Microinjection into Xenopus oocytes: a precise semi-automatic instrument and optimal parameters for injection of mRNAs

A new apparatus for the injection of Xenopus oocytes is described which provides semi-automatic cell handling together with highly accurate and reproducible volume delivery. Using the system requires very little skill, yet it gives 6.3% average reproducibility in the 5 to 70 nl volume range. The instrument uses a fixed injector system driven by an Inchworm piezoelectric positioner or, in a low-cost version, by a Rainin EDP-2 battery-operated motorized pipette. A movable, vacuum-operated oocyte holder minimizes lateral movement of the oocyte during injection. Oocytes injected with the system show better survival and enhanced expression of mRNA compared with those injected with a widely used type of manual injector (Coleman, 1984).     

栅板源源棒间散射和吸收对辐射场分布的影响

栅板源源棒间对＾６０Ｃｏ－γ射线的散射和吸收,是影响辐射场分布的重要因素,本工作在栅板辐射分布规律研究的基础上,对散射和吸收的影响进行理论描述,利用自行设计的计算机件,给出考虑源棒间散射和吸收后的等照射量率曲线,研究表明：理论描述与Ｆｒｉｃｋｅ剂量计的测点数据相吻合,在靠近Ｙ轴的场空间散射和吸收影响尤为明显,Ｙ轴上的遮挡效率可达３０－５０％,且随着距源架中心距离的增加,这一影响表现的愈加显著,。     

Design of an automated temperature mapping system for ultrasound or microwave hyperthermia

An automated temperature mapping system was designed to accomplish the following goals: remote control mapping; a maximum position error of 0.5 mm; mapping simultaneously on several channels; real-time screen display on a dedicated computer; to be inexpensive; and have a simple patient interface and set up. A four channel, microstepper system was fabricated for less than $1000 and controlled by an IBM-AT computer. The system utilizes direct drive of Luxtron fibre-optic probes fed through thin flexible Teflon^® tubing which allows for patient movement. The driving and control software were written in the programming language “C”. Mapping parameters for each independent channel include start and stop positions and map increment. The software permits the user to automatically find the maximum temperature along a track in three passes of 2.0, 1.0 and 0.5 mm steps. The latter two passes take five or seven readings centred about the maximum of the previous pass. A high resolution monitor plots the temperatures in real time, overlaying the previous map in a new colour. A screen dump was written to drive a colour printer with the plot information. The computer evaluates each plot to safeguard against any shift in the maximum location. Visualization of orthogonal pullbacks provides rapid feedback and aids in the repositioning of superficial hyperthermia transducers. The time saved over the previous manual mapping methods easily justifies the additional set up time.     

Interfacing a flow cytometer computer with a PC-based patient information system

We have established an interface between our flow cytometer's computer and the personal computer (PC) which supports our patient database system. The PC has been equipped with a commercially available IEEE-488 bus interface board which is connected to the interface bus of the cytometer's Hewlett-Packard 9000/300 computer (HP). The PC is set as a bus device with the same address as that of the HP's printer. It is programmed to examine the stream of data sent to the printer and extract from it and store in an MS-DOS text file selected information which subsequently may be transferred to the database system.     

Skin temperature changes induced by strong static magnetic field exposure

High intensity static magnetic fields, when applied to the whole body of the anesthetized rat, have previously been reported to decrease skin temperature. The hypothesis of the present study was that in diamagnetic water, molecules in the air play significant roles in the mechanism of skin temperature decrease. We used a horizontal cylindrical superconducting magnet. The magnet produced 8 T at its center. A thermistor probe was inserted in a subcutaneous pocket of the anesthetized rats to measure skin temperature. Animals (n=10) were placed in an open plastic holder in which the ambient air was free to move in any direction (group I). Animals (n=10) were placed in a closed holder in which the air circulation toward the direction of weak magnetic field was restricted (group II). Each holder was connected to a hydrometer to measure humidity around the animal in the holder. The data acquisition phase consisted of a 5 min baseline interval, followed by inserting the animal together with the holder into the center of the magnet bore for a 5 min exposure and a 5 min postexposure period outside the bore. In group I, skin temperature and humidity around the animal significantly decreased during exposure, followed by recovery after exposure. In group II, skin temperature and humidity did not decrease during the measurement. The skin temperature decrease was closely related to the decrease in humidity around the body of the animal in the holder, and the changes were completely blocked by restricting the air circulation in the direction of the bore entrance. Possible mechanisms responsible for the decrease in skin temperature may be associated with magnetically induced movement of water vapor at the skin surface, leading to skin temperature decrease.     

Specimen Holder to Critical-Point Dry Microorganisms for Scanning Electron Microscopy

Critical-point drying of microorganisms for scanning electron microscopy can be rapidly and effectively accomplished by use of a newly described specimen holder. Up to eight different samples of spores or vegetative cells are placed between polycarbonate membrane filters in the holder and processed through solvent dehydration and critical-point drying using carbon dioxide without loss or cross contamination of microorganisms. Yeasts, molds, bacteria, and actinomycetes have been successfully processed.     

Adaptation of laser-Doppler flowmetry to measure cerebral blood flow in the fetal sheep.

The purpose of this study was to devise a means to use laser-Doppler flowmetry to measure cerebral perfusion before birth. The method has not been used previously, largely because of intrauterine movement artifacts. To minimize movement artifacts, a probe holder was molded from epoxy putty to the contour of the fetal skull. A curved 18-gauge needle was embedded in the holder. At surgery, the holder, probe, and skull were fixed together with tissue glue. Residual signals were recorded after fetal death and after maternal death 1 h later. These averaged <5% of baseline flow signals, indicating minimal movement artifact. To test the usefulness of the method, cerebral flow responses were measured during moderate fetal hypoxia induced by giving the ewes approximately 10% oxygen in nitrogen to breathe. As fetal arterial PO(2) decreased from 21.1 +/- 0.5 to 10.7 +/- 0.4 Torr during a 30-min period, cerebral perfusion increased progressively to 56 +/- 8% above baseline. Perfusion then returned to baseline levels during a 30-min recovery period. These responses are quantitatively similar to those spot observations that have been recorded earlier using labeled microspheres. We conclude that cerebral perfusion can be successfully measured by using laser-Doppler flowmetry with the unanesthetized, chronically prepared fetal sheep as an experimental model. With this method, relative changes of perfusion from a small volume of the ovine fetal brain can be measured on a continuous basis, and movement artifacts can be reduced to 5% of measured flow values.     

Electron Microscopic Study of a Slime Layer

Slime layers are being studied in our laboratories in an attempt to understand their functions in the control of pollution in natural streams. A method for fixing, staining, and embedding microorganisms in the intact slime has been developed. In this method, epoxy resin discs are placed in a holder and are introduced into a simulated stream. After various periods of time the discs are punched out of the holder into the fixative. The disc with the attached slime is fixed, stained (4% osmium tetroxide plus ruthenium red), dehydrated, and embedded in epoxy resin so that thin sections can be cut through the vertical plane of the slime mass. Such thin sections permit detailed examination of the attached layer, the surface-slime interface, the spatial relationships between cells in the vertical slime structure, and the strands of extracellular material between and around cells. No special attachment structures were noted as the cells appeared to be attached to the surface by extracellular material alone. This material was observed in strands and netlike forms between cells which are positioned 1 to 4 mum apart in the slime.     

A Filter Method for Processing Small Samples of Marine Ova Through Fixation, Dehydration, and Embedding Without Changing Containers

Free-floating cells can be fixed, dehydrated, and embedded in a single container. The container was constructed from stainless steel, and the paraffin block formed by the shape and size of a container was perfect for microtoming. Eight containers were embedded in a fiberglass holder. This holder was designed so that it could be used with a 47 mm Millipore filter. Cells were pipetted into the top of a container while the Millipore filter sealed the bottom; thus the cells were retained on the filter while fluids were allowed to pass through it. The exposure of the cells to histological reagents was regulated by applying a vacuum to control the rate of flow through the filter.     

Technique for the Determination of the Rate of Ethylene Production by Pseudomonas solanacearum

A tube culture system was designed for measurement of ethylene evolved by the phytopathogenic bacterium, Pseudomonas solanacearum. The system consisted of 10 glass tubes joined together in series and coated on the inside surface with a dextrose-peptone-casamino acids agar medium. The system provided a large surface for bacterial growth in relation to the volume of air. The system was seeded with a bacterial suspension (7 × 10⁸ cells/ml) drawn through all the tubes by vacuum applied at one end and was then placed in a water bath at 30 C. Air was pumped through the system at 3 ml/min; the outlet was connected directly to the inlet port of a gas sampling loop and ethylene in the sample was determined by gas chromatography.     

Automatic data recording of circadian rhythms

This paper illustrates a method for automatic data recording using the printer port of personal computer and software designed ad hoc. The system was tested by measuring circadian rhythms of activity in the subterranean rodent Ctenomys talarum. Data is recorded in a text-only comma-delimited file, and displayed on screen.     

Modelling Biological Gel Contraction by Cells: Mechanocellular Formulation and Cell Traction Force Quantification

Traction forces developed by most cell types play a significant role in the spatial organisation of biological tissues. However, due to the complexity of cell-extracellular matrix interactions, these forces are quantitatively difficult to estimate without explicitly considering cell properties and extracellular mechanical matrix responses. Recent experimental devices elaborated for measuring cell traction on extracellular matrix use cell deposits on a piece of gel placed between one fixed and one moving holder. We formulate here a mathematical model describing the dynamic behaviour of the cell-gel medium in such devices. This model is based on a mechanical force balance quantification of the gel visco-elastic response to the traction forces exerted by the diffusing cells. Thus, we theoretically analyzed and simulated the displacement of the free moving boundary of the system under various conditions for cells and gel concentrations. This modelis then used as the theoretical basis of an experimental device where endothelial cells are seeded on a rectangular biogel of fibrin cast between two floating holders, one fixed and the other linked to a force sensor. From a comparison of displacement of the gel moving boundary simulated by the model and the experimental data recorded from the moving holder displacement, the magnitude of the traction forces exerted by the endothelial cell on the fibrin gel was estimated for different experimental situations. Different analytical expressions for the cell traction term are proposed and the corresponding force quantifications are compared to the traction force measurements reported for various kind of cells with the use of similar or different experimental devices. This revised version was published online in June 2006 with corrections to the Cover Date.     

Two- and three-dimensional image reconstructions from stained and autoradiographed histological sections

A complete system has been developed to utilize histologicalserial sections for two- and three-dimensional image reconstructions.Eighty to 120 sections are digitized using a personal computingsystem augmented with a imaging board and CCD camera. The imagefiles are transmitted to a VAX computer for processing and imagereconstruction, and the processed images are transmitted backto the personal computer for display and recording using a filmrecorder or PostScript printer. The software developed for thesystem allows serial sections to be placed into proper registrationin a 256³ array, 256 grey levels. Autoradiographs of the sectionsare obtained in the presence of appropriate standards whichare used to recalibrate grey levels to represent linearly theradioactivity of each pixel in the sections and scale the valuesto allow maximum use of the grey scale. Starting from coronallysectioned material the system has been used to analyse and reconstructrat nasal turbinates. In two dimensions horizontal and sagittalsections have been obtained while in three dimensions back-to-frontand surface-rendered images have been constructed. Useful renderingof differential metabolic activity within an organ of complexgeometry has been obtained, and there appears to be no reasonwhy the system cannot be used for any material for which serialsectioning is appropriate. Received on November 29, 1989; accepted on February 28, 1990     

Open-Source Syringe Pump Library

This article explores a new open-source method for developing and manufacturing high-quality scientific equipment suitable for use in virtually any laboratory. A syringe pump was designed using freely available open-source computer aided design (CAD) software and manufactured using an open-source RepRap 3-D printer and readily available parts. The design, bill of materials and assembly instructions are globally available to anyone wishing to use them. Details are provided covering the use of the CAD software and the RepRap 3-D printer. The use of an open-source Rasberry Pi computer as a wireless control device is also illustrated. Performance of the syringe pump was assessed and the methods used for assessment are detailed. The cost of the entire system, including the controller and web-based control interface, is on the order of 5% or less than one would expect to pay for a commercial syringe pump having similar performance. The design should suit the needs of a given research activity requiring a syringe pump including carefully controlled dosing of reagents, pharmaceuticals, and delivery of viscous 3-D printer media among other applications.     

The Automatic Spreading of Bacterial Culture over a Solid Agar Plate

Inoculated culture plates are automatically transported from a holder, into which they are placed manually, to a unit in which the culture is spread, by means of an electrically sterilizable loop drawn along a radius, and subsequently to another holder.     

An Apparatus for Rapid Attachment of Frozen Tissue to Cryostat Tissue Carriers

A brass cylindrical container 11.5 cm high and 7.5 cm in diameter was housed in an insulated wooden box. A 2.2 cm diameter hole was drilled in the centre of the removable brass lid on the underside of which a holder was attached for a cryostat tissue carrier. The container was filled with a mixture of acetone and solid CO₂ to within 1.5 cm of the lid. The frozen tissue was placed in a drop of water on a tissue carrier which was then lowered into the holder through the hole in the lid. The tissue carrier was rapidly cooled by the acetone-solid CO₂ mixture thus freezing the water and attaching the tissue to the carrier.     

Compact optical microfluidic uric acid analysis system

We designed, fabricated and tested a novel compact fluorescence analysis system for quantification of uric acid (UA) in clinical samples at the point-of-care. To perform an analysis, diluted saliva, urine or blood samples are simply placed in a disposable thin-film sample holder using a dropper. A new enzyme immobilization technique was developed to retain within the sample holder two enzymes and a molecule, which transforms into a fluorescer in amounts depending on the UA concentration. The small instrument (7.5 cm × 5 cm × 5 cm) into which the sample holder is placed for analysis contains an LED, a narrow-band filter and an amplified photodiode. The analysis time is 30s, and the dynamic range of the system is 4-400 μM of UA. The calibration curve for transparent saliva and urine was made using solutions of UA. The calibration curve for opaque blood was obtained with spiked samples of blood. The three different types of clinical samples were collected from three subjects and simply diluted before their measurements. Analysis with our instrument yielded UA concentrations within the expected concentration ranges. Development of instruments based on the current laboratory prototype is expected to result in products for clinical trials and point-of-care.     

DIALYSIS WITH STIRRING

Substances to be purified by dialysis are placed in collodion bags together with a toy "marble" or a bubble of air. The bags are stoppered and placed in glass tubes of a rocking machine. Distilled water of the desired temperature is circulated through the tubes (around the bags) at a rate of about 8 cc. per minute per bag while the machine is in motion. The rolling of the marbles or bubbles causes stirring which makes it possible to remove the salts from a protein solution in 24 to 48 hours.     

An automated system for detection and analysis of locomotor behavior in crustaceans

An efficient and simple system is presented for the analysis of crustacean locomotor behavior. The system is composed by six dual-compartment actographic chambers with photocoupling circuits for movement detection, and a device for acquisition and analysis of data. Such device is made by a digital interface which feeds into a microcomputer with disc unit and printer. Information is processed in real time during the experiment, with a simultaneous printout and storage in a floppy disc.     

Accelerated sliding of pollen tube organelles along Characeae actin bundles regulated by Ca2+

Pollen tubes show active cytoplasmic streaming. We isolated organelles from pollen tubes and tested their ability to slide along actin bundles in characean cell models. Here, we show that sliding of organelles was ATP-dependent and that motility was lost after N-ethylmaleimide or heat treatment of organelles. On the other hand, cytoplasmic streaming in pollen tube was inhibited by either N-ethylmaleimide or heat treatment. These results strongly indicate that cytoplasmic streaming in pollen tubes is supported by the "actomyosin"-ATP system. The velocity of organelle movement along characean actin bundles was much higher than that of the native streaming in pollen tubes. We suggested that pollen tube "myosin" has a capacity to move at a velocity of the same order of magnitude as that of characean myosin. Moreover, the motility was high at Ca2+ concentrations lower than 0.18 microM (pCa 6.8) but was inhibited at concentration higher than 4.5 microM (pCa 5.4). In conclusion, cytoplasmic streaming in pollen tubes is suggested to be regulated by Ca2+ through "myosin" inactivation.