Soft-X-ray damage to biological samples

X-ray damage to biological samples was investigated in the wavelength region of 2.7–5 nm, which overlaps the so-called 'water window', the wavelength range of 2.4–4.3 nm usually used in X-ray microscopy. Yeast cells and myofibrils were chosen as representatives of whole cell samples and motile protein systems, respectively. The samples were exposed to X-rays using an apparatus composed mainly of a laser-plasma X-ray source, a Wolter mirror condenser, and a sample cell. The yeast cells lost their dye exclusion ability when the X-ray flux was higher than 1 × 10⁶ photons μm⁻², while the myofibrils lost contractility when the X-ray flux was higher than 4 × 10⁵ photons μm⁻². These X-ray fluxes are lower than the flux required for the X-ray microscope observation of biological samples at a resolution higher than that of light microscopes.     

一种毫微米级进给机构几个问题的研究

通过建模的方法对所研制的一种毫微米级微进给机构的原理进行了分析,结合实验结果提出了有关结构参数的设计要点。并从动静态的角度分析了其定位精度的影响因素,提出了减少误差、提高系统稳定性的措施。     

生物检材中有机磷农药的检验概述

从有机磷农药的性质、生物检材中有机磷农药的提取净化和分析检测等方面概述生物检材中有机磷农药的检验研究现状。     

Rapid combined light and electron microscopy on large frozen biological samples

The use of large unfixed frozen tissue samples (10 × 10 × 5 mm³) for combined light microscopy (LM) and electron microscopy (EM) is described. First, cryostat sections are applied for various LM histochemical approaches including in situ hybridization, immunohistochemistry and metabolic mapping (enzyme histochemistry). When EM inspection is needed, the tissue blocks that were used for cryostat sectioning and are stored at −80 °C, are then fixed at 4 °C with glutaraldehyde/paraformaldehyde and prepared for EM according to standard procedures. Ultrastructurally, most morphological aspects of normal and pathological tissue are retained whereas cryostat sectioning at −25 °C does not have serious damaging effects on the ultrastructure. This approach allows simple and rapid combined LM and EM of relatively large tissue specimens with acceptable ultrastructure. Its use is demonstrated with the elucidation of transdifferentiated mouse stromal elements in human pancreatic adenocarcinoma explants grown subcutaneously in nude mice. Combined LM and EM analysis revealed that these elements resemble cartilage showing enchondral mineralization and aberrant muscle fibres with characteristics of skeletal muscle cells.     

A laboratory apparatus for streaming potential and resistivity measurements on soil samples

We describe an apparatus designed to perform streaming potential and resistivity measurements on unconsolidated soil samples. The apparatus enables the use of both unidirectional and oscillatory flow methods to measure the streaming potential coupling coefficient (C); the direct current resistivity method is used to measure the bulk resistivity (rho) of the soil sample. Measuring both of these parameters on the same sample under the same conditions enables us to properly characterize the streaming current cross-coupling coefficient (L). The apparatus is designed to test reconstituted saturated soil samples up to a maximum grain size of 9.5 mm, and hydraulic gradients from less than 0.1 up to a maximum of 4 m of H(2)Om in flow-through experiments. Excellent agreement between C values measured using the unidirectional and oscillatory flow methods validates the oscillatory flow method for unconsolidated samples.     

X-ray microanalytical resolution in frozen-hydrated biological bulk samples

In coated frozen-hydrated gelatin gels the backscattered electron yield does not increase during electron irradiation as it does in uncoated samples. Neither is the backscattered electron yield greater from coated frozen-hydrated gels than that from more conductive organic samples. This is interpreted as indicating that a significant distortion of the electron interaction volume, due to the development of a space charge, does not occur in electron irradiated frozen-hydrated gelatine gels when they are coated with a conducting coat. The depth resolution as estimated from models of biological samples in the form of frozen-hydrated photographic film and frozen-hydrated sections of gelatine gel is consistent with that computed from X-ray depth distribution curves, i.e. close to 2.0 μm at 15 kV. Lateral resolution was estimated from photographic film to be close to 2.0 μm also, at 15 kV.     

Quantitative elemental X-ray imaging of frozen-hydrated biological samples

It is shown that quantitative X-ray imaging of planed, frozen-hydrated, biological bulk samples that have not been etched is possible. X-ray imaging represents a better alternative to static beam (selected area) analysis of fractured frozen-hydrated samples. This procedure avoids the undesirable necessity of etching planed frozen-hydrated samples to provide an interpretable electron image. Qualitative oxygen and carbon X-ray images, which can be acquired in a short time, can be used for distinguishing morphological features and remove the requirement for electron images. In test samples of frozen-hydrated albumin, containing salts, analyses by X-ray images compared well with static beam (selected area) analyses from the same samples. An example of an analysis of frozen-hydrated insect Malpighian tubules is given in which the response to ouabain treatment was analysed. In this example X-ray imaging showed that ouabain resulted in a significant increase in cytoplasmic and luminal Na and a significant decrease in cytoplasmic and luminal K. X-ray imaging also showed that there was a significant increase in cellular water content. The presence of a potassium gradient in soybean root nodules was also demonstrated. The use of standard deviation images for processing low count images increases analytical precision but results in underestimates of the true concentrations.     

Gradiometric micro-SQUID susceptometer for scanning measurements of mesoscopic samples

We have fabricated and characterized micro-SQUID susceptometers for use in low-temperature scanning probe microscopy systems. The design features the following: a 4.6 mum diameter pickup loop; an integrated field coil to apply a local field to the sample; an additional counterwound pickup-loop/field-coil pair to cancel the background signal from the applied field in the absence of the sample; modulation coils to allow setting the SQUID at its optimum bias point (independent of the applied field), and shielding and symmetry that minimizes coupling of magnetic fields into the leads and body of the SQUID. We use a SQUID series array preamplifier to obtain a system bandwidth of 1 MHz. The flux noise at 125 mK is approximately 0.25 mu Phi 0/ sqrt Hz above 10 kHz, with a value of 2.5 mu Phi 0/ sqrt Hz at 10 Hz. The nominal sensitivity to electron spins located at the center of the pickup loop is approximately 200 muB/ sqrt Hz above 10 kHz, in the white-noise frequency region.     

Energy-filtered transmission electron microscopy of biological samples on highly transparent carbon nanomembranes

Ultrathin carbon nanomembranes (CNM) comprising crosslinked biphenyl precursors have been tested as support films for energy-filtered transmission electron microscopy (EFTEM) of biological specimens. Due to their high transparency CNM are ideal substrates for electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI) of stained and unstained biological samples. Virtually background-free elemental maps of tobacco mosaic virus (TMV) and ferritin have been obtained from samples supported by ∼1 nm thin CNM. Furthermore, we have tested conductive carbon nanomembranes (cCNM) comprising nanocrystalline graphene, obtained by thermal treatment of CNM, as supports for cryoEM of ice-embedded biological samples. We imaged ice-embedded TMV on cCNM and compared the results with images of ice-embedded TMV on conventional carbon film (CC), thus analyzing the gain in contrast for TMV on cCNM in a quantitative manner. In addition we have developed a method for the preparation of vitrified specimens, suspended over the holes of a conventional holey carbon film, while backed by ultrathin cCNM.     

底质、生物样品微波消化法研讨

通过对上海新料微波技术应用研究志研制的ＭＫ－１型压力自控微波溶样系统进行微波消化法的空白测定、标准物质贮产等测定、重现性测定等以及与常规消化法的对比实验来探讨微波消化法的优缺点。     

Observation of biological samples using a scanning microwave microscope

We present the application of a scanning microwave microscope technique to biological samples. Since dielectric properties of most biological samples originate mainly from the water they contain, we were able to obtain microscope images of biological samples by our scanning microwave microscope technique. As a model system, we have measured the electrical properties of water in the microwave region. The high dielectric constant and the large loss tangent of water were verified. Furthermore, we have measured the properties of water with differing amounts of sodium chloride concentration ranging from de-ionized water to the saturated solution. We have observed a significant change in the resonant frequency and Q value of the resonator as a function of sodium chloride concentration. The concentration dependence of the signals shows that our scanning microwave microscope technique can be useful for investigating the local electric behavior of biological samples with a simple model of ionic conduction.     

Scanning probe microscopy of biological samples and other surfaces

Scanning probe microscopes derived from the scanning tunnelling microscope (STM) offer new ways to examine surfaces of biological samples and technologically important materials. The surfaces of conductive and semiconductive samples can readily be imaged with the STM. Unfortunately, most surfaces are not conductive. Three alternative approaches were used in our laboratory to image such surfaces. 1. Crystals of an amino acid were imaged with the atomic force microscope (AFM) to molecular resolution with a force of order 10^?8 N. However, it appears that for most biological systems to be imaged, the atomic force microscope should be able to operate at forces at least one and perhaps several orders of magnitude smaller. The substitution of optical detection of the cantilever bending for the measurement by electron tunnelling improved the reliability of the instrument considerably. 2. Conductive replicas of non-conductive surfaces enabled the imaging of biological surfaces with an STM with a lateral resolution comparable to that of the transmission electron microscope. Unlike the transmission electron microscope, the STM also measures the heights of the features. 3. The scanning ion conductance microscope scans a micropipette with an opening diameter of 0·04-0·1 μm at constant ionic conductance over a surface covered with a conducting solution (e.g., the surface of plant leaves in saline solution).     

Manipulating biological samples for environmental scanning electron microscopy observation

Biological samples having different characteristics were observed by environmental scanning electron microscopy (ESEM). The environmental conditions for untreated biological samples was determined by optimizing sample temperature and chamber pressure. When the temperature was at 4 degrees - 6 degrees C and chamber pressure was 5.2-5.9 Torr, the relative humidity in the specimen chamber was about 85%. Under these conditions, the surface features of the sample were completely exposed and did not exhibit charging. The images obtained from the untreated samples at different ESEM conditions were also compared with fixed and coated samples observed under high vacuum.     

Electron beam-induced changes in vitreous sections of biological samples

Nonpretreated high pressure frozen samples of Zea mays, cartilage and human erythrocytes were cryosectioned and observed at 110 K in a cryoelectron microscope. Changes induced by medium doses of electron irradiation (< 10 ke nm^?2) are described. After some ke nm^?2, the most conspicuous cutting artefacts are erased to a large extent and the visibility of the cell organelles is improved. The sections, compressed in the cutting direction by the sectioning process, shrink once more, in the same direction, when irradiated. This shrinkage depends on the section support and on how the section is adsorbed to it. Shrinkage is not uniform; it is most pronounced in mitochondria, condensed chromatin and nucleolus. This differential shrinkage improves the visibility of major structures on the section and, as a result, ‘nicer’ images are recorded. However, this apparent improvement is a beam-induced artefact that must be paired with a loss of high resolution information.     

Imaging of metal-coated biological samples by scanning tunneling microscopy

A method for imaging biological samples by scanning tunneling microscopy (STM) is presented. There are two main difficulties in imaging biological samples by STM: (1) the low conductivity of biological material and (2) finding a method of reliably depositing the sample on a flat conducting surface. The first of these difficulties was solved by coating the samples with a thin film of platinum-carbon. The deposition problem was solved by a method similar to a procedure used to deposit biological molecules onto field ion microscope (FIM) tips. STM images of bacteriophage T7 and filamentous phage fd are shown. The substrate on which the samples were absorbed was atomically flat gold. The images do not show molecular detail due to the metal coating, but the gross dimensions and morphology are correct for each type of virus. Also, the surface density of virus particles increases and decreases in the way expected when the conditions of deposition are changed. These methods allow reliable and reproducible STM imaging of biological samples.     

Simple procedures for evaluating the cryofixation of biological samples

Ultra-rapid cooling of biological material can be achieved in the absence of cryoprotectants by using thin samples. Three methods now employed to prepare thin samples for freeze-fracture electron microscopy are compared: contacting the sample against a liquid helium-cooled copper surface (Heuser et al., 1979), spraying the sample with a jet of propane (Mueller et al., 1980), and plunging a streamlined copper ‘sandwich’ into liquid propane (Costello, 1980). In the first method a thin surface layer of the sample is ultra-rapidly cooled while in the other methods the entire sample sandwiched between sheets of conducting metals is cooled. The morphology of fracture-faces of dilauryllecithin-water systems is used to evaluate the effectiveness of cooling methods. At optimum cooling rates the initial disordered arrangement of lipid in the lamellar (L_α) phase is preserved, giving smooth fracture faces. At slower cooling rates a worm-like texture appears which signals the formation of molecular ordering characteristic of the P_β, phase. All three methods are capable of cooling these lipid-water phases as well as other more dilute aqueous suspensions without evidence of ice crystal growth or damage. Measurement of cooling rates employing miniature thermocouples embedded in samples indicates that rates for all three methods are in excess of 10,000 K/s. The propane jet (32 times 10³ K/s, slope at 273 K) exposes the sample to coolant more rapidly than the sandwich plunging method (10 times 10³ K/s, slope at 273 K) and therefore produces slightly higher cooling rates for samples of equivalent mass and thickness. Each method has its advantages. The contact method is well suited for tissues; the sandwich method is simple and inexpensive; the jet method can potentially produce the highest cooling rates. The last two methods yield complementary replicas.     

限进填料及其在生物样品分析中的应用

限进填料是一类特殊的萃取介质,允许生物基质直接、反复进样分析,实现待测物质的纯化和富集。限进填料萃取是生物分析领域的一种快速、简便、准确的在线样品制备方法。本文就限进填料的原理、种类及其在生物样品分析中的应用进行了综述。     

Progress in quantitative X-ray microanalysis of frozen-hydrated bulk biological samples

The analysis of bulk frozen-hydrated biological samples has developed now to a level where practical application of the technique is possible. Provided the sample is carefully coated with a conductive metal, the development of a space charge capable of causing a significant distortion of the electron diffusion volume does not seem to occur, and analytical resolution can be conveniently held to approximately 2 μm (both depth and lateral resolution). Two valid quantitative methods are available, and two methods of determining dry weight fractions are also available. An area where further research could lead to improvement in analysis of frozen-hydrated bulk samples is in the investigation of fracturing methods. If fracture planes that were flat and reproducible could be easily obtained, some of the difficulties of analysing frozen-hydrated bulk samples would be considerably reduced.     

Personal-computer-based system for electron beam X-ray microanalysis of biological samples

A system based on a personal computer has been developed which provides a relatively inexpensive way to equip an electron microscopy laboratory for quantitative elemental analyses of cryosectioned biological samples. This system demonstrates the feasibility of making an X-ray analyser from a personal computer, together with commercially available hardware and software components. Hardware and software have been assembled to drive the beam in a scanning electron microscope, collect and analyse X-ray spectra, and save, retrieve, and analyse data. Our software provides a menu-controlled user interface to direct spectra acquisition and analysis. Spot analyses, video images, and quantitative elemental images may be obtained and results transferred in ASCII format to other computers. Wet weight, as well as dry weight, concentrations are calculated, if measurements were made of areas of the hydrated sample before it was freeze-dried. Grey-level copies of video and quantitative elemental images may be made on a laser printer.     

Thermodynamic measurements of submilligram bulk samples using a membrane-based "calorimeter on a chip"

Calorimetry offers a direct measurement of thermodynamic properties of materials, including information on the energetics of phase transitions. Many materials can only be prepared in thin film or small crystal (submilligram) form, negating the use of traditional bulk techniques. The use of micromachined, membrane-based calorimeters for submilligram bulk samples is detailed here. Numerical simulations of the heat flow for this use have been performed. These simulations describe the limits to which this calorimetric technique can be applied to the realm of small crystals (1-1000 microg). Experimental results confirm the feasibility of this application over a temperature range from 2 to 300 K. Limits on sample thermal conductivity as it relates to the application of the lumped and distributed tau 2 models are explored. For a typical sample size, the simulations yield 2.5% absolute accuracy for the heat capacity of a sample with thermal conductivity as low as 2 x 10(-5) W/cm K at 20 K, assuming a strong thermal link to the device. Silver paint is used to attach (both thermally and physically) the small samples; its heat capacity and reproducibility are discussed. Measurements taken of a submilligram single crystal of cobalt oxide (CoO) compare favorably to the results of a bulk calorimetric technique on a larger sample.