A simple cryogenic holder for tensile testing of soft biological tissues

To overcome the difficulty of gripping soft biological materials for tensile test, a simple inexpensive cryogenic holder was developed which allows rapid (3 min) preparation of samples. It is made of 6 parts, built in a bakelite cloth, which is an excellent thermal isolant, and is used with rectangular (8x10(-2)x10(-2)x10(-2)m) samples. The holder with the sample inside is completely immersed in liquid nitrogen for 50 s. This duration allows the freezing of the sample ends on a 10(-2)m length and gives a very flat freezing surface throughout the sample cross section. The 6x10(-2)m central part of the sample remained at ambient temperature. Two parts of the holder help maintain the sample until its ends are vertically gripped in the tensile machine thus avoiding any sample deformation during this step. No pressure was applied on the frozen part of the sample by grips of the tensile machine and this avoids breaks in this region. The sample is fixed by adhesion forces (>1 kN) between its frozen parts and 2 pieces of the holder. The procedure has been successfully tested with bovine and salmon muscle samples and results show tensile breaks randomly distributed in the unfrozen region of the samples. Particular attention has been paid to obtain a very flat freezing surface so that the axial strain is equal throughout the sample and therefore any strain-related mechanical parameters can be accurately determined. The dimensions of the holder can be easily modified to fit other sample geometries and can be used with other biological materials.     

An improved method of sample introduction in gas chromatography-mass spectrometry of biological materials

In the GC-MS analysis of biological materials the method of sample introduction is an important factor affecting the value of the information obtained. A method has been developed which utilizes small glass beads as sample holders which are introduced into the GC column by way of a gas-tight, Teflon stopcock. This glass sample holder introduction system is very simple to construct and, in GC-MS analysis, offers advantages over other methods of sample introduction.     

Modification of the drop dialysis technique for use with larger volume samples

Desalting of nucleic acids by the drop dialysis method is limited by the fact that only small volume samples can be used due to the lack of sample containment on the membrane filters. A specially modified Styrafoam cup can be used as a membrane filter holder which serves to contain the sample, thus permitting dialysis of larger sample volumes.     

A handy impact corer for sampling lake surface sediment

A handy impact corer for sampling of surface sediment (< 50 cm) in lakes is described. The coring apparatus consists of a sample tube, tube holder, stopper, stopper holder, shaft with wing, shaft holder, hammer, and pushing rod. The total weights are 3.8 kg (Type I: 2.3 cm inside diameter of the tube) and 6.7 kg (Type II: 5.5 cm inside diameter of the tube). The system enables confirmation of the vertical landing of the corer onto the lake bottom and effective entering of sediment into the sample tube using hammer, and prevention of falling out of the core using stopper and pushing rod.     

A Simple and Inexpensive Device for Freeze Substitution at 183 K/-90 C

The freeze substitution device described here consists of commonly available materials, is easy to construct and to handle and provides maximal working safety. An adequate mass of copper and/or brass, serving as holder for 16 cryovials. is chilled to liquid nitrogen temperature. The vial holder is placed in a precooled thermos flask filled with ethanol and stored in an ultrafreezer at 193 K/-90 C. This ensures that the vial holder maintains temperatures of about 183 K/-90 C for more than 12 hr to minimize the risk of ice recrystallization.     

The use of an internal standard for semiquantitative analysis of low temperature (77°K) fluorescence of photosynthetic cells

A simple method is described which permits quantitative estimation of chlorophyll fluorescence emission intensity relative to the chlorophyll concentration in sample material at 77°K. A fluorescent standard excited by light of a similar wavelength as that of the sample material but emitting at a slightly shorter wavelength is mixed with the sample in known proportions. The fluorescence yield of the various peaks of the sample are normalized to the fluorescence yield of the internal standard. The spectra are recorded using an inexpensive attachment consisting of a Dewar holder, mounted instead of the light source of any double beam or split beam spectrophotometer. A glass rod is immersed in the sample standard mixture and then placed in the liquid nitrogen containing Dewar. Exciting light is provided by a light pipe mounted at 90° to the sample holder, connected with a tungsten-hallogen lamp provided with an appropriate filter. This method has been found to be particularly useful for the quantitation of chlorophyll fluorescence emission at 77°K of photosynthetic cells or chlorophyll containing membrane fractions. For this purpose, phycocyanin was found to be a suitable internal standard.     

Staining and Washing Ultrathin Sections; a Device for Handling Various Materials Simultaneously

To eliminate individual manipulation, as many as 10 grids, each held firmly by a small notched bar of polyethylene plastic, are simultaneously stained, then washed. If the stain used is reactive with atmospheric CO₂ it can be forced through a Millipore filter into a small chamber made of glass tubing which contains the grid holder. The stain, cleared of any solid particles, has very little contact with air and remains free of lead carbonate contamination. Washing is carried out by submerging the chamber and removing the grid holder under water (Feldman, D. G., J. Cell Biol., 15: 592-5, 1962). Washing is minimized because there is not the risk of contaminating grids and wash water with stain trapped between the points of forceps. The polyethylene is nonadherent to the wash water, and the grids can therefore be dried quickly on the holder. With this method, the relative stainability of different materials may be observed because each grid within a batch receives identical treatment.     

A novel technique for efficient multiple sample dialysis

Conventional methods for dialyzing numerous samples are either expensive or tedious and inefficient. These disadvantages were overcome through the construction and use of a Plexiglas dialysis sample holder (DSH). Large numbers of dialysis samples having 0.5 to 2.0-ml volumes may be attached to numbered positions on the DSH. Sample identification is greatly simplified and considerable savings in time and material are achieved. Furthermore, the risk of sample spill or mixing during filling or emptying of dialysis sacks, and the risk of leaks in dialysis tubing, are minimized.     

A Filter-based Surface Enhanced Raman Spectroscopic Assay for Rapid Detection of Chemical Contaminants

We demonstrate a method to fabricate highly sensitive surface-enhanced Raman spectroscopic (SERS) substrates using a filter syringe system that can be applied to the detection of various chemical contaminants. Silver nanoparticles (Ag NPs) are synthesized via reduction of silver nitrate by sodium citrate. Then the NPs are aggregated by sodium chloride to form nanoclusters that could be trapped in the pores of the filter membrane. A syringe is connected to the filter holder, with a filter membrane inside. By loading the nanoclusters into the syringe and passing through the membrane, the liquid goes through the membrane but not the nanoclusters, forming a SERS-active membrane. When testing the analyte, the liquid sample is loaded into the syringe and flowed through the Ag NPs coated membrane. The analyte binds and concentrates on the Ag NPs coated membrane. Then the membrane is detached from the filter holder, air dried and measured by a Raman instrument. Here we present the study of the volume effect of Ag NPs and sample on the detection sensitivity as well as the detection of 10 ppb ferbam and 1 ppm ampicillin using the developed assay.     

Automatic Sampling and Analysis of Organics and Biomolecules by Capillary Action-Supported Contactless Atmospheric Pressure Ionization Mass Spectrometry

Contactless atmospheric pressure ionization (C-API) method has been recently developed for mass spectrometric analysis. A tapered capillary is used as both the sampling tube and spray emitter in C-API. No electric contact is required on the capillary tip during C-API mass spectrometric analysis. The simple design of the ionization method enables the automation of the C-API sampling system. In this study, we propose an automatic C-API sampling system consisting of a capillary (∼1 cm), an aluminium sample holder, and a movable XY stage for the mass spectrometric analysis of organics and biomolecules. The aluminium sample holder is controlled by the movable XY stage. The outlet of the C-API capillary is placed in front of the orifice of a mass spectrometer, whereas the sample well on the sample holder is moved underneath the capillary inlet. The sample droplet on the well can be readily infused into the C-API capillary through capillary action. When the sample solution reaches the capillary outlet, the sample spray is readily formed in the proximity of the mass spectrometer applied with a high electric field. The gas phase ions generated from the spray can be readily monitored by the mass spectrometer. We demonstrate that six samples can be analyzed in sequence within 3.5 min using this automatic C-API MS setup. Furthermore, the well containing the rinsing solvent is alternately arranged between the sample wells. Therefore, the C-API capillary could be readily flushed between runs. No carryover problems are observed during the analyses. The sample volume required for the C-API MS analysis is minimal, with less than 1 nL of the sample solution being sufficient for analysis. The feasibility of using this setup for quantitative analysis is also demonstrated.     

A simple sandwich-cryogen-jet procedure with high cooling rates for cryofixation of biological materials in the native state

Summary We evaluate a method for improved, ice-crystal-free cryofixation of various biological materials (macromolecules, subcellular fractions, whole cells in suspensions or as monolayers) without any chemical pretreatments. The principle of the method (as introduced byPscheid, Schudt, andPlattner 1981 for cell monolayer cultures) is as follows. Firstly, a thin sample layer is sandwiched between a thin copper object holder and a thermally insulating plastic sheet (Thermanox, which is also used as a cell culture substratum); secondly, a jet of melting propane is shot from a simple, inexpensive brass vessel onto the copper object holder. We now ascertained that this simple and inexpensive method allows cooling rates up to > 18,000 °C/sec and, thus, gives better results than comparable but more sophisticated and expensive procedures. The method also avoids all the disadvantages of some other rapid freezing methods. Specifically, the sample mass required is minimal, the sample remains fully hydrated and no changes of ionic or other conditions can occur. There is no hazard of passing a cold gas phase (cooling curves being very steep right at the beginning) or of impairment by mechanical impact (as it could be with cold metal block freezing). The expedient of using a thermal insulator as part of a sandwich sample allows cooling rates which are superior or at least equivalent to those obtained with a commercial double propane-jet device (and double copper sandwiches). The reason for this is that in practice a two-side propane jet onto a copper-copper sandwich can not reach both sides precisely at the very same time (as implied from our cooling rate measurements). The tentative transformation of our mono-jet into a double-jet variation (with a branched nozzle piece) did also not improve the results, when compared with a single propane-jet onto a copper-Thermanox sandwich.This work is taken in part from a Ph.D. thesis by G.Knoll.     

An Apparatus Designed for Holding Cover Slips During Fixation and Staining

The need of an apparatus for fixing and staining cover slips upon which cell cultures are grown, led to the construction of the glass cover-slip holder here described. The holder is made with grooves on the sides and on the bottom into which the cover slips fit. The holder illustrated is for eight cover-slips, but may of course be made for any number.     

Modification of a scanning transmission electron microscope specimen holder for large section viewing.

A modification of a scanning transmission electron microscope specimen holder which permits full viewing of large plastic embedded tissue sections is discussed. The method for producing one-centimeter diameter "giant" grids is explained and the procedure for sample preparation is outlined. The modification aids the microscopist in his evaluation of tissue structural relationships by providing large areas of tissue for examination and reduces significantly the time required to prepare and examine standard 1-2 mm2 electron microscopy tissue sections. Light and electron microscopic evaluations can be made on the same tissue sections.     

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.     

Modification of a Scanning Transmission Electron Microscope Specimen Holder for Large Section Viewing

A modification of a scanning transmission electron microscope specimen holder which permits full viewing of large plastic embedded tissue sections is discussed. The method for producing one-centimeter diameter “giant” grids is explained and the procedure for sample preparation is outlined The modification aids the microscopist in his evaluation of tissue structural relationship by providing large areas of tissue for examination and reduces significantly the time required to prepare and examine standard 1-2 mm² electron microscopy tissue sections. Light and electron microscopic evaluations can be made on the same tissue sections.     

A Simplified Autoradiographic Dipping Procedure–-Slides Handled in Groups of Five

The dipping method of autoradiography (Joftes 1963) can be carried out rapidly with inexpensive materials by processing slides in a plastic holder. Autograms of good resolution are obtained, and a direct comparison between test and control is assured. The procedure can be done at normal room temperature and humidity, either in darkness or with dim, red illumination.     

A Simplified Autoradiographic Dipping Procedure--Slides Handled in Groups of Five

The dipping method of autoradiography (Joftes 1963) can be carried out rapidly with inexpensive materials by processing slides in a plastic holder. Autograms of good resolution are obtained, and a direct comparison between test and control is assured. The procedure can be done at normal room temperature and humidity, either in darkness or with dim, red illumination.     

Modular Coils with Low Hydrogen Content Especially for MRI of Dry Solids

IntroductionRecent advances have enabled fast magnetic resonance imaging (MRI) of solid materials. This development has opened up new applications for MRI, but, at the same time, uncovered new challenges. Previously, MRI-invisible materials like the housing of MRI detection coils are now readily depicted and either cause artifacts or lead to a decreased image resolution. In this contribution, we present versatile, multi-nuclear single and dual-tune MRI coils that stand out by (1) a low hydrogen content for high-resolution MRI of dry solids without artifacts; (2) a modular approach with exchangeable inductors of variable volumes to optimally enclose the given object; (3) low cost and low manufacturing effort that is associated with the modular approach; (4) accurate sample placement in the coil outside of the bore, and (5) a wide, single- or dual-tune frequency range that covers several nuclei and enables multinuclear MRI without moving the sample.

Materials and Methods

The inductors of the coils were constructed from self-supporting copper sheets to avoid all plastic materials within or around the resonator. The components that were mounted at a distance from the inductor, including the circuit board, coaxial cable and holder were manufactured from polytetrafluoroethylene.

Results and Conclusion

Residual hydrogen signal was sufficiently well suppressed to allow ¹H-MRI of dry solids with a minimum field of view that was smaller than the sensitive volume of the coil. The SNR was found to be comparable but somewhat lower with respect to commercial, proton-rich quadrature coils, and higher with respect to a linearly-polarized commercial coil. The potential of the setup presented was exemplified by ¹H / ²³Na high-resolution zero echo time (ZTE) MRI of a model solution and a dried human molar at 9.4 T. A full 3D image dataset of the tooth was obtained, rich in contrast and similar to the resolution of standard cone-beam computed tomography.     

A rapid controller of temperature for use in determining Arrhenius profiles in biomembrane systems

To minimize artifacts in temperature-velocity (Arrhenius) profiles due to aging of preparations of biological membranes, a rapid controller of temperature was developed for spectrophotometric or polarographic (O₂ electrode) measurements. The reaction mixture is cooled or heated through contact with Peltier elements. One Pt temperature sensor in the cuvette or electrode holder controls current flow into the Peltier units, and another Pt temperature sensor in the reaction mixture is used to read out the sample temperature on a meter or recorder, and to provide feedback control. The sample temperature can be reproducibly set to within 0.1°C, with a noise level of 0.04°C or less; a change of 4°C takes 1 min.On leave at the Arrhenius Laboratory, University of Stockholm.     

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.