A Simple Holder for Efficient Mass Staining of Thin Sections for Electron Microscopy

An inexpensive simply-constructed gridholder for reproducible staining of sections and storage of grids is made of a platelet of dental wax (Ladd No. 2460, or Belladi, rosa, normal) mounted on a glass slide. The grids are simply inserted perpendicularly into the wax and are held securely at their edges by the slightly sticky wax. Staining solutions may be applied directly to the standing grids or the gridholder may be inverted in a trough containing the staining solution, thus avoiding excessive contact with the air. Washing is carried out by dipping the gridholder in a series of beakers with distilled water or by flushing gently. The grids may be labeled directly on the slide and are therefore identified easily. The wax-gridholder eliminates excessive handling of the grids with tweezers not only during the staining procedure but as well as during storage and thus helps to prevent mechanical damage of the delicate sections mounted on the grids. Waxes other than those specified should not be used because of a tendency for particles to adhere to the grids, with the attendant possibility of column contamination.     

Can demographic parameters be estimated from capture-recapture data on small grids? A test with forest rodents

This preliminary study proposes small trapping grids as an alternative to traditional large grids for the simultaneous monitoring of several rodent populations by capture--recapture. Monthly trapping sessions of wood mouse Apodemus sylvaticus (Linnaeus, 1758) and bank vole Clethrionomys glareolus (Schreber, 1780) were carried on over a small area (0.015 ha, 21 traps). The coherence of demographic parameter estimates on such small grids with those obtained on classical large grids was checked by performing two trapping sessions on a larger grid (0.9 ha, 110 traps, 10 m mesh) surrounding the small grid. We compared the two grid designs on the basis of sex ratio upon first capture, trap saturation rate, minimum number alive (MNA), monthly survival, and trappability. These demographic parameters proved to be non-biased by the trapped area, even though the precision was lower on the small grid. Small grids seem therefore to give the same picture of population dynamics as classical large grids except for parameters sensitive to an edge effect (eg density). By decreasing significantly the trapping effort, small grids will be of particular interest whenever the simultaneous operation of several trapping grids is needed (eg to compare different environmental conditions).     

Grid staining--a useful device.

A simple and disposable holder for electron microscope grids can be constructed from discs of silicon rubber. The discs (10 mm thick X 25 mm diameter) are scored 0.4 mm deep with a razor. Flexing the discs opens the scores for insertion of grids. Staining wells can be made from polyethylene snap-in vial tops by cutting away the nipple-like portion. After impaling the holders on the tips of a pair of forceps the grids can be washed by immersion. This holder is rigid, inert and inexpensive. It is of particular advantage for handling single-slotted grids.     

Grid Staining—A Useful Device

A simple and disposable holder for electron microscope grids can be constructed from discs of silicon rubber. The discs (10 mm thick × 25 mm diameter) arc scored 0.4 mm deep with a razor. Flexing the discs opens the scorns for insertion of grids. Staining wells can be made from polyethylene snap-in vial tops by cutting away the nipple-like portion. After impaling the holders on the tips of a pair of forceps the grids can be washed by immersion. This holder is rigid, inert and inexpensive. It is of particular advantage for handling single-slotted grids.     

Association of an axonally transported polypeptide (H) with 100-A filaments. Use of immunoaffinity electron microscope grids

Polypeptide H (mol wt 195,000) is axonally transported in rabbit retinal ganglion cells at a velocity of 0.7--1.1 mm/d, i.e., in the most slowly moving of the five transport groups described in these neurons. To identify the organelle with which H is associated, we purified H, prepared antibodies directed against it, and adsorbed the antibodies onto Formvar-coated electron microscope grids. When the resulting "immuno-affinity grids" were incubated with extracts of spinal cord and then examined in the electron microscope, they contained as many as 100 times more 100-A filaments than did grids coated similarly with nonimmune IgG. The ability of the anti-H IgG to specifically adsorb filaments to grids was completely blocked by incubating the IgG with polypeptide H. The 100-A filaments adsorbed to anti-H immunoaffinity grids could be specifically decorated by incubating them with anti-H IgG. These observations demonstrate that H antigens (and most likely H itself) are associated with 100-A neurofilaments. In addition, they suggest that the use of immunoaffinity grids may be a useful approach for determining the organelle associations of polypeptides.     

An optimized device for staining electron microscopy grids

Proper staining of grids is critical for transmission electron microscopy (TEM). Staining must be done as quickly as possible using minimal reagents and with consideration for the environment. We developed a new device for efficient staining of multiple TEM grids. We studied reagent evaporation, rinsing volume, flow rate and re-use of uranyl acetate, and provide here a procedure for efficient staining using the new device. Our device permits TEM grids to be stained with less reagent than alternative staining apparatuses; staining requires a total volume of 260 μl for five grids. Reagent evaporation is less than 6% even if used at 37° C. Moreover, our staining apparatus reduces chemical waste and shortens experiment time by staining several grids simultaneously. Our staining device is a compromise between time-consuming single grid processing and expensive commercial devices that consume large amounts of reagents.     

Supported phospholipid bilayers for two-dimensional protein crystallization

Phospholipid bilayers, supported on UV irradiated carbon shadowed nitrocellulose electron microscope grids, have been used to induce two-dimensional crystal growth of IgE and IgG anti-DNP monoclonal antibodies. The UV irradiation renders the grids hydrophilic in a very uniform fashion and allows for the transfer of phospholipid monolayers from an air/water interface in a sequential dipping procedure. The surface coverage achieved was nearly 100% as measured by antibody binding and by the formation of protein arrays on the bilayer covered grids. The supported bilayers appear to be stably held and are appropriate for slow binding conditions and long incubation times with low concentrations of binding protein.     

Nickel Grids Cause Astigmatism in the Transmission Electron Microscope

Nickel grids are used in various methods (e.g., in immunocytochemistry) where chemically inert grids are required. Recently (Neiss 1983) we have used formvar-coated nickel grids when removing osmium from mounted ultrathin sections with 10% periodic acid (Lewis and Knight 1977). This is possible because nickel, unlike copper, adequately withstands the oxidation necessary for osmium removal. Handling sections on nickel grids avoids the disadvantages of free-floating sections, whose use for osmium removal has been recommended by Lewis and Knight (1977, chapter 2.2.3).     

Fabrication of carbon films with ∼500 nm holes for cryo-EM with a direct detector device

Single particle electron cryomicroscopy (cryo-EM) is often performed using EM grids coated with a perforated or holey layer of amorphous carbon. Regular arrays of holes enable efficient cryo-EM data collection and several methods for the production of micropatterned holey-carbon film coated grids have been described. However, a new generation of direct detector device (DDD) electron microscope cameras can benefit from hole diameters that are smaller than currently available. Here we extend a previously proposed method involving soft lithography with a poly(dimethylsiloxane) (PDMS) stamp for the production of holey-carbon film coated EM grids. By incorporating electron-beam (e-beam) lithography and modifying the procedure, we are able to produce low-cost high-quality holey-carbon film coated EM grids with ∼500 nm holes spaced 4 μm apart centre-to-centre. We demonstrate that these grids can be used for cryo-EM. Furthermore, we show that by applying image shifts to obtain movies of the carbon regions beside the holes after imaging the holes, the contrast transfer function (CTF) parameters needed for calculation of high-resolution cryo-EM maps with a DDD can be obtained efficiently.     

Demographic attributes of dispersing southern bog lemmings (Synaptomys cooperi) in eastern Kansas

Summary Demographic attributes of southern bog lemmings colonizing two removal grids were compared with those of residents on neighboring control grids over a two year period in eastern Kansas. There was a positive association between the number of lemmings colonizing the removal grids and density of the control grids. Overall, 41% of the losses from the two control grids was accounted for by dispersal. The following differences were observed comparing residents with dispersers: 1) a greater proportion of males colonized the removal grids, 2) a lower percentage of adult females colonizing the removal grids were in breeding condition, whereas the reverse was true for subadult females, and 3) a greater proportion of subadults colonized the removal grids. These results are consistent with those obtained for other microtine species.I don't understand, said the scientist, why you lemmings all rush down to the sea and drown yourselves. How curious, said the lemming. The one thing I don't understand is why you human beings don't — from Intervie with a Lemming by James Thurber.     

A Simple Method for Handling Grids

A simple method for handling electron microscope grids is described here. While assuring their identification and safety, this method provides improved handling, temporary storage, and identification of grids bearing ultra-thin sections, and in addition, provides a novel method for preparing bulk samples. Grids are attached at their edges to the weakly adhesive surface of a “Post-it” note pad which sits in a petri dish. The grids are safely immobilized on the pad, classified based on their location, and identified by convenient pad notation. Grid manipulation and identification are simplified using this device, which is easily assembled from readily available and inexpensive materials.     

Production and Cleaning of Freeze-Etch Replicas which Show Complementary Surfaces of Fractured Fungus Spores and Hyphae

London finder grids (H-2) and 3 mm copper discs were flared on opposite sides by use of a die and punch. The grids were registered on the discs and attached with vaseline. The specimen was squeezed through the grid apertures by the 2 copper discs. The complete sandwich was frozen in freon 22 and placed in a hinged specimen holder which fractured the layer of the specimen between the 2 finder grids. Platinum-carbon and carbon replicas were made immediately after fracturing. After drying the replicas, the vaseline was removed by chloroform, and replicas were cleaned with 70% H₂SO₄ for 1-18 hr at 52 C, followed by 6% commercial sodium hypochlorite (Chlorox) for 1-2 hr at 23-25 C.     

Accurate Placement of Ultrathin Sections on Grids; Control by Sol-Gel Phases of a Gelatin Flotation Fluid

A 3% solution of gelatin in a petri dish at 25-30 C. provides a liquid, viscous surface upon which ultrathin sections can be floated. On cooling, the gelled substratum immobilizes the sections, allowing grids to be placed on them with any desired grid-to-section orientation. When the gelatin is remelted, the sections remain attached to the grids. After draining, traces of gelatin adhering to the grids are removed by flotation (section side down) for 30 min on 2% acetic acid at 60 C. This is followed by flotation for 3-5 min on Tris buffer, pH 7.1, and then on distilled water for 30 min—both treatments at 60 C. The technique is particularly useful for mounting serial sections.     

Simulations of airflow and substance concentration around a human body

In order to predict airflow and suspended substance concentration around a human body, we developed a geometric model of the human form and generated grids around it for Computational Fluid Dynamics (CFD). According to a CFSV model proposed by us we made a domain that included a geometric human model and generated the grids within this domain. By using this model with the grids and the developed CFD program, it is possible to simulate the airflow and the transfer of a suspended substance around the body. The simulated airflow provided a different velocity profile for each region of the body due to the characteristics of the body shape. The simulated distribution of the suspended substance concentration demonstrates how usable the present model is for quantifying a substance in any exposed region of the body.     

Towards automated screening of two-dimensional crystals

Screening trials to determine the presence of two-dimensional (2D) protein crystals suitable for three-dimensional structure determination using electron crystallography is a very labor-intensive process. Methods compatible with fully automated screening have been developed for the process of crystal production by dialysis and for producing negatively stained grids of the resulting trials. Further automation via robotic handling of the EM grids, and semi-automated transmission electron microscopic imaging and evaluation of the trial grids is also possible. We, and others, have developed working prototypes for several of these tools and tested and evaluated them in a simple screen of 24 crystallization conditions. While further development of these tools is certainly required for a turn-key system, the goal of fully automated screening appears to be within reach.     

Preparing Mixed Cellulose Ester or Polycarbonate Membrane Filter Replicas for Transmission Electron Microscopy

Simple methods for preparing large numbers of grids exhibiting excellent coverage of intact replicas on mixed cellulose ester or polycarbonate membrane filters are described. The techniques ensure that grids and carbon replicas receive identical treatment and are not rearranged or lost during processing. The techniques permit grids and filter sections to be handled en masse rather than individually. Also, replica section squares remain centered on the grids. A temporary grid storage method (“grid- pad”) is also described, which facilitates grid identification and handling.     

毛乌素沙地群落动态中克隆和非克隆植物作用的比较

毛乌素沙地的克隆植物适应当地的沙化环境 ,并对沙化景观具有积极的改造作用。对克隆植物群落和非克隆植物群落内裸地斑块植物入侵的野外对比观测实验表明 ,克隆植物群落内裸地斑块入侵植物所占的格子数显著地大于非克隆植物群落 ,克隆植物可以提高沙化景观的自我恢复能力 ,在沙化景观的恢复重建中是一种值得利用的生物资源。     

基于红外相机技术对广东车八岭国家级自然保护区大中型兽类与雉类的编目清查与评估

近年来, 红外相机技术已被广泛应用于国内外自然保护地内地栖鸟兽的物种编目和动态评估。本文以广东车八岭国家级自然保护区为例, 探讨基于红外相机技术如何进行保护区全境大中型兽类和雉鸡类的物种编目清查与评估。通过对车八岭保护区全境为期1年的调查, 共记录兽类和雉鸡类18种, 其中兽类15种, 鸡形目鸟类3种。基于物种累计曲线, 采用全年数据所需的最小调查网格数、最少调查相机日均要少于雨季或旱季, 而旱季调查需要的最小调查网格数和最少调查相机日比雨季更少。通过红外相机图像数据获得了车八岭保护区的大中型兽类和地栖雉鸡类物种名录、物种丰富度、每个物种的相对多度、分布图和凭证标本等重要内容。     

Differential trappability of small mammals in three habitats of Southeastern Brazil.

We compared the trappability of marked and unmarked individuals in species of marsupials and rodents of three tropical assemblages of small mammals in Brazil. Two studies used trapping grids, one in cerrado and the other in an Atlantic forest reserve, whereas the study in a rural area used transects. In the two studies using trapping grids, marked animals were frequently more trappable than unmarked ones, but in some species this difference was not significant. In the rural area, marked and unmarked animals did not differ significantly. The number of recaptures per resident animal was higher in the two studies using trapping grids than in the rural area where transects were used. Differences in trappability between the three studies might have been caused by differences not only in trapping design (grids vs. transects), but also in the type of trap used, bait, and habitat. Although differential trappability tends to be considered the rule in small mammals, these results suggest that trappability of marked and unmarked animals may be specific for the particular combination of sampling design, field methods, and habitat under study.     

MICROGEOGRAPHIC VARIATION IN MITOCHONDRIAL DNA OF MEADOW VOLES (MICROTUS PENNSYLVANICUS) IN RELATION TO POPULATION DENSITY

We examined mitochondrial-DNA (mtDNA) sequence heterogeneity on four adjacent trapping grids in an island population of meadow voles (Microtus pennsylvanicus) at two different population densities. Four restriction endonucleases revealed 20 different mtDNA composite phenotypes in samples totaling 198 meadow voles. There were significant heterogeneities in the distribution of four common mtDNA composite phenotypes among the four trapping grids, suggesting that there is population subdivision on a fine scale. Genetic distances between grids, mtDNA diversity within grids, and G_ST also varied during the study period. We found a decrease in genetic distance and an increase in diversity when the population density was high and vice versa when the population density was low. When population density was high, the coefficient of gene differentiation was smaller than the same coefficient observed when the population density was low. These changes in population subdivision and diversity are consistent with theoretical expectations of population structure in which effective female population size and dispersal are the critical variables. The data also support the hypothesis of maintenance of mtDNA diversity by population subdivision, rapid population growth rate, and dispersal.