix aligning the membrane arm. On the basis of this finding, it was proposed that the energy released by the redox reaction is transmitted to the membrane arm via a conformational change in the horizontal helix. The helix corresponds to the C-terminal part of the most distal subunit NuoL. To investigate its role in proton translocation, we characterized the electron transfer and proton translocation activity of complex I variants lacking either NuoL or parts of the C-terminal domain. Our data suggest that the H+/2e- stoichiometry of the ΔNuoL variant is 2, indicating a different stoichiometry for proton translocation as proposed from structural data. In addition, the same H+/e- stoichiometry is obtained with the variant lacking the C-terminal transmembraneous helix of NuoL, indicating its role in energy transmission.     

The role of glycine residues 140 and 141 of subunit B in the functional ubiquinone binding site of the Na+-pumping NADH:quinone oxidoreductase from Vibrio cholerae

The Na(+)-pumping NADH:quinone oxidoreductase (Na(+)-NQR) is the main entrance for electrons into the respiratory chain of many marine and pathogenic bacteria. The enzyme accepts electrons from NADH and donates them to ubiquinone, and the free energy released by this redox reaction is used to create an electrochemical gradient of sodium across the cell membrane. Here we report the role of glycine 140 and glycine 141 of the NqrB subunit in the functional binding of ubiquinone. Mutations at these residues altered the affinity of the enzyme for ubiquinol. Moreover, mutations in residue NqrB-G140 almost completely abolished the electron transfer to ubiquinone. Thus, NqrB-G140 and -G141 are critical for the binding and reaction of Na(+)-NQR with its electron acceptor, ubiquinone.