Transducer of the coordinates of a biological object for the Thermogradient device

A device designed to determine the position of a biological object within the experimental field used to investigate behavioral reactions has been described. By means of light sources focusing the photodetector objective lens the square experimental field is divided into 9 equal sections. The movement of an animal in question from one section of the experimental field to another excites electric signals at the output of the corresponding photodetectors. These signals are processed and stored in the location unit made of integrated circuits. Further data processing for their convey to the pen-and-ink recorder is accomplished in the write pulse forming unite which is also made of microcircuits and discontinuous semiconductors. The device permits to document all the movement parameters of animals within the experimental field throughout the experiment.     

A Mechanical Device for Retrieving Ribbons of Ultrathin Sections Without Folds

An improved version of a mechanical device for retrieving ribbons of ultrathin sections free of wrinkles is described. The device is simple and easy to use and can be used by both right- and left-handed operators. A grid is grasped by forceps which are held in position by a magnet in such a way that it can be lowered into the water well behind the floating sections. After a ribbon has been properly aligned, moved to the grid and fastened by an end-section, the grid with the sections is slowly drawn up and out of the water with a rack-and-pinion mechanism.     

A new tool based on two micromanipulators facilitates the handling of ultrathin cryosection ribbons

A close to native structure of bulk biological specimens can be imaged by cryo-electron microscopy of vitreous sections (CEMOVIS). In some cases structural information can be combined with X-ray data leading to atomic resolution in situ. However, CEMOVIS is not routinely used. The two critical steps consist of producing a frozen section ribbon of a few millimeters in length and transferring the ribbon onto an electron microscopy grid. During these steps, the first sections of the ribbon are wrapped around an eyelash (unwrapping is frequent). When a ribbon is sufficiently attached to the eyelash, the operator must guide the nascent ribbon. Steady hands are required. Shaking or overstretching may break the ribbon. In turn, the ribbon immediately wraps around itself or flies away and thereby becomes unusable. Micromanipulators for eyelashes and grids as well as ionizers to attach section ribbons to grids were proposed. The rate of successful ribbon collection, however, remained low for most operators. Here we present a setup composed of two micromanipulators. One of the micromanipulators guides an electrically conductive fiber to which the ribbon sticks with unprecedented efficiency in comparison to a not conductive eyelash. The second micromanipulator positions the grid beneath the newly formed section ribbon and with the help of an ionizer the ribbon is attached to the grid. Although manipulations are greatly facilitated, sectioning artifacts remain but the likelihood to investigate high quality sections is significantly increased due to the large number of sections that can be produced with the reported tool.     

Colloidal gold immunostaining on deplasticized ultra-thin sections

We localized tissue antigens on ultra-thin sections by deplasticizing the sections while on the grid, incubating in primary antiserum followed by immunoglobulin-conjugated colloidal gold, and ultimately re-embedding in dilute Epon. This procedure permitted ultrastructural localization of tissue antigens that were previously masked by the embedding plastic surrounding tissue components. In addition, replacement of the plastic matrix on the thin section after immunostaining prevented development of the drying artifacts that occur in unsupported tissue sections. Optimal preservation of components in the tissue sections was achieved despite extensive steps involved in plastic removal and immunostaining. This method may be useful in situations where the number of exposed epitopes on the surface of a thin section is low. The procedure also allows the use of antisera at greater dilutions and provides enhanced immunostaining specificity with low background.     

The Membrane Method of Electron Microscope Autoradiography

Thin sections of methacrylate and Araldite embedded tissues labelled with radioactive isotopes were transferred with a wire loop or brush from the knife edge onto thin formvar membranes which covered 7 mm holes in 76 × 25 × 1.5 mm or 76 × 38 × 1.5 mm plastic slides. To facilitate the mounting of sections, a platform supported the plastic slides close to the ultramicrotome knife. Photographic emulsion diluted 1:5 or 1:10 with water was applied with a pipette to the upper surface of each formvar membrane to cover the mounted sections. Excess emulsion was drained off and the remaining thin film was dried on a warm plate at 45 C to produce a uniform layer over the sections. After storing in the dark for several weeks, preparations were processed in photographic solutions and washed, and sometimes stained, before applying electron microscope grids to the underside of each formvar membrane. To detach each grid with its adherent formvar, section and emulsion, the membrane was pierced around the perimeter of the grid. Grain counts made over nuclei of cells labelled with tritiated thymidine indicate that emulsion is uniformly distributed over each section and that quantitative comparison is possible between labelled areas.     

A new approach to improve the quality of ultrathin cryo-sections; its use for immunogold EM and correlative electron cryo-tomography

Cryo-ultramicrotomy can be used to obtain ultrathin cryo-sections from cryo-fixed or aldehyde-fixed cryo-protected vitreous biologic samples. For immuno-gold EM, cryo-sections are retrieved from the cryo-chamber on a droplet of a pick-up solution (paste-like and almost frozen) to which the sections attach. The sections are then placed on an EM specimen grid at room temperature. This procedure compromises the ultrastructure, resulting in folds, holes, and loss of the original material. In this paper we show the critical influence of humidity, stretching, and relief of compression during thawing of the sections. We show a new lift-up hinge device for semi-automated retrieval of cryo-sections that results in significantly improved section quality. This approach was also applied successfully to vitreous sections from high pressure frozen samples. An important advance is that these vitreous cryo-sections can now successfully be post-fixed and immunolabelled after thawing; this allows cryo-EM comparison with adjacent ribbons of sections still in the frozen hydrated state. These findings call for technical innovations aiming at automated cryo-ultramicrotomy in a fully controlled environment for improved localization of proteins within their 'close to native' cellular context and correlative electron cryo-tomography of consecutive ribbons of sections of one frozen hydrated sample.     

A Device for the Precise Transfer of Serial Sections for Electron Microscopy

A device based on a standard stereotaxic instrument, in which a Formvar-dipped loop carrying serial thin sections, and a rotatable grid holder are mounted in separate electrode holders, thus permitting accurate positioning of each with respect to the other and the precise transfer of the sections to the grid, is described and illustrated.     

~(16)O~(8+)离子辐照普通小麦引起幼苗可溶性蛋白质的不同变化

利用不同能量、不同剂量的1 6 O8+ 离子辐照春麦。处理方式为离子贯穿种子 (75MeV u)以及离子注入胚和胚乳等种子的不同部位。然后分析其幼苗的可溶性蛋白质 (solubleprotein) ,根据其分子量大小不同 ,将其划分为五个区段并计算各组分的相对含量 ,结果表明 :1 同对照相比 ,随着辐照剂量的增加 ,所有辐照材料 (包括贯穿和注入 )第二区段 (分子量较高区段 )蛋白质组分的相对含量下降 ;而第五区段 (分子量最小区段 )蛋白质组分的相对含量升高。 2 种子的贯穿处理 ,同时也引起第一区段 (分子量最高区段 )和第三区段蛋白质组分相对含量的下降以及第四区段的升高 ;其中第三、四区段的变化明显区别于注入效应。 3 注入胚和胚乳的区别在于后者第一区段蛋白质组分相对含量的较大下降和第五区段的较大提高。 4 小剂量辐照的材料 ,可溶性蛋白质组分的变化异常 ,可能与低剂量辐射兴奋效应有关。在讨论中提出了注入胚的即时效应与注入胚乳的后期效应。     

Impact of photon cross section uncertainties on Monte Carlo-determined depth-dose distributions

This work studies the impact of systematic uncertainties associated to interaction cross sections on depth dose curves determined by Monte Carlo simulations. The corresponding sensitivity factors are quantified by changing cross sections by a given amount and determining the variation in the dose. The influence of total and partial photon cross sections is addressed. Partial cross sections for Compton and Rayleigh scattering, photo-electric effect, and pair production have been accounted for. The PENELOPE code was used in all simulations. It was found that photon cross section sensitivity factors depend on depth. In addition, they are positive and negative for depths below and above an equilibrium depth, respectively. At this depth, sensitivity factors are null. The equilibrium depths found in this work agree very well with the mean free path of the corresponding incident photon energy. Using the sensitivity factors reported here, it is possible to estimate the impact of photon cross section uncertainties on the uncertainty of Monte Carlo-determined depth dose curves.     

An Ocular Grid-Planimeter Method for Enumerating Nerve Fibers

The estimation of numbers of nerve fibers in cross sections of peripheral nerves containing both fine and large fiber components can be accomplished by using an ocular grid and selectively counting a known area. With the use of a projecting apparatus and planimeter, the total cross section area is determined.

The following proportion expresses the principle involved:

total number of fibers in the nerve area of ass section of entire nerve number counted in the sample area area of sample

I f the planimeter calibration and the magnification of the tracing remain the same, a constant factor may be used for successive estimates. This factor is equal to the value of the planimeter reading of 1.000 divided by the area of the grid times the magnification squared. The final calculations are made by multiplying the number of fibers counted times the planimeter reading times the constant and dividing by the number of grid squares counted.

Counts of some nerves, using high magnification in enumerating the sample areas, can be finished in less than an hour after the preparation of slides. In comparing numbers obtained by complete counts with estimated numbers, the error was determined to be approximately ± 5%     

Gap junction structures. VIII. Membrane cross-sections.

Profiles of negatively stained gap junctions have been measured by grid sectioning. After normal levels of electron irradiation, the membrane thickness shrinks to about half that of unirradiated controls, but no shrinkage occurs in the hexagonal lattice plane. Even under low irradiation conditions, there is significant thinning of the membranes. Edge views, in which rows of connexons are aligned parallel to the beam, were obtained from grid sections, folds in normal negatively stained specimens, and sections of a positively stained specimen. Averaging these micrographs with the translational and mirror symmetry of the projected lattice image displays conserved and variable features in the stain distribution of different specimens. Variations in the relative amount of negative stain in the gap at the surfaces and in the channel are uncorrelated with the irradiation but appear to depend on the local staining conditions and the integrity of the connexons. The dimensions measured from previously unirradiated grid sections, folds, and positively stained sections are in accord with x-ray diffraction measurements. Radiation-induced shrinkage can be accounted for by mass loss principally from the membrane bilayer. Disordering of the surface structure appears to be correlated with the radiation sensitivity of the bilayer; in contrast, the gap structure is well preserved under a variety of conditions.     

Your “Third Hand” in Mounting Serial Sections on Grids for Electron Microscopy

A heavy base 12 cm in diameter supports a vertical rod 40 × 3 cm. To this rod is clamped a horizontal rod 24 × 1.5 an, which bears on its outer end a rack-and-pinion mechanism that carries a clamp. Slow motion can be imparted to the clamp by a knurled pinion knob. For use, a grid is grasped at its edge by a watchmaker's forceps, and the forceps secured in the clamp. The rack and pinion is adjusted to give motion at about 45° and thus allow the grid to be lowered at an angle into the flotation water. While observed under magnification, the ribbon of sections can be manipulated into proper alignment with the grid, the end section fastened to the grid, and the grid raised mechanically to cause the remainder of the ribbon to adhere to it.     

The Use of Ethylenediamine in Softening Hard Plant Structures for Paraffin Sectioning

Ethylenediamine has been used as an agent for softening very hard woods prior to sectioning on a sliding microtome. The use of ethylenediamine is recommended for two additional uses: for preparing 1) soft woods in which wide, thin-walled tracheids or vessels tend to collapse during sliding microtome sectioning and 2) plant tissues with sclerenchyma mixed with soft-walled cells (bark, leaves, fruits, etc.) which frequently fail to section well. After softening in ethylenediamine, material is washed, infiltrated, and embedded in paraffin. Preliminary sections are made with a rotary microtome, just exposing the cut surface of the material; this exposed surface is soaked overnight in water. Sectioning is then continued. Sections produced in this fashion are considerably improved. The wood and pith of Podocarpus ustus, a parasitic conifer from New Caledonia, is used as an object to demonstrate improvements in sectioning by the ethylenediamine-paraffin method. Thinner sections with minimal tearing, cell collapse, and unevenness are produced. Sections can be handled easily and stained more effectively than unmounted sections. Variations in timing and in treatment are recommended to suit different materials. Ethylenediamine, used with reasonable caution, is much less hazardous than hydrofluoric acid and is more effective in softening plant material. The ethylenediamine method may be used routinely on any material difficult to section because of hardness.     

Cesarean section: trends and morbidity.

A review of 1683 cesarean sections performed at one hospital in a 3-year period (1977-79) showed that the cesarean section rate had trebled since 1967-79, the rates being 16.9% and 5.8%. The main indications for cesarean section responsible for this rise were dystocia, breech presentation and a previous cesarean section. AFter the operation 23.3% of received antibiotics. If the cesarean section rate is to fall, the biggest impact can be made by planning vaginal delivery in selected patients with a previous cesarean section and by improving the management of nonprogressive labour.     

Method for Staining and Washing Thin Sections on Support Films

A procedure is described for staining large numbers of thin sections on support films for use with one-hole grids. The film is picked up, carried and protected using easily made plastic blocks. Loop-tipped forceps are then used to transfer tissue ribbons from the knife boat to the support film. A large number of tissue sections can then be stained and washed simultaneously in a modified Pyrex dish without damaging the film. After staining, the slot in the one-hole grid is centered over the tissue ribbon, and the grid is attached to the film. The method is suitable for serial reconstruction and the unobstructed viewing of large thin sections in the TEM.     

藏中联网工程生态系统服务功能重要性评价

对电网工程沿线区域的生态系统服务功能进行评价是规避和降低工程对沿线区域生态破坏的重要前提。本研究选取水源涵养、水土保持、防风固沙、生物多样性保护等4项生态系统服务功能为主体,基于遥感与GIS技术对藏东南地区生态系统服务功能的重要性进行评价,在此基础上分析了不同电网工程标段影响区的生态系统服务功能重要性。结果表明:藏东南地区生态系统服务功能重要性总体上呈现出"东西部高、中部低"的空间分布格局,极重要区面积为74843 km²,占区域总面积的18.47%,主要分布在藏东南的森林区,包括澜沧江沿线及其以东地区、雅鲁藏布江沿线及错那县、墨脱县南部。藏中联网工程东、西两端标段影响区内的生态系统服务功能重要性相对较高,在施工过程中应根据影响区内主导的生态系统服务功能分布状况采取相应措施加强生态环境保护。本研究方法可为其他输电工程的设计和施工提供生态标准及科学依据。     

Improving the technique of vitreous cryo-sectioning for cryo-electron tomography: Electrostatic charging for section attachment and implementation of an anti-contamination glove box

Cryo-electron tomography of vitreous cryo-sections is the most suitable method for exploring the 3D organization of biological samples that are too large to be imaged in an intact state. Producing good quality vitreous cryo-sections, however, is challenging. Here, we focused on the major obstacles to success: contamination in and around the microtome, and attachment of the ribbon of sections to an electron microscopic grid support film. The conventional method for attaching sections to the grid has involved mechanical force generated by a crude stamping or pressing device, but this disrupts the integrity of vitreous cryo-sections. Furthermore, attachment is poor, and parts of the ribbon of sections are often far from the support film. This results in specimen instability during image acquisition and subsequent difficulty with aligning projection images.Here, we have implemented a protective glove box surrounding the cryo-ultramicrotome that reduces the humidity around and within the microtome during sectioning. We also introduce a novel way to attach vitreous cryo-sections to an EM grid support film using electrostatic charging. The ribbon of vitreous cryo-sections remains in place during transfer and storage and is devoid of stamping related artefacts. We illustrate these improvements by exploring the structure of putative cellular 80S ribosomes within 50 nm, vitreous cryo-sections of Saccharomyces cerevisiae.     

冰冻切片与石蜡切片对乳腺肿瘤的诊断价值研究

目的:分析和比较冰冻切片与石蜡切片对乳腺肿瘤的诊断价值。方法:选取480例新鲜乳腺标本,将其制成冰冻切片以及石蜡切片,根据诊断结果进行对比分析,评价乳腺肿瘤的冰冻切片与石蜡切片的对乳腺肿瘤的诊断价值。结果:经石蜡切片诊断乳腺良性肿瘤277例,占57.71%,良性肿瘤中以乳腺纤维瘤诊断居多;经石蜡切片诊断乳腺恶性肿瘤203例,占42.29%,以乳腺浸润性导管癌居多。冰冻切片诊断乳腺良性肿瘤279例,占58.13%;恶性肿瘤195例,占40.62%;延迟诊断6例,占1.25%。以石蜡切片诊断结果为金标准,冰冻切片诊断乳腺良性肿瘤的准确率为98.56%(273/277),诊断恶性肿瘤的准确率为95.07%(193/203),假阳性率为0.72%(2/277),假阴性率为2.96%(6/203),冰冻切片与石蜡切片诊断乳腺肿瘤的结果具有显著一致性,K值为0.965(P0.05)。结论:冰冻切片与石蜡切片诊断乳腺肿瘤的符合率较高,可作为术中快速病理检测的手段,但该种切片方式存在少量延迟诊断,多与术者操作经验有关,故术中应注重制片过程,提高冰冻切片质量。     

Technical note: A stereological analysis of the cross‐sectional variability of the femoral osteon population

Unbiased selection of regions of interest (ROIs) and unbiased definition of histological structures are needed to improve the repeatability of microscopic methods for age at death determination and to reduce operator subjectivity. We present results obtained by selecting ROIs according to stereological principles on a sample of 28 femoral cross sections of Caucasoid males aged 20–89 years. A regular grid was overlaid on the cross section, and the ROIs were selected as close as possible to the periosteum in the anterior, lateral, and medial regions. The areas consisting of all intact secondary osteons plus fragments were outlined and osteon population density, percent osteon population, area, and perimeter were calculated using stereological methods and software. Overall, the analyses of intra‐ and inter‐section variability showed no significant difference between the ROIs, i.e., the location within the cross section of the ROIs does not affect the outcome of the analyses. The individual variability was found to be higher in adults aged 30–55 years than in other age ranges. ranges. Am J Phys Anthropol 2010. © 2010 Wiley‐Liss, Inc.     

Electron tomographic analysis of frozen-hydrated tissue sections

Electron tomography of frozen-hydrated tissue sections enables analysis of the 3-D structure of cell organelles in situ and in a near-native state. In this study, 160-200-nm-thick sections were cut from high-pressure frozen rat liver, and improved methods were used for handling and mounting the sections. Automated data collection facilitated tilt-series recording at low electron dose (approximately 4000 e(-)/nm(2) at 400 keV). Higher doses (up to 10,000 e(-)/nm(2)) were found to increase contrast and smooth out surface defects, but caused section distortion and movement, with likely loss of high-resolution information. Tomographic reconstruction showed that knife marks were 10-40 nm deep and located on the "knife face" of the section, while crevices were 20-50 nm deep and found on the "block face." The interior of the section was normally free of defects, except for compression, and contained useful structural information. For example, the topology of mitochondrial membranes in tissue was found to be very similar to that in frozen-hydrated whole mounts of isolated mitochondria. In rare cases, a 15-nm banding pattern perpendicular to the cutting direction was observed in the interior of the section, most evident in the uniformly dense, protein-rich material of the mitochondrial matrix.