Preparing micro sections of entire (dry) conifer increment cores for wood anatomical time-series analyses

The type of samples most commonly used in dendro sciences are increment cores of conifers. These cores allow for an easy determination and measurement of ring-width variations over long time periods. For wood anatomical analyses, the cores have to be split into pieces to enable the preparation of high quality micro sections for detailed measurements of cell properties. A major drawback of this procedure is the fact that it is labor intensive and time consuming. We present a new technique enabling the preparation of micro sections of entire increment cores up to a length of 40 cm. For that purpose we combined standard wood-anatomical techniques with the application of Mowiol glue and common Tesa tape. We tested the introduced method on increment cores of Larix decidua Mill. sampled years ago for ring-width analyses to reanalyze them on a microscopic level. The ability to cut these long sections will tremendously reduce the time needed to prepare micro sections. This is of special interest for wood anatomical image analyses of cores used before to create long ring-width chronologies for any kind of environmental reconstructions.     

The core-microtome: A new tool for surface preparation on cores and time series analysis of varying cell parameters

A microtome designed for the surface preparation of entire increment cores allows cutting plane surfaces on cores up to a length of 40 cm. Compared to the common sanding procedure, the wood cells of the annual rings remain open, not filled with swarf, and the cell walls are smooth and hence clearly visible. This article aims at describing the functionality of the microtome and the procedures needed for an accurate surface preparation to achieve a good contrast for subsequent image analysis. Possible applications for a more detailed analysis of variations in the tracheid structure of conifers and vessel sizes of oak are presented, which can be included in time series analyses.     

Quantitation of phosphotyrosine signals in human prostate cell adhesion sites

A simple method is described for the quantitation of phosphotyrosine signaling in human prostate cell cultures. The phosphotyrosine signals are observed by standard immunohistochemistry techniques, and the resulting digital images are analyzed using the Scion image software program. The signals within the cell adhesion sites are quantitated using the density slice and particle analysis features of the software. The immunohistochemistry results are compared with detection of phosphotyrosine signals using a standard Western blotting procedure with whole cell lysates. The resulting data is converted into graphs using the Sigma Plot Program. This method is illustrated using damage-induced signaling within cell adhesion sites after a low dose of ionizing radiation.     

The effect of missing rings on stand-age estimation of even-aged forests in northern Hokkaido,Japan

The effectiveness of estimating stand age from increment core samples taken at stump height (20 cm above ground) was tested in an even-aged stand ofBetula ermanii that had regenerated after a forest fire in 1945 in Hokkaido, Japan. Careful cross-dating revealed that annual rings were missing in 11 cores out of a total of 42 cores sampled, and that all these missing rings occurred in the outermost part of the core. These facts indicate that precaution has to be taken in selecting trees from which cores are to be sampled. The present work also revealed that those trees with missing rings have a characteristic appearance, with a thinner stem and less crown foliage than normal trees with complete rings. It was also found that even if this appearance test failed, the possibility of missing rings could be detected from a declining growth pattern, with extremely narrow rings on the increment core which normal trees did not show.     

BAI BAI bias – An evaluation of uncertainties in calculating basal area increments from cores

Basal area increment (BAI) is increasingly used in tree-ring based studies as it provides a direct measure of wood production and thus allows for the interpretation of growth trends. BAIs from increment cores are generally calculated while assuming circular growth patterns. However, observation of stem discs shows that many ring shapes are characterized by some form of eccentricity, not only characterized by deviations from circular ring shapes but also by piths not being centrally located. This observation poses to the question with what accuracy BAIs are calculated from increment cores. To quantify the estimation bias in BAI, we have developed a method that mimics eccentric tree growth by simulation. Various aspects of eccentricity are incorporated to created ‘stem discs’ with realistic appearances. Since BAI time series for our simulated discs are known, we can evaluate the accuracy of BAI calculation methods from cores. The ‘coring’ is simulated by taking cores at the thickest and thinnest sides of the simulated discs, whereby the number of cores is varied from one to four. In our simulations, we choose two calculation methods, namely the traditional circular approach and one that is based on the assumption of elliptical growth shapes. We find that bias in calculated BAI values is highly influenced by the number of cores taken, with a dramatic decrease from one to four cores. Furthermore, trend patterns in BAI series might be misleading in case of highly eccentric growth patterns. Based on these findings, we discuss the consequences for the interpretation of existing literature, where BAI analyses are based on one or two cores (along with the assumption of circular ring shapes). Such consequences are, however, difficult to quantify since we have no eccentricity statistics of tree growth within a forest stand. Therefore, we do not know the randomness of eccentricity within a stand, and thus to what extent chronology building (i.e. averaging BAI estimates over multiple trees) may reduce estimation bias. To lower BAI bias, we recommend to base BAI calculations on as many cores as possible. For individual trees with high levels of eccentricity, taking four increment cores seems necessary to reasonably estimate their basal area increments.     

In vitro development of core cells of the inner cell mass of the mouse blastocyst: effects of conditioned medium.

Blastocysts submitted to two rounds of immunosurgery give rise to cores of presumptive ectoderm cells, many of which do not survive for more than 48 hours when cultured individually. Precoating of the culture plates with conditioned medium (CM) from PYS-2 cells increases the incidence with which cores regenerate an outer layer. This procedure also improves the survival frequency of the cores, but only for a limited period of time. The small number of cores which survive for two weeks or more, either in uncoated or CM-coated plates, give rise to any array of cell types, including giant cells resembling trophoblast.     

Fluorescent metal nanoshell and CK19 detection on single cell image

In this article, we report the synthesis strategy and optical properties of a novel type of fluorescence metal nanoshell when it was used as imaging agent for fluorescence cell imaging. The metal nanoshells were made with 40 nm silica cores and 10 nm silver shells. Unlike typical fluorescence metal nanoshells which contain the organic dyes in the cores, novel metal nanoshells were composed of Cy5-labelled monoclonal anti-CK19 antibodies (mAbs) on the external surfaces of shells. Optical measurements to the single nanoparticles showed that in comparison with the metal free labelled mAbs, the mAb-Ag complexes displayed significantly enhanced emission intensity and dramatically shortened lifetime due to near-field interactions of fluorophores with metal. These metal nanoshells were found to be able to immunoreact with target cytokeratin 19 (CK19) molecules on the surfaces of LNCAP and HeLa cells. Fluorescence cell images were recorded on a time-resolved confocal microscope. The emissions from the metal nanoprobes could be clearly isolated from the cellular autofluorescence backgrounds on the cell images as either individuals or small clusters due to their stronger emission intensities and shorter lifetimes. These emission signals could also be precisely counted on single cell images. The count number may provide an approach for quantifying the target molecules in the cells.     

Quantitative densitometry of autoradiograms: digital images representative of optical density

Microcomputer software has been developed to control video acquisitionof 1024 x 1024 digital autoradiogram images which representtrue optical density (OD). Since video cameras are sensitiveto intensity (I), background correction and conversion to ODmust be accomplished in software. The software linearizes cameraoutput against an optical step tablet of known ODs and createsa ‘look-up table’ through which captured imagesare passed. This procedure allows the accurate and rapid conversionof a large number of images. The user is directed through thecalibration and capture procedures; safeguards are includedto ensure that the resultant images are correctly calibrated.The algorithms used in these programs accommodate the limitedcomputing power available in microcomputers. The use of a commerciallyavailable graphics library will enhance the portability to multiplehardware configurations. This software is a low-cost alternativefor the capture of digital images needed for quantitative densitometry. Received on June 20, 1991; accepted on February 17, 1991     

枸杞体细胞胚发生中蛋白质代谢动态的立体计量

以宁夏枸杞无菌苗叶片为材料,离体培养并诱导体细胞胚胎发生。根据细胞形态计量学原理,应用数字图像处理软件计量由光学底片经Ａ／Ｄ转换成的数字图像中的蛋白质大分子,对于枸杞体细胞胚发生过程中蛋白质分子的代谢动态进行了量化处理,并对量化结构分析了蛋白质代谢动态与体细胞胚发生、发育的关系。     

A power-driven increment borer for sampling high-density tropical wood

High-density hardwood trees with large diameters have been found to damage manually operated increment borers, thus limiting their use in the tropics. Therefore, we herein report a new, low-cost gasoline-powered sampling system for high-density tropical hardwood trees with large diameters. This system provides increment cores 15 mm in diameter and up to 1.35 m in length, allowing minimally invasive sampling of tropical hardwood tree species, which, up to the present, could not be collected by conventional 5 or 10 mm increment borers. This system provides a single core sample with ample amount of wood for multidisciplinary analyses, including ring width, stable isotope and wood anatomical measurements. The borer never gets stuck inside stems, even in hollowed trees, cores will never twist during coring, and the gasoline drill gives ample flexibility in the field. It is anticipated that the dendrochronological community will find our technique very useful in the pursuit of tropical tree ring research.     

Tree-Ring Dating of the Reshui-1 Tomb in Dulan County,Qinghai Province,North-West China

Tuyuhun and Tubo were two important states that thrived in north-western China during AD 311-900 in parallel with the Han Chinese dynasties of Sui and Tang periods. The Reshui Tomb Cluster located in Dulan County of the north-eastern Tibetan Plateau is an important cultural relic of the Tuyuhun-Tubo age. The official excavations of the Reshui tombs were regarded as top events in archaeology in the 1980s and 1990s in China. The Reshui-1 Tomb is the largest one among the tombs in the area. Since its excavation, there have been debates on whether the owner of the tomb belonged to the Tuyuhun or Tubo ethnicity. Therefore, accurately dating the Reshui-1 Tomb has a critical place in studying the Tubo and Tuyuhun histories. We collected 7 discs and 11 increment cores of Qilian juniper (Juniperus przewalskii Kom.) from the exposed and fallen beams of the roof of the Reshui-1Tomb. The lengths of the 16 tree-ring records are between 69 and 152 years. Based on a previously developed master dating chronology using Qilian juniper samples from the eastern Qaidam Basin, the calendar dates of the 16 specimens were determined by the COFECHA program and visual dating procedure. The average inter-series correlation among the dated sample series is 0.696, indicating good quality of cross-dating. The year of the outermost rings is AD 715 for the 7 discs and 4 out of the 9 increment cores. Moreover, the ring-width variations of the samples are consistent with the existing chronologies from the region. The presence of late-wood of AD 715 in the samples indicated that the Reshui-1 Tomb was completed in late AD 715 or early 716, which means that the Reshui-1 Tomb was finished in the Tubo age. This date provides direct evidence for archaeologists to determine the owner’s ethnicity and identify of the Reshui-1 Tomb.     

Commitment to division in Paramecium: effect of nutrient level on the macronuclear DNA increment

In Paramecium, a fixed macronuclear DNA increment is associated with commitment to cell division. This study shows that this threshold DNA increment is about 70% of the final DNA increment in well-fed cells. The DNA increment is reduced when growth rate is decreased and decreases in parallel with growth rate to a minimum of 30% of the normal DNA increment. This minimum value is obtained when the growth rate is 20% of its normal level or lower. Further reduction in the growth rate produces no further reduction in the DNA increment. Following abrupt nutrient-level shifts, both the threshold DNA increment and the final DNA increment change progressively as the time of the shift is moved to later stages of the cell cycle. The threshold DNA increment is reset following nutrient-level shifts up to the point of commitment to division. These observations are consistent with the notion that the magnitude of the threshold DNA increment is strongly correlated with the rate of growth and is rapidly reset by factors which alter the growth rate. The implications of these observations for growth-driven regulation of the cell cycle are discussed.     

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology¹ even in complex tissue sections². Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells³, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgenössische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.     

Combining qualitative and quantitative imaging evaluation for the assessment of genomic DNA integrity: The SPIDIA experience

In this note, we present an ad hoc procedure that combines qualitative (visual evaluation) and quantitative (ImageJ software) evaluations of Pulsed-Field Gel Electrophoresis (PFGE) images to assess the genomic DNA (gDNA) integrity of analyzed samples. This procedure could be suitable for the analysis of a large number of images by taking into consideration both the expertise of researchers and the objectiveness of the software. We applied this procedure on the first SPIDIA DNA External Quality Assessment (EQA) samples. Results show that the classification obtained by this ad hoc procedure allows a more accurate evaluation of gDNA integrity with respect to a single approach.     

Raster image correlation spectroscopy in live cells

Raster image correlation spectroscopy (RICS) is a noninvasive technique to detect and quantify events in a live cell, including concentration of molecules and diffusion coefficients of molecules; in addition, by measuring changes in diffusion coefficients, RICS can indirectly detect binding. Any specimen containing fluorophores that can be imaged with a laser scanning microscope can be analyzed using RICS. There are other techniques to measure diffusion coefficients and binding; however, RICS fills a unique niche. It provides spatial information and can be performed in live cells using a conventional confocal microscope. It can measure a range of diffusion coefficients that is not accessible with any other single optical correlation-based technique. In this article we describe a protocol to obtain raster scanned images with an Olympus FluoView FV1000 confocal laser scanning microscope using Olympus FluoView software to acquire data and SimFCS software to perform RICS analysis. Each RICS measurement takes several minutes. The entire procedure can be completed in ～2 h. This procedure includes focal volume calibration using a solution of fluorophores with a known diffusion coefficient and measurement of the diffusion coefficients of cytosolic enhanced green fluorescent protein (EGFP) and EGFP-paxillin.     

Freeze-fracture study of the rat parathyroid gland under hypo- and hypercalcemic conditions,with special reference to secretory granules

Summary Freeze-fracture images of the parenchymal cells in the parathyroid gland of rats were observed after vitamin D₂ plus calcium chloride-suppression and EGTA-activation of secretion. In cells of the suppressed glands, large bulges protruded from the Golgi cisternae, and large granules with a stalk, which are identified as storage granules, suggest that, during maturation, some storage granules may be connected by long tubules with the Golgi cisternae and supplied with secretory products from the Golgi cisternae via these tubules.In the activated glands, presumptive exocytotic and endocytotic specializations of intramembranous particles of the parenchymal cell plasma membrane were frequently observed. In addition, elevations and complementary shallow depressions of various shape and extent were occasionally encountered in the intercellular space. From their morphological characteristics it was concluded that these originated from secretory granule cores, which are discharged from the parenchymal cells into the intercellular space by exocytosis, and it was suggested that discharged granule cores may retain their spherical shape until they fuse to form a flat conglomerate.     

Measuring orientation of actin filaments within a cell: orientation of actin in intestinal microvilli.

Orientational distribution of actin filaments within a cell is an important determinant of cellular shape and motility. To map this distribution we developed a method of measuring local orientation of actin filaments. In this method actin filaments within cells are labeled with fluorescent phalloidin and are viewed at high magnification in a fluorescent microscope. Emitted fluorescence is split by a birefringent crystal giving rise to two images created by light rays polarized orthogonally with respect to each other. The two images are recorded by a high-sensitivity video camera, and polarization of fluorescence at any point is calculated from the relative intensity of both images at this point. From the value of polarization, the orientation of the absorption dipole of the dye, and thus orientation of F-actin, can be calculated. To illustrate the utility of the method, we measured orientation of actin cores in microvilli of chicken intestinal epithelial cells. F-actin in microvillar cores was labeled with rhodamine-phalloidin; measurements showed that the orientation was the same when microvillus formed a part of a brush border and when it was separated from it suggesting that "shaving" of brush borders did not distort microvillar structure. In the absence of nucleotide, polarization of fluorescence of actin cores in isolated microvilli was best fitted by assuming that a majority of fluorophores were arranged with a perfect helical symmetry along the axis of microvillus and that the absorption dipoles of fluorophores were inclined at 52 degrees with respect to the axis. When ATP was added, the shape of isolated microvilli did not change but polarization of fluorescence decreased, indicating statistically significant increase in disorder and a change of average angle to 54 degrees. We argue that these changes were due to mechanochemical interactions between actin and myosin-I.     

Automatic leukocyte classification using cytochemically stained smears.

A leukocyte classification algorithm suitable for automated differential counting has been developed for blood smears stained with a new three-component cytochemical stain which has relatively narrow absorption bands centered at 460, 540 and 640 nm, respectively. The classification procedure is the result of a pattern recognition experiment using a sample of 223 leukocytes distributed evenly over the five normal cell types. The basic data for each cell were three digital microscopic images obtained with narrow band illumination at the above central wavelengths using a TV-digitizer system interfaced to a PDP-15 computer. The classification algorithm involves a sequential decision procedure utilizing five pattern features computed from the intensity histograms of the green and blue digital images. Thus the number of arithmetic operations and the number of computer memory words necessary to perform the classification into one of the five normal white blood cell types are both proportional to n where n is the number of gray levels into which the intensity scale is divided. In this experiment, n equals 256. Comparison of our results with work of others on smears prepared with Romanowski-type stains indicates that such narrow-band, spectrally well separated cytochemical multiple stains can permit the use of algorithms which are approximately ten times faster.     

Fluorescence Microscopy for Measuring Fibril Angles in Pine Tracheids

Observation and measurement of fibril angles in increment cores or similar small samples from living pine trees was facilitated by the use of fluorescence microscopy. Although some autofluorescence was present, brighter images could be obtained by staining the specimens with a 0.1% aqueous solution of a fluoro-chrome (Calcozine Ha vine TG extra concentrated, Calcozine red 6G extra, rhodamine 6G, rhodamine 6GD extra, or a succession of flavine and rhodamine). Staining for 2—5 min followed by a 5-10 sec washing in distilled water and drying 15 min at 190-195°C prepared radially split surfaces of specimens for microscopic observation with ultraviolet light. Measurement of fibril angles, important for the determination of wood strength and the properties of its pulp, was made with a protractor eyepiece. Photomicrography was feasible also, and the need of preparing microtome sections was obviated.     

Dating fire events in Pinus heldreichii forests by analysis of tree ring cores

Bosnian pine (Pinus heldreichii Christ, also known as Pinus leucodermis Antoine) is a relict species found in isolated locations in the mountains of the Balkan Peninsula and Southern Italy. The forests are of high conservational value because they are extremely rich in rare and endemic species of plants and fungi. Yet, the natural history and disturbance regime of P. heldreichii ecosystems is not well understood. Fire traces show that fires played a major role, but there is very limited historical data. Therefore proxy methods to reconstruct past events have to be used. The analysis of tree rings provides such an opportunity. To our knowledge, there have been no attempts to use tree ring cores from P. heldreichii trees to date fire events. Our aim was therefore to test if tree ring cores collected with an increment borer could successfully be used to date fires and verify other tree ring indicators caused by the fire events. We tested an approach that was based on extracting multiple cores from fire-scarred trees and nearby standing trees without injuries. A total of 136 cores from 99 trees were collected from which we dated all 34 cores with fire scars. We found the exact fire years for 29 of the samples, and the remaining 5 samples were approximately dated. Up to 83% of all sampled trees had additional growth reactions, mostly suppressions lasting 5–10 years after the fire years.