Application of Fine Grain Processing and Condenser Illumination Enlarging to Photomicrography

Photomicrographs involving great resolution are usually made by means of long initial projection. This involves the use of heavy, often cumbersome, apparatus designed to eliminate vibration. This paper evaluates the possibility of using an intermediate projection distance equal to or greater than 160 mm., which is considered the minimum efficient distance, and recording the initial negative image in very fine grain. Then by critical enlarging a positive image is obtained which closely approaches the resolution obtainable by the finest long projection equipment. The initial short projection permits elimination of vibration difficulties attending long exposures so that critical printing at magnifications above 2000 × will give consistently good results, provided fine grain technic is mastered by the operator.     

Crystalline tubes of myosin subfragment-2 showing the coiled-coil and molecular interaction geometry

We have produced crystalline tubes of chicken breast myosin long subfragment-2 that show order to resolutions better than 2 nm. The tubes were formed from a thin sheet in which the myosin long subfragment-2 molecules were arranged on an approximately rectangular crystalline lattice with a = 14.1 +/- 0.2 nm and b = 3.9 +/- 0.1 nm in projection. Shadowing indicated that the tube wall was approximately 7 nm thick and that the sheets from which it was formed followed a right-handed helix. Superposition of the lattices from the top and bottom of the tube produced a moire pattern in negatively stained material, but images of single sheets were easily obtained by computer image processing. Although several molecules were superimposed perpendicular to the plane of the sheet, the modulation in density due to the coiled-coil envelope was clear, indicating that the coiled-coils in these molecules were in register (or staggered by an even number of quarter pitches). In projection the coiled-coil had an apparent pitch of 14.1 nm (the axial repeat of the unit cell), but the small number of molecules (probably four) superimposed perpendicular to the plane of the sheet meant that pitches within approximately 1 nm of this value could have shown a modulation. Therefore, a more precise determination of the coiled-coil pitch must await determination of the sheet's three-dimensional structure. The coiled-coils of adjacent molecules within the plane of the sheet were staggered by an odd number of quarter pitches. This arrangement was similar to that between paramyosin molecules in molluscan thick filaments and may have features in common with other coiled-coil protein assemblies, such as intermediate filaments. Each molecule in the crystal had two types of neighbor: one staggered by an odd number of quarter pitches and the other by an even number of quarter pitches, as has been proposed for the general packing of coiled-coils (Longley, W., 1975, J. Mol. Biol., 93:111-115). We propose a model for the detailed packing within the sheet whereby molecules are inclined slightly to the plane of the sheet so that its thickness is determined by the molecular length.     

Presynaptic Inputs to Any CNS Projection Neuron Identified by Dual Recombinant Virus Infection

Although neuroanatomical tracing studies have defined the origin and targets of major projection neurons (PN) of the central nervous system (CNS), there is much less information about the circuits that influence these neurons. Recently, genetic approaches that use Cre recombinase-dependent viral vectors have greatly facilitated such circuit analysis, but these tracing approaches are limited by the availability of Cre-expressing mouse lines and the difficulty in restricting Cre expression to discrete regions of the CNS. Here, we illustrate an alternative approach to drive Cre expression specifically in defined subsets of CNS projection neurons, so as to map both direct and indirect presynaptic inputs to these cells. The method involves a combination of Cre-dependent transneuronal viral tracers that can be used in the adult and that does not require genetically modified mice. To trigger Cre-expression we inject a Cre-expressing adenovirus that is retrogradely transported to the projection neurons of interest. The region containing the retrogradely labeled projection neurons is next injected with Cre-dependent pseudorabies or rabies vectors, which results in labeling of poly- and monosynaptic neuronal inputs, respectively. In proof-of-concept experiments, we used this novel tracing system to study the circuits that engage projection neurons of the superficial dorsal horn of the spinal cord and trigeminal nucleus caudalis, neurons of the parabrachial nucleus of the dorsolateral pons that project to the amygdala and cortically-projecting neurons of the lateral geniculate nucleus. Importantly, because this dual viral tracing method does not require genetically derived Cre-expressing mouse lines, inputs to almost any projection system can be studied and the analysis can be performed in larger animals, such as the rat.     

X-ray absorption and phase contrast imaging to study the interplay between plant roots and soil structure

Plant performance is, at least partly, linked to the location of roots with respect to soil structure features and the micro-environment surrounding roots. Measurements of root distributions from intact samples, using optical microscopy and field tracings have been partially successful but are imprecise and labour-intensive. Theoretically, X-ray computed micro-tomography represents an ideal solution for non-invasive imaging of plant roots and soil structure. However, before it becomes fast enough and affordable or easily accessible, there is still a need for a diagnostic tool to investigate root/soil interplay. Here, a method for detection of undisturbed plant roots and their immediate physical environment is presented. X-ray absorption and phase contrast imaging are combined to produce projection images of soil sections from which root distributions and soil structure can be analyzed. The clarity of roots on the X-ray film is sufficient to allow manual tracing on an acetate sheet fixed over the film. In its current version, the method suffers limitations mainly related to (i) the degree of subjectivity associated with manual tracing and (ii) the difficulty of separating live and dead roots. The method represents a simple and relatively inexpensive way to detect and quantify roots from intact samples and has scope for further improvements. In this paper, the main steps of the method, sampling, image acquisition and image processing are documented. The potential use of the method in an agronomic perspective is illustrated using surface and sub-surface soil samples from a controlled wheat trial. Quantitative characterization of root attributes, e.g. radius, length density, branching intensity and the complex interplay between roots and soil structure, is presented and discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

A constrained linear regression optimization algorithm for diaphragm motion tracking with cone beam CT projections

PurposeWe presented a feasibility study to extract the diaphragm motion from the inferior contrast cone beam computed tomography (CBCT) projection images using a constrained linear regression optimization algorithm.MethodsThe shape of the diaphragm was fitted by a parabolic function which was initialized by five manually placed points on the diaphragm contour of a pre-selected projection. A constrained linear regression model by exploiting the spatial, algebraic, and temporal constraints of the diaphragm, approximated by a parabola, was employed to estimate the parameters. The algorithm was assessed by a fluoroscopic movie acquired at anterior-posterior (AP) fixed direction and kilovoltage CBCT projection image sets from four lung and two liver patients using the Varian 21iX Clinac. The automatic tracing by the proposed algorithm and manual tracking were compared in both space and frequency domains for the algorithm evaluations.ResultsThe error between the results estimated by the proposed algorithm and those by manual tracking for the AP fluoroscopic movie was 0.54 mm with standard deviation (SD) of 0.45 mm. For the detected projections the average error was 0.79 mm with SD of 0.64 mm for all six enrolled patients and the maximum deviation was 2.5 mm. The mean sub-millimeter accuracy outcome exhibits the feasibility of the proposed constrained linear regression approach to track the diaphragm motion on rotational fluoroscopic images.ConclusionThe new algorithm will provide a potential solution to rendering diaphragm motion and possibly aiding the tumor target tracking in radiation therapy of thoracic/abdominal cancer patients.     

A cost-effective transparency-based digital imaging for efficient and accurate wound area measurement

Wound measurement is an objective and direct way to trace the course of wound healing and to evaluate therapeutic efficacy. Nevertheless, the accuracy and efficiency of the current measurement methods need to be improved. Taking the advantages of reliability of transparency tracing and the accuracy of computer-aided digital imaging, a transparency-based digital imaging approach is established, by which data from 340 wound tracing were collected from 6 experimental groups (8 rats/group) at 8 experimental time points (Day 1, 3, 5, 7, 10, 12, 14 and 16) and orderly archived onto a transparency model sheet. This sheet was scanned and its image was saved in JPG form. Since a set of standard area units from 1 mm(2) to 1 cm(2) was integrated into the sheet, the tracing areas in JPG image were measured directly, using the "Magnetic lasso tool" in Adobe Photoshop program. The pixel values/PVs of individual outlined regions were obtained and recorded in an average speed of 27 second/region. All PV data were saved in an excel form and their corresponding areas were calculated simultaneously by the formula of Y (PV of the outlined region)/X (PV of standard area unit) × Z (area of standard unit). It took a researcher less than 3 hours to finish area calculation of 340 regions. In contrast, over 3 hours were expended by three skillful researchers to accomplish the above work with traditional transparency-based method. Moreover, unlike the results obtained traditionally, little variation was found among the data calculated by different persons and the standard area units in different sizes and shapes. Given its accurate, reproductive and efficient properties, this transparency-based digital imaging approach would be of significant values in basic wound healing research and clinical practice.     

The shadow response of the cockroach periplaneta americana

We show that the “escape response” (running) of the cockroach Periplaneta americana is interrupted when the insect runs through a shadow. Shadows were cast by holding an opaque plastic sheet so that the cockroach ran beneath it. In control experiments, a similarly positioned clear plastic sheet was ineffective. Removal of the shadow stimulus allowed running to continue. The likelihood that cockroaches would resume running after removal of the shadow stimulus decreased with the time they were allowed to remain in shadow.     

A PHOTOGRAPHIC ANALYSIS OF PROTOPLASMIC MOVEMENT DURING CLEAVAGE IN THE SEA URCHIN EGG

The movement of the protoplasm during cleavage was analyzed by tracing the movements of particles in the protoplasm by time-lapse microcinematography of the eggs of the heart-urchin, Clypeaster japonicus .
Three methods of analysis are used. The first is to trace protoplasmic particles in the projected image frame by frame. The second is to record the displacements of protoplasmic particles at various regions of the egg within a definite period by printing several images of the same egg on the same sheet of photographic paper. The third is to record protoplasmic movement in the cleavage plane or along the spindle axis by projecting the film at a constant frame rate through a narrow slit on a sheet of photographic paper moving at a constant speed in a direction perpendicular to the slit.
As a result of the analysis, which confirms the result of a previous study (H iramoto , 1958), it is concluded that during cleavage of the sea urchin egg there is deformation of the preexisting cortex rather than the formation of a new cortex from endoplasmic materials.     

In vitro rearing of the parasitoid Exorista larvarum (Diptera: Tachinidae) from eggs laid out of host

We evaluated the possibility of using the eggs laid out of host to rear Exorista larvarum (L.) (Diptera: Tachinidae), a larval parasitoid of Lepidoptera, on artificial media. In a first experiment, eggs oviposited on a plastic sheet (either by inexperienced or experienced females) showed the same in vitro hatching capability as those removed from the larvae of the factitious host Galleria mellonella L. Subsequently, eggs laid on the host integument or out of host, either by inexperienced or experienced females, were removed from the oviposition substrate and placed on a skimmed milkbased artificial medium. The percentages of hatched eggs, of puparia and adults, as well as the puparial weights did not differ significantly among the three treatments. These findings suggested that E. larvarum may be successfully reared in vitro with a total exclusion of the host insect. In a further test, no difference for the in vitro hatching time was found between the eggs oviposited either on a plastic sheet or on G. mellonella larvae in the same length of time (60 min). This result suggested that at oviposition the out-of-host eggs were unincubated, similarly to those that had been laid on the host larvae.     

An innovative coating technique for light and electron microscopic autoradiography.

We describe a modified nuclear emulsion coating technique for both electron and light microscopic autoradiography. We propose that by reversing the application of formvar film so that it adheres to and covers thin sections placed on grids, we have developed a technically accessible methodology that produces optimal conditions for the tracing of specific nuclear activity. A smooth, continuous base is formed over the sections on which a monolayer of evenly packed silver halide crystals can be applied by dip-coating. The same principle is applied to pre-stained 1-micron plastic sections of glass slides. We suggest that the application of formvar film over thin sections does not impede or interfere with the exposure of the emulsion by the labeled tissue. On the contrary, it virtually eliminates contamination and background radiation, enhancing the specificity and quality of resolution at even low magnifications. This technical modification, which facilitates the application of the emulsion, could render electron microscopic autoradiography a routine laboratory procedure, allowing for easily reproducible results and quantitative evaluation. At the light microscopic level, this technique prevents chemical fogging caused by certain stains, and thus allows routine pre-staining before coating with emulsion.     

Olfactory maps and odor images

The understanding of neuronal processing of olfactory stimuli has been furthered by genetic studies and specialized imaging of particular neuronal populations. Selective optical imaging of odor-induced presynaptic and postsynaptic glomerular activity in the olfactory bulb/antennal lobe has visualized odorant-responsive receptor repertoires and shown a more confined odor image at the level of projection neurons compared to their olfactory receptor neuron input. Genetic tracing of projection neurons connected to particular glomeruli has revealed a somewhat dispersed spatial map of termination areas for these neurons both in insects and in vertebrates. Modifications of the glomerular odor map have resulted in altered percepts of the corresponding odors.     

基于低成本聚甲基丙烯酸甲酯(PMMA)电泳芯片的微型DNA分析仪

在低成本的聚甲基丙烯酸甲酯(PMMA)电泳芯片上,利用双通道共聚焦激光诱导荧光检测系统实现了单链DNA快速高效的分离检测．选用0.8 mm厚度的PMMA薄片加工微管道,一方面降低了检测限,另一方面提高了散热性能．通过线性聚丙烯酰胺筛分胶液以及使用纤维素衍生物对微管道表面进行动态修饰等条件的优化,芯片完成了高分辨率、高重现性的短串联重复序列(STR)等位基因的快速分型检测．两个STR位点D13S317 和CSF1PO的等位基因分型标准物(allelic ladder)和实际样本的PCR扩增产物均在3 min内达到了基线分离,表明低成本的PMMA电泳芯片在法医学及临床医学领域的基因分析方面具有良好的发展潜能．     

A study of oesophageal structure in a nematode, Aphelenchoides blastophthorus (Aphelenchida), using computer graphics reconstruction from serial thin sections

It can be difficult to visualize the shape and disposition of components in biological material from serial sections. They can be reconstructed by tracing outlines from sections onto an acetate sheet and stacking the sheets, but this method provides only a static model in which the elements cannot be manipulated or accurately measured. A more versatile method uses an interactive computer program. Data are entered via a digitizing tablet and the model may be displayed in any desired orientation and with any combination of elements presented. In this two-part paper we describe this technique and explain how it was used to elucidate the cellular composition of part of the feeding apparatus of a plant-infesting nematode. The oesophageal metacorpus of Aphelenchoides blastophthorus was found to consist of 43 cell bodies: 24 muscle, six epithelial and 13 nerve cells. The wide biological applicability of the technique is discussed and, in particular, its use in studying taxonomy and phylogeny of nematodes.     

An economical bioreactor for evaluating biogas potential of particulate biomass

An economical bioreactor designed for evaluating the biogas potential of particulate biomass is described. The bioreactor uses a simple stirring apparatus, called the Bordeaux stirrer, to enable gas-tight mixing of fermentation cultures. The apparatus consists of a low-rpm motor connected to a bent steel stir rod, which is placed in a length of flexible plastic tubing inserted through a rubber stopper in a gas-tight manner. This stirrer is suitable for providing intermittent or continuous mixing in bench-scale anaerobic cultures containing particulate biomass. The reactor system may be operated as a batch-fed or semi-continuously fed digester. This communication documents the advantages of the stirring apparatus, describes the details of reactor fabrication and operation, and outlines the type of experimental work for which the bioreactor is suitable.     

A hydroponic culture system for growing<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> plantlets under sterile conditions

We developed a hydroponic cultivation system for growingArabidopsis plantlets under sterile, controlled environmental conditions. The system consists of a piece of stainless-steel wire cloth (125 μm mesh size) that is fixed between 2 flat rings and held in place by 3 legs, placed in a commercially-available glass jar, and covered by the original glass lid or a sheet of sterilized cellophane. Sterilized seeds were distributed evenly across the mesh piece, the size of which allowed root growth and kept the seeds in place. After 3 weeks of cultivation, shoot and root tissues were easily harvested without mechanical damage. Proteome and metabolite analyses were performed on root and shoot tissues and demonstrated excellent reproducibility, indicating that the system is advantageous when biological variation is minimized. Induction experiments can be performed by transferring the apparatus (with plants) to a new jar containing a different nutrient solution. The apparatus is reusable and can easily be sterilized by autoclaving or dry heat. The system can be adapted to other small-seed plants by varying the mesh size.     

Structure of the regular surface layer of Aquaspirillum serpens MW5.

The structure of the regular surface layer of Aquaspirillum serpens MW5 has been investigated by electon microscopy supplemented by computer image processing and least-squares analysis. The layer has a ribbed appearance, both on the bacterium and in isolated, negatively stained fragments. However, detailed analysis indicated that the layer was composed of two hexagonal sheets having p6mm symmetry and a = 16 nm. One sheet was staggered by one half repeat along a (1,0) line of the p6nm lattice relative to the second so that, in projection, the pattern of the composite layer was a translational moiré, characterized by a series of ribs spaced 16 nm apart. The ribbed layer had cmm symmetry with a = 32 nm and b = 18.5 nm. Analysis of this pattern indicated that the two p6nm hexagonal sheets were unevenly stained, and this was confirmed by using least-squares methods to simulate the observed pattern by combining two hexagonal patterns. The general structure of the layer was consistent with a role as a selective and protective barrier on the cell surface.     

A Direct Projection from Mouse Primary Visual Cortex to Dorsomedial Striatum

The mammalian striatum receives inputs from many cortical areas, but the existence of a direct axonal projection from the primary visual cortex (V1) is controversial. In this study we use anterograde and retrograde tracing techniques to demonstrate that V1 directly innervates a topographically defined longitudinal strip of dorsomedial striatum in mice. We find that this projection forms functional excitatory synapses with direct and indirect pathway striatal projection neurons (SPNs) and engages feed-forward inhibition onto these cells. Importantly, stimulation of V1 afferents is sufficient to evoke phasic firing in SPNs. These findings therefore identify a striatal region that is functionally innervated by V1 and suggest that early visual processing may play an important role in striatal-based behaviors.     

小麦/玉米间作优势及地上部与地下部因素的相对贡献

 明确种间地上部相互作用和地下部相互作用对间作优势的贡献,优化资源配置对提高间作体系产量具有重要意义。该文采用田间微区试验以及地下部种间根系分隔技术研究了玉米(Zea mays)覆膜与不覆膜两种情况下小麦(Triticum aestivum)/玉米间作优势及地上部和地下部因素对间作优势的相对贡献, 以期为间作体系的增产提供理论依据。结果得出,玉米不覆膜时小麦/玉米间作具有明显的产量间作优势(籽粒产量和生物学产量土地当量比(Land equivalent ration, LER)分别为 1.30和1 .29),玉米覆膜能显著增加小麦/玉米间作系统产量间作优势（LER分别为1.41和1.40）;玉米不覆膜时小麦/玉米间作系统具有明显的氮、磷、钾养分吸收优势,玉米覆膜也能显著增加小麦/玉米间作系统养分吸收优势。小麦/玉米间作系统籽粒产量和生物产量间作优势来自地上部种间相互作用和地下部种间相互作用两个方面,但其相对贡献以地上部大于地下部, 当玉米不覆膜时均以地上部占75%、地下部占25%,当玉米覆膜时均为地上部占67%、地下部占33%,玉米覆膜能明显增加小麦/玉米间作系统产量间作优势地下部的相对贡献。地上部和地下部因素对小麦/玉米间作系统养分吸收优势也都具有重要贡献。玉米不覆膜时地上部和地下部因素对氮、磷养分吸收优势的相对贡献均分别为67%与33%,钾则相等（各占50%）;玉米覆膜能显著增加小麦/玉米间作系统氮、磷养分吸收优势地下部贡献率,但对钾养分吸收优势贡献影响不明显。表明,间作优势不仅可以通过作物组合来获得,也可通过地膜覆盖等措施进行调控,玉米覆膜能显著增加小麦/玉米间作系统产量间作优势和养分吸收优势及其地下部的相对贡献。可见,该研究对小麦／玉米间作模式持续增产和发展具有十分重要的指导作用。