Cryo X-ray microscope with flat sample geometry for correlative fluorescence and nanoscale tomographic imaging

X-ray imaging offers a new 3-D view into cells. With its ability to penetrate whole hydrated cells it is ideally suited for pairing fluorescence light microscopy and nanoscale X-ray tomography. In this paper, we describe the X-ray optical set-up and the design of the cryo full-field transmission X-ray microscope (TXM) at the electron storage ring BESSY II. Compared to previous TXM set-ups with zone plate condenser monochromator, the new X-ray optical layout employs an undulator source, a spherical grating monochromator and an elliptically shaped glass capillary mirror as condenser. This set-up improves the spectral resolution by an order of magnitude. Furthermore, the partially coherent object illumination improves the contrast transfer of the microscope compared to incoherent conditions. With the new TXM, cells grown on flat support grids can be tilted perpendicular to the optical axis without any geometrical restrictions by the previously required pinhole for the zone plate monochromator close to the sample plane. We also developed an incorporated fluorescence light microscope which permits to record fluorescence, bright field and DIC images of cryogenic cells inside the TXM. For TXM tomography, imaging with multi-keV X-rays is a straightforward approach to increase the depth of focus. Under these conditions phase contrast imaging is necessary. For soft X-rays with shrinking depth of focus towards 10nm spatial resolution, thin optical sections through a thick specimen might be obtained by deconvolution X-ray microscopy. As alternative 3-D X-ray imaging techniques, the confocal cryo-STXM and the dual beam cryo-FIB/STXM with photoelectron detection are proposed.     

Influence of Illumination on Settlement of Diatom <Emphasis Type="Italic">Navicula</Emphasis> sp.

Diatoms are responsible for biofouling, which causes many problems in various marine industries. This study examined the effects of different light conditions (intensity, incident direction, time of illumination) on the settling behavior of the marine diatom Navicula sp. on glass surfaces. The density of this diatom’s settlement on glass was strongly influenced by light conditions. Moreover, very weak light emitted on the bottom of the culture dish could also rapidly inhibit diatom settlement. These phenomena were explained by spatial interference between chloroplast and holdfast-like structures inside the thecae. The holdfast-like structure is observed to be responsible for diatom locomotion and hence the settlement behavior. It was proposed that the interrelation of illumination and attachment of diatoms allowed them to better adapt to the habitat with higher efficiency of attachment and successive reproduction.     

Contributions to Semithin Sextioning on A Conventional Rotary Microtome

Procedures for obtaining sections 1 μ thick on a conventional rotary microtome are described. Hydrophilic resin blocks with adequate hardness and elasticity for semithin sectioning are made by addition of divinylbenzene and methylmethacrylate to a commercial embedding kit. The blocks are pinched between two simple adapters and mounted in the specimen bolder of a microtome. A glass knife of the Ralph type with an effective blade length of 25 mm is made from a glass slide and attached to a metal bar with paraffin. The low cost assembly is set in the steel knife holder of a conventional rotary microtome. Sections I micron in thickness can be cut from the resin embedded blocks. Staining with the usual staining solutions may be weak due to the thinness of the sections, but the fine resolution and low distortion achieved are compensating gains.     

A Plexiglas Isolation-Transfer Chamber for Use with the Fonbrune Micromanipulator

The chamber is made with two pieces of clear Plexiglas, both 1 inch wide and 3 inches long; the top piece, 1/16 inch thick; the bottom, 1/4 inch. A recess 14 mm wide, 37 mm long and 5 mm deep is cut into the bottom piece leaving a margin 1.5 mm wide, this margin is then cut down to a height of 2.5 mm for the length of the recess; a piece of moistened filter paper I1/2 inches long and 5 mm deep is attached to the rear wall, leaving the bottom clear for the transmitted light; two notches 13 × 15 mm and 12 mm apart are cut from the top piece. The top is superimposed on the bottom and held by two screws to form a chamber that is accessible to microdissection instruments through its open side. Two cover glasses, one bearing the specimen and the other, the medium to which the transfer is to be made, are placed over the notches in the top (agar side down). The actual transfer of material is made by shifting the position of the cover glasses with the mechanical stage of the microscope while the specimen is held by the micromanipulator.     

Stain Historadiography

Fresh bone specimens were dehydrated in acetone and embedded in Ward's Bio-Plastic. Sections 10-15μ thick were cut with a special bone-cutting microtome and subjected first to radiography and then stained with safranin-fast green. The radiograph was made with a Machlett AEG-50-A X-ray tube on a fine-grained, spectroscopic plate; which, after processing, was mounted (dry) on a glass slide adjacent to the stained specimen. By these means, it was possible to make a correlated study of the calcium-bearing parts of the tissue and determine accurately their relationship to the bone itself. The method serves well for investigations of growing bone and for bone that is undergoing pathological involution. It has been named “Stain Historadiography” because it combines a stained specimen with its radiograph.     

New chamber for holding and sketching copepods and other small zoological specimens

The chamber is a simple device which makes it possible to hold copepods of length range between 1 and 15 mm or other bilateral animals of similar size for visual examination, sketching or dissection. The chamber consists of four parts: a base (a standard slide); the chamber proper which is a cylindrical cavity 16 mm in diameter cut into a rectangular (or square), transparent, 3 mm thick perspex (plexiglass) plate whose maximum width is the same as the width of the base; one or more lengths of thin synthetic (nylon) threads; and a standard coverglass. The perspex plate (the chamber proper) is attached to the base (slide) using a thin coat of plasticine (or any suitable dense lubricant) applied to the bottom surface of the plate. One or several nylon threads are set in the plasticine coat and cross the plate and its cavity. The ends of threads are outside the plate; one end of each thread is fixed, and one is left free. The chamber is filled with a suitable liquid medium, the specimen is placed in the desired position beneath the thread(s) and its position is fixed by pulling the free end of thread(s) so that the thread(s) press slightly against the specimen, holding it to the surface of the slide. The chamber then is covered with the coverglass.     

Staining Osteoid Seams in Thin Slabs of Undecalcified Trabecular Bone

For qualitative and quantitative study of osteoid seams in trabecular bone, 5-7 mm thick slabs were cut from the bodies of fresh, frozen, undecalcified human vertebrae. After washing out the bone marrow and soft tissue in a jet stream of water, the slabs were stained in 0.5% aqueous basic fuchsin for 30-40 hr at 18-20 C. The specimens were then trimmed by sawing off both overstained surfaces, to make a 2 mm slab which was submerged in 50% ethanol (2 or 3 changes of 10-30 min each), until the nonosteoid trabeculae became pale pink. The slab was allowed to dry in air. Osteoid seams are stained dark red and are well differentiated under a dissecting microscope with reflected illumination, either dry or immersed in water. This method permits the various types of trabculae to be separately studied in the same specimen     

Design and fabrication of a simple,versatile cryomicroscopy stage

A new cell freezing stage for cryomicroscopy is described with the intention of providing information adequate to enable easy duplication by other investigators. The stage is designed to be constructed from commonly available stock materials and to be fabricated with a minimal investment of time using simple techniques with standard machine tools. Provisions are made both for convective cooling with a chilled refrigerant gas and for heating with a transparent electrical resistance coating applied to a glass plate on which the specimen is mounted. The specimen temperature and its time rate of change can be regulated independently by continuously adjusting the balance between the cooling and heating fluxes. A temperature controller may be interfaced to the stage to regulate the specimen temperature automatically according to a preprogrammed protocol.Thermal performance capabilities of the stage satisfy most general cryomicroscopy requirements. Cooling and warming rates can be effected up to several hundred degrees Kelvin per minute across a temperature range in excess of 120 to 375 K. Thermal gradients within the viewing aperature of the stage are kept small by creating a tortuous convective flow path for the refrigerant fluid.     

Growth of fruit-bodies inFavolus arcularius

The fruiting ofFavolus arcularius in culture is described. When the cultures, which have been pre-incubated in darkness to allow the inoculum mycelia to become thick and white wooly in texture, are exposed to light, fruit-body primordia, 1 mm in height, are formed about 4 days after the start of illumination. The primordium develops into a cylindrical stipe, the growth of which mainly occurs in the final 1 mm of the terminal region. Hyphal elongation in the region within 3 mm of the apex is predominant in the growth of the pileate stipe. With maturation of the stipe, changes in hyphal orientation occur on the periphery of the subapical region, and then the pileus-primordium is formed. The differentiation into the inner layer and the outer layer (pre-hymenial layer) in the pileus tissue is completed at this stage. The early growth of the pileus may be due to rapid elongation of the hyphae on the margin in addition to gradual expansion of the hyphae in the preformed pseudo-tissue. When the pileus has grown to about 3 mm in diameter, the subsequent three to four fold increase in size may be due to parallel expansion of the hyphae constituting the young pileus tissue.     

Deep and clear optical imaging of thick inhomogeneous samples

Inhomogeneity in thick biological specimens results in poor imaging by light microscopy, which deteriorates as the focal plane moves deeper into the specimen. Here, we have combined selective plane illumination microscopy (SPIM) with wavefront sensor adaptive optics (wao). Our waoSPIM is based on a direct wavefront measure using a Hartmann-Shack wavefront sensor and fluorescent beads as point source emitters. We demonstrate the use of this waoSPIM method to correct distortions in three-dimensional biological imaging and to improve the quality of images from deep within thick inhomogeneous samples.     

A Plexiglas Isolation-Transfer Chamber for Use with the Fonbrune Micromanipulator

The chamber is made with two pieces of clear Plexiglas, both 1 inch wide and 3 inches long; the top piece, 1/16 inch thick; the bottom, 1/4 inch. A recess 14 mm wide, 37 mm long and 5 mm deep is cut into the bottom piece leaving a margin 1.5 mm wide, this margin is then cut down to a height of 2.5 mm for the length of the recess; a piece of moistened filter paper I1/2 inches long and 5 mm deep is attached to the rear wall, leaving the bottom clear for the transmitted light; two notches 13 × 15 mm and 12 mm apart are cut from the top piece. The top is superimposed on the bottom and held by two screws to form a chamber that is accessible to microdissection instruments through its open side. Two cover glasses, one bearing the specimen and the other, the medium to which the transfer is to be made, are placed over the notches in the top (agar side down). The actual transfer of material is made by shifting the position of the cover glasses with the mechanical stage of the microscope while the specimen is held by the micromanipulator.     

Fully automated and fast image analysis of autoradiographs with a TAS-Leitz. Determination of size, Feulgen fluorescence and grain counts of individual nuclei and their evaluation by a simplified cluster analysis

A fully automatic analysis system based on television image analysis was developed to measure simultaneously three parameters in individual nuclei of microscopic autoradiographs prepared from mouse jejunal crypt cell squashes and ascites tumor cell smears: size, Feulgen fluorescence and reflection from silver grains. A dark light camera with an image intensified silicon tube (RCA-ISIT), an automatic scanning stage and an autofocus device were fitted to a Leitz-TAS microscope. The camera permitted localization of Feulgen stained nuclei and measurement of area and light intensity by means of incident of light fluorescence in the red. After automatic changes of the Opak-illuminator silver grains were determined by means of polarized incident light reflected from the grains in the blue. A 25 X oil objective (aperture 0.75) yielded sufficient resolution for measurements. The nadir between the proportions of labeled and unlabeled nuclei was calculated from the data of one specimen on a PDP-computer using a new algorithm based on the minimal variance of the logarithm of reflected light per nucleus. Labeling indices determined by visual grain counting and by automatic analysis of the autoradiographs were well correlated (r = 0.87 to 0.92). Visual grain counts/nucleus and reflected light/nucleus correlated well when individual nuclei were compared (r = 0.92 to 0.97) or means of labeled nuclei of various specimens prepared during a 5 year period (r = 0.90 to 0.93). Quenching of nuclear Feulgen fluorescence was minimal. The optimal labeling range is 30-100 grain counts/nucleus. The time interval between measurements of two specimens was 25 min for a squash of approximately 350 crypt cells within a 3 mm X 3 mm field, and 20 min for a meandering scan with 1,000 ascites tumor cells.     

四川自贡中侏罗世璧山上龙一新种

2001年春,自贡市永安乡村民王新民在自家花园附近的紫红色沙质泥岩里发现一批脊椎动物化石。自贡恐龙博物馆接到报告后,由舒纯康同志前往调查处理,并将这批化石发掘回馆。该化石为一具不完整蛇颈龙类骨架,因其左后肢带骨较完整,有必要对它进行报道。     

Contributions to semithin sectioning on a conventional rotary microtome.

Procedures for obtaining sections 1 micrometer thick on a conventional rotary microtome are described. Hydrophilic resin blocks with adequate hardness and elasticity for semithin sectioning are made by addition of divinylbenzene and methylmethacrylate to a commercial embedding kit. The blocks are pinched between two simple adapters and mounted in the specimen holder of a microtome. A glass knife of the Ralph type with an effective blade length of 25 mm is made from a glass slide and attached to a metal bar with paraffin. The low cost assembly is set in the steel knife holder of a conventional rotary microtome. Sections 1 micron in thickness can be cut from the resin embedded blocks. Staining with the usual staining solutions may be weak due to the thinness of the sections, but the fine resolution and low distortion achieved are compensating gains.     

光照对苦瓜形态可塑性及生物量配置的影响

在人为遮阴条件下,对苦瓜的生长动态、形态特征以及生物量分配进行了研究。结果表明,不同遮阴处理下苦瓜植株的生长有较大的差异,弱光照不利于苦瓜构件数量的增加和生物量的积累;植株在弱光下形成较少的分枝,较薄的叶片以及较细长的主茎和叶柄,表现出较强光生长环境下更强的形态可塑性;植株在生长早期较生长晚期有较大的形态可塑性;生物量对叶片和主茎的分配随光照的减弱而增大,分枝的生物量分配随光照的减弱而降低,光照对分枝茎的生物量分配影响不大;在光资源充足的情况下,外界支持物的缺乏不会对苦瓜的生长造成太大的影响。     

Growth of Spirulina platensis enhanced under intermittent illumination

The growth characteristics of microalgae under different light conditions (continuous or intermittent) are essential information for photobioreactor design and operation. In this study, we constructed a thin-layer (10 mm) flat plate photobioreactor device with a light/dark (L/D) alternation system to investigate the growth of Spirulina platensis under two different light regimes: (1) continuous illumination in a wide range of light intensities (1.00-77.16 mW cm⁻²); (2) intermittent illumination in medium frequency (0.01-20 Hz). Specific growth rate and light efficiency based on biomass production were determined for each round of experiment. Four regions (light limited region, intermediate region, light saturated region and light inhibition region) were recognized according to the results under continuous illumination. Under intermittent illumination, when L/D frequency increased from 0.01 Hz to 20 Hz, specific growth rate and light efficiency were enhanced. However, the enhancement was different, depending on the applied light intensity and light fraction. The higher the light intensity, the greater the enhancement would be when L/D frequency increased from 0.01 Hz to 20 Hz; and the higher the light intensity, the lower the light fractions is needed to maintain light efficiency as high as that under continuous illumination in light limited region.     

A Simplified Procedure for Preparation of Undecalcified Human Bone Sections

A new type of apparatus for sectioning samples of hard, undecalcified bone is described. Slices of fresh or archeological human bone 4-5 mm thick are dehydrated and then embedded in epoxy resin. The apparatus used to prepare sections from the resulting blocks consists of a low-speed rim-type diamond cut-off wheel and a slowly advancing table carrying the specimen held in a rotating mount. Sections may be cut at a thickness of 80 μm ± 1%. After cleaning in an ultrasonic bath, these can be mounted on slides for quantitative microscopic examination with transmitted light. Grinding and polishing are not necessary. The results obtained are illustrated.     

Field Enhancement by Shaping Nanocavities in a Gold Film

We report on 2D plasmonic crystals composed of a hexagonal lattice of polymeric nanopillars embedded in an optically thick gold film on a glass substrate. A tapered shape of the polymeric pillars is proved to localize the electric field distribution close to the free surface of the device and to determine a significant increase in the electric field intensity particularly when the incident light comes from the glass side. These effects significantly improve the sample sensitivity to a refractive index change occurring at the free surface of the device.     

The Ultramicrotome Superstage: A Versatile Aid for Section Manipulation

The design and properties of a rigid, box-like device to be placed on the knife stage of a Porter-Blum MT-2 ultramicrotome are described. This “superstage” acts as a hand and tool rest, and a wind screen for the knife boat. The superstage easily permits tight rafts of ultrathin sections to be centrally located on grids. Additionally, it aids in placing 1 μm thick sections at the same relative location on glass slides to facilitate their examination in the light microscope.     

一种对甲螨无形态损伤的DNA提取技术

甲螨是一类重要的土壤动物,体型微小,一般具有较厚的体壁。本研究针对甲螨这一特定类群,探讨了一种无形态特征损伤的DNA提取技术。通过结合试剂盒DNA提取法,并适当改进实验条件,设计出一套行之有效的DNA提取流程。通过对提取DNA之后的标本进行形态学观察,发现其主要的分类学特征均保存完好,可以作为凭证标本长期保存。本研究所提供的DNA提取技术既可以提取出足够的DNA又可以保留凭证标本,因此能有效促进甲螨分子分类学相关研究。