Application of high-resolution scanning electron microscopy to biological macromolecules

The development of ultrahigh-resolution scanning electron microscopes (SEMs) has made the observation of biological macromolecules feasible, but adequate preparation methods have not yet been established. Although it has been possible to observe some molecules after they have been spread on a carbon substrate, this method has not proved suitable for other molecules which exhibit lower contrast, or are more susceptible to damage by the electron beam. In this study we have applied heavy-metal impregnation methods using phosphotungstic acid, uranyl acetate, or osmium tetroxide mordanted by tannic acid. In addition, contamination due to the electron beam was reduced by improving the vacuum in the specimen chamber, and by the use of a heated specimen stage. Using these measures, haemocyanin, ferritin, apoferritin, thyro-globulin and immunoglobulin M were successfully imaged. Ultrahigh-resolution SEM seems likely to become an important means for studying the morphology of biological macromolecules.     

Cathodoluminescence of biological molecules, macromolecules and cells.

Cathodoluminescence observations on biological compounds are compared with previously established data from ultra violet and visible light excitation studies. The comparison demonstrates that the same molecules are responsible for the luminescence properties of macromolecules independent of the type of exciting radiation. Natural cathodoluminescence was also observed from cells. Moreover, advantages gained by the absorption of strongly cathodoluminiscent dyes into cells are demonstrated.     

Imaging biological macromolecules by STM: quantitative interpretation of topographs

Methods are discussed which permit the calibration of x-, y-, z-sensitivities, non-linearities and frequency responses of the scanning device of a scanning tunneling microscope (STM) either by interferometry or directly from STM topographs. A technique is presented to measure the frequency response of the complete STM feedback unit and to derive a maximum speed in z direction which allows one to estimate the maximum scanning speed still permitting one to track surface corrugations. The signal transfer characteristics of a STM are evaluated in a direct comparison with high resolution transmission electron microscopy on an identical specimen area. The various effects of contaminants between tip and specimen and the finite tip radius receive special attention.     

Detection, classification and 3D reconstruction of biological macromolecules on hypercube computers.

In this work we present results of the mapping on hypercube computers of some of the key steps involved in the procedure for 3D structural determination from transmission electron microscopy images. The goal is the introduction of parallel processing tools in the field of electron microscopy image processing. We show how the rich topology of the hypercube, combined with an efficient programming strategy, allows for order-of-magnitude increase in computational capacity for such time-consuming tasks as calculation of multidimensional FFT's, cross-correlation coefficients, fuzzy partitioning functionals and the filtered back-projection 3D reconstruction method.     

Comparative mass measurement of biological macromolecules by scanning transmission electron microscopy

A method is described for measuring the mass/length or mass of molecular assemblies by comparative electron scattering in the STEM. Standard particles whose mass is well established (e.g. TMV or fd bacteriophage) are deposited on the electron microscope grid together with the sample to be measured. Images containing at least one sample and standard and with a clean, contamination-free background are chosen and stored on computer disc and then directly integrated. Use of a comparative technique does not require accurate determination of scattering parameters or instrumental geometry and requires only that the limits of linearity be established. The results of the mass/length measurements on phage pf 1, pili, muscle thick filaments and actin are in good agreement with existing molecular weight data and generally have a standard deviation of about 10%. The results for the total mass measurement of the multisubunit enzymes glutamate dehydrogenase and glutamine synthetase are also close to the literature values for their molecular weights. The results for the spherical, Semliki forest and tomato bushy stunt viruses are lower than expected, possibly reflecting some dissociation during preparation.     

Use of multivariate statistics in analysing the images of biological macromolecules

We have developed a new technique of analysis that allows automatic classification of molecule images according to subtle differences. Computer alignment and multivariate statistical methods were used to analyze electron micrographic images of horseshoe crab hemocyanin half-molecules. The molecule projections fell into four distinct classes related to four different positions of the molecule on the grid. Averages obtained for each images subset are interpreted in terms of a three-dimensional model arrangement for the four subunits forming the half-molecule.     

Quantitative STEM mass measurement of biological macromolecules in a 300 kV TEM

For almost four decades, the scanning transmission electron microscope (STEM) has made significant contributions to structural biology by providing accurate determinations of the molecular masses of large protein assemblies that have arbitrary shapes and sizes. Nevertheless, STEM mass mapping has been implemented in very few laboratories, most of which have employed cold field‐emission gun (FEG) electron sources operating at acceleration voltages of 100 kV and lower. Here we show that a 300 kV commercial transmission electron microscope (TEM) equipped with a thermally assisted Shottky FEG can also provide accurate STEM mass measurements. Using the recently published database of elastic‐scattering cross sections from the National Institute of Standards and Technology, we show that the measured absolute mass values for tobacco mosaic virus and limpet hemocyanin didecamers agree with the known values to within better than 10%. Applying the established approach, whereby tobacco mosaic virus is added to a specimen as a calibration standard, we find that the measured molecular weight of the hemocyanin assemblies agrees with the known value to within 3%. This accuracy is achievable although only a very small fraction (∼0.002) of the incident probe current of 300 kV electrons is scattered onto the annular dark‐field STEM detector. FEG TEMs operating at intermediate voltages (200–400 kV) are becoming common tools for determining the structure of frozen hydrated protein assemblies. The ability to perform mass determination with the same instrument can provide important complementary information about the numbers of subunits comprising the protein assemblies whose structure is being studied.     

Transcytosis of plasma macromolecules in endothelial cells: a cell biological survey

The modern exploration of endothelial cell biology is a largely interdisciplinary exercise. Cell biological, physiological, and more recently molecular biology approaches were used to study the pathways and the organelles involved in transcytosis of macromolecules in endothelial cell (EC). Here we discuss mainly the cell biological findings that revealed that EC have the attributes to fulfill the transport function. They are polarized cells, heterogeneous, and, thus, structurally and functionally adapted to the vascular bed in which they reside. The structural heterogeneity involves the number and distribution of plasmalemmal vesicles (caveolae), their generated channels, and the organization of intercellular junctions. The closely related functional heterogeneity comprises the degree of permeability for plasma molecules that vary as a function of organ. The EC are endowed with the cellular machinery to perform (1) endocytosis, that is to take up plasma proteins and the molecules they carry to be used for themselves (cholesterol-carrying low density lipoproteins, fatty acid carrying albumin, iron carrying transferrin, etc.), and (2) transcytosis, which implies to transport plasma proteins to the subjacent cells and tissues. The possible pathways for transport of molecules are transcellular, via caveolae and channels, and paracellular via intercellular junctions. Most of the results obtained, so far, indicate that transcytosis of albumin, low-density lipoproteins, metaloproteases, and insulin, is performed by cargo-vesicles and their generated channels. The paracellular pathway can be used for water and ions; in postcapillary venules, at the level of which approximately 30% of junctions are open to a space of 6 nm, small molecules may take this route. Recent data obtained by molecular biology techniques revealed that caveolae are endowed with the molecular machinery for fusion/fission, docking, and movement across cells. Moreover, the various and numerous molecules that have been detected in the caveolae membrane and the different functions assumed by this differentiated microdomain strengthen the postulate that there are at least two or more types of vesicles molecularly tailored for the local physiological requirements.     

Multivariate statistical classification of noisy images (randomly oriented biological macromolecules)

Multivariate Statistical Analysis (MSA) methods have recently been introduced for analyzing images of biological macromolecules [Van Heel and Frank, Ultramicroscopy 6 (1981) 187]. With these techniques, the significant characteristics of each molecular image can be expressed in merely 2 to 8 factorial coordinate values rather than in the typical 64 X 64 = 4096 pixel grey values that originally described the image. This very large reduction in total amount of data facilitates the understanding of the general behavior of a set of molecular images in terms of classes or of general trends in the data set. The (artificial) intelligence of the procedure, however, lies in the decision-making or classification phase. The theory and philosophy of multivariate statistical classification are reviewed using generalized metrics. Problem-dependent classification rationales are proposed. A set of computer-generated "randomly oriented molecular images" are used to test the classification schemes. This model experiment is a step towards 3D structure analysis of macromolecules based on large numbers of (noisy) electron microscopical images of randomly oriented biological macromolecules.     

Characterization of biological macromolecules by combined mass mapping and electron energy-loss spectroscopy

The combination of scanning transmission electron microscopy (STEM) and parallel-detection energy-loss spectroscopy (EELS) was used to detect specific bound elements within macromolecules and macromolecular assemblies prepared by direct freezing. After cryotransferring and freeze-drying in situ, samples were re-cooled to liquid nitrogen temperature and low-dose (about 10³ e/nm²) digital dark-field images were obtained with single-electron sensitivity using a beam energy of approximately 100 keV and a probe current of approximately 5 pA. These maps provided a means of characterizing the molecular weights of the structures at low dose. The probe current was subsequently increased to about 5 nA in order to perform elemental analysis. The 320 copper atoms in a keyhole limpet haemocyanin molecule (mol.wt = 8 MDa) were detected with a sensitivity of ± 30 atoms in an acquisition time of 200 s. Phosphorus was detected in an approximately 10-nm length of single-stranded RNA contained in a tobacco mosaic virus particle (mol.wt = 130 kDa/nm) with a sensitivity of ± 25 atoms. Near single-atom sensitivity was achieved for the detection of iron in one haemoglobin molecule (mol.wt = 65 kDa, containing four Fe atoms). Such detection limits are only feasible if special processing methods are employed, as is demonstrated by the use of the second-difference acquisition technique and multiple least-squares fitting of reference spectra. Moreover, an extremely high electron dose (about 10¹⁰ e/nm²) is required resulting in mass loss that may be attributable to ‘knock-on’ radiation damage.     

Image contrast in high-resolution electron microscopy of biological macromolecules: TMV in ice.

It is shown that the contrast in high-resolution electron micrographs of biological macromolecules, illustrated by a study of TMV in ice, falls considerably below the level which should theoretically be attained. The factors which contribute to the low contrast include radiation damage, inelastic scattering, specimen movement and charging. Future progress depends on improved understanding of their contributions and relative importance. Contrast is defined as the amplitude of a particular Fourier component extracted from an image in comparison to that expected by extrapolation from separate electron or X-ray diffraction measurements. The fall in contrast gets worse with increased resolution and is particularly serious at 10 A and beyond for specimens embedded in vitreous ice, a method of specimen preparation which is otherwise particularly desirable because of the expectation that the embedded molecules should be well preserved in a near-native environment. This low contrast at high resolution is the principal limitation to atomic-resolution structure determination by electron microscopy. In spite of good progress in the direction of better images, it remains a major problem which prevents electron microscopy from becoming a simple and rapid method for biological atomic structure determination.     

Fuzzy sets-based classification of electron microscopy images of biological macromolecules with an application to ribosomal particles

Pattern recognition methods based on the theory of fuzzy sets are tested for their ability to classify electron microscopy images of biological specimens. The concept of fuzzy sets was chosen for its ability to represent classes of objects that are vaguely described from the measured data. A number of partitional clustering algorithms and an extensive set of cluster-validity functionals (some already reported and some newly developed) have been applied to a test-data set and to two real-data sets of images. One of the real-data sets corresponded to images of the Escherichia coli 50S ribosomal subunits depleted of proteins L7/L12 and the other set to images of the E. coli 70S monosome in the range of overlap views. These two latter sets had been previously studied by another clustering methodology. The new results obtained by the application of fuzzy clustering techniques will be compared to those previously obtained and some conclusions about the consistency of these classifications will be drawn from this comparison.     

Structural and functional aspects of biological freezing techniques.

The cooling procedures used to prepare samples for ultrastructural examination at low temperatures often differ markedly from those used to recover optimal function of cells on thawing. The implications of these differences are reviewed. Damage and alteration to the structure and function of the cells may be caused by the high concentrations of cryoprotective agents such as glycerol or dimethyl sulphoxide (DMSO) often added to reduce ice crystal artefacts. Under the rapid cooling conditions commonly employed for structural studies, these additives are not cryoprotective; low rates of cooling are necessary for them to be effective. Rapidly cooled cells that contain intracellular ice are only injured during rewarming so their structure may be as yet unaltered by any damaging effects at low temperatures. Most cells able to recover on thawing are grossly shrunken at low temperatures but since they are potentially functional they are of interest structurally. These cryobiological principles are illustrated with freeze-fracture, freeze substitution and functional assays. The cell types chosen were Chlorella sp. and mammalian tissue culture cells.     

不同部位的功能性磁刺激对于脊髓损伤所致高反应性膀胱临床疗效研究

目的 应用不同部位功能性磁刺激(S1、S3)治疗脊髓损伤高反应性膀胱,评估治疗疗效。方法 此次研究选取2019.1.1—2021.12.31我院脊髓损伤高反应性膀胱功能障碍患者55例,按照不同部位功能性刺激为两组,S1部位治疗组及S3部位治疗组。入组时进行膀胱功能评定,在完成10次治疗后,再次进行膀胱功能评定,对比治疗前后及各组间的疗效差异。结果 两组治疗前膀胱的最大容量(ml)、最大压力(cmH_2O)、残余尿量(ml)进行分析,数据无差异。治疗前后,两组组内比较均有统计学差异(P<0.05)。两组患者治疗过程中及治疗后患者无不良反应。结论 S1、S3部位功能性磁刺激及常规康复治疗脊髓损伤高反应性膀胱功能障碍均有临床治疗效果,S3组功能性磁刺激治疗效果优于S1组,且治疗方案安全。     

Interfacial energies and surface-tension forces involved in the preparation of thin,flat crystals of biological macromolecules for high-resolution electron microscopy

It is generally agreed that surface-tension forces and the direct interaction between the specimen and either the air-water interface or the water-substrate interface can influence significantly the preparation of biological materials for electron microscopy. Even so, there is relatively little systematic information available that would make it possible to control surface-tension forces and interfacial energies in a quantitative fashion. The main objective in undertaking the present work has been to understand somewhat better the factors that influence the degree of specimen flatness of large, monolayer crystals of biological macromolecules. However, the data obtained in our work should be useful in understanding the preparation of specimens of biological macromolecules in general. Data collection by electron diffraction and electron microscopy at high resolution and high tilt angles requires thin crystals of biological macromolecules that are flat to at least 1°, and perhaps less than 0·2°, over areas as large as 1 μm² or more. In addition to determining empirically by electron diffraction experiments whether sufficiently flat specimens can be prepared on various types of modified or unmodified carbon support films, we have begun to use other techniques to characterize both the surfaces involved and the interaction of our specimen with these surfaces. In the specific case of large, monolayer crystals of bacteriorhodopsin prepared as glucose-embedded specimens on hydrophobic carbon films, it was concluded that the initial interfacial interaction involves adsorption of the specimen to the air-water interface rather than adsorption of the specimen to the substrate. Surface-tension forces at the air-water interface and an apparently repulsive interaction between the specimen and the hydrophobic carbon seem to be major factors influencing the specimen flatness in this case. In the more general case it seems likely that interfacial interactions with either the substrate or the air-water interface can be variously manipulated in the search to find desirable conditions of specimen preparation.     

Cellular and subcellular coherent dynamics,biological functional properties,and system-environment interaction

In this view point paper, we briefly summarize some of the clinical, biochemical and biophysical results obtainedin our research on Relaxation Response. We also qualitatively describe the theoretical biophysical model that could linkthem. Our work points to a unified view of the human biological system activity, joining the dynamics ruling theinteractions and correlations of the microscopic components to the knowledge of their specific individual propertiesin the effort of going beyond a purely atomistic approach.     

科学计算可视化智能映射技术的研究

分析了可视化映射的特点,提出了一种在可视化映射问题进行方案设计时,将神经网络用于类比和联想,在可视化映射方案进行选择与调整时,将专家系统用于逻辑推理的可视化智能映射技术。     

A novel method for the prediction of functional biological activity of polyethylene wear debris

The comparative performance of artificial hip joints has been extensively investigated in vitro through measurements of wear volumes. In vivo a major cause of long-term failure is wear-debris-induced osteolysis. These adverse biological reactions are not simply dependent on wear volume, but are also controlled by the size and volumetric concentration of the debris. A novel model is presented which predicts functional biological activity; this is determined by integrating the product of the biological activity function and the volumetric concentration function with the wear volume over the whole particle size range. This model combines conventional wear volume measurements with particle analysis and the output from in vitro cell culture studies to provide a new indicator of osteolytic potential. The application of the model is demonstrated through comparison of the functional biological activity of wear debris from polyethylene acetabular cups articulating under three different conditions in a hip joint simulator.