Calibration of x-ray densitometry for the measurement of coral skeletal density

A new method is proposed for the measurement of coral skeletal density by x-radiography. X-radiographs were made of sections cut from skeletons of massive corals of the genus Porites. Included on the x-ray film with each specimen were an aluminium step-wedge, a set of aragonite standards and several aluminium bars, all of measured thickness and density. The images on the developed x-ray film were scanned with a microdensitometer. Semilogarithmic plots of microdensitometer output voltage vs. thickness of the aluminium and aragonite standards provided characteristic curves, with initial linear slopes which were defined as relative linear absorption coefficients. These coefficients varied with the x-ray exposure and microdensitometer measurement conditions; however, they were consistent within one set of conditions. The relative linear absorption coefficients and densities of the standards, together with data for thickness of standard vs. film exposure, can be used to determine the density of coral specimens at points along microdensitometer traverses of their x-ray images. The bars of aluminium can be used to correct for the non-uniform irradiation of the x-ray film which is characteristic of x-ray machines.Contribution No. 299 from the Australian Institute of Marine Science     

Densitometry from digitized images of X-radiographs: Methodology for measurement of coral skeletal density

Skeletons of massive coral colonies contain annual density bands that are revealed by X-radiography of slices cut along growth axes. These bands allow measurement of skeletal growth parameters such as annual extension rate and annual calcification rate. Such measurements have been important in understanding coral growth, in assessing environmental impacts and in recovering proxy environmental information. Measurements of coral calcification rate from annual density banding require measurements of skeletal density along tracks across skeletal slices and, until now, such density measurements have depended upon specialized and expensive equipment. Here, we describe a straightforward, inexpensive and accurate technique for measuring skeletal density from digitized images of X-radiographs of coral skeletal slices. An aragonitic step-wedge was included in each X-radiograph of a coral slice together with two aluminium bars positioned along the anode-cathode axis. Optical density was measured along tracks across the X-ray images of these different objects. The aragonite step-wedge provided a standard for converting optical density to skeletal density. The aluminium bars were used to correct for the heel effect—a variation in the intensity of the X-ray beam along the anode-cathode axis that would, otherwise, introduce large errors into measurements of skeletal density. Exposure was found to vary from X-radiographs to X-radiograph, necessitating the inclusion of the calibration standards in each X-radiograph of a coral slice. Results obtained using this technique compared well with results obtained by direct gamma densitometry of skeletal slices.     

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     

The measurement of skeletal maturity

A new approach to the measurement of skeletal maturity has been described. This was applied to the distal end of the femur in boys aged one month to four years. It is planned to extend this study to girls and to other ages and sites and to other bones in the knee area. The study began with the testing of reported indicators of skeletal maturity in respect of replicability, validity, discrimination and universality. As a result, seven qualitative graded indicators and three quantitative ratios were selected for further investigation. Maturity scores based on each qualitative indicator can be estimated using parameters from a simple probit analysis with chronological age as the independent variable. The statistical factors that influence the usefulness of these indicators have been discussed. The quantitative indicators investigated appear very useful as measures of maturity although some are redundant. The planned extension of the present study should allow the formulation of an appropriate model for the measurement of skeletal maturity by combining qualitative and quantitative data.     

The density of graptoloid skeletal tissue, and its implication for the volume and density of the soft tissue

It has been proposed by many authors that a mechanism is necessary to achieve neutral buoyancy in graptoloids. Calculations are given to show the range of densities needed for both soft and skeletal tissues to achieve this, and it is concluded that a large volume of soft tissue, containing low density fats, is the most likely mechanism. This is not readily compatible with the rhabdopleuran model of the graptolite mid.     

Gamma 1 subunit interactions within the skeletal muscle L-type voltage-gated calcium channels

Voltage-gated calcium channels mediate excitationcontraction coupling in the skeletal muscle. Their molecular composition, similar to neuronal channels, includes the pore-forming alpha(1) and auxiliary alpha(2)delta, beta, and gamma subunits. The gamma subunits are the least characterized, and their subunit interactions are unclear. The physiological importance of the neuronal gamma is emphasized by epileptic stargazer mice that lack gamma(2). In this study, we examined the molecular basis of interaction between skeletal gamma(1) and the calcium channel. Our data show that the alpha(1)1.1, beta(1a), and alpha(2)delta subunits are still associated in gamma(1) null mice. Reexpression of gamma(1) and gamma(2) showed that gamma(1), but not gamma(2), incorporates into gamma(1) null channels. By using chimeric constructs, we demonstrate that the first half of the gamma(1) subunit, including the first two transmembrane domains, is important for subunit interaction. Interestingly, this chimera also restores calcium conductance in gamma(1) null myotubes, indicating that the domain mediates both subunit interaction and current modulation. To determine the subunit of the channel that interacts with gamma(1), we examined the channel in muscular dysgenesis mice. Cosedimentation experiments showed that gamma(1) and alpha(2)delta are not associated. Moreover, alpha(1)1.1 and gamma(1) subunits form a complex in transiently transfected cells, indicating direct interaction between the gamma(1) and alpha(1)1.1 subunits. Our data demonstrate that the first half of gamma(1) subunit is required for association with the channel through alpha(1)1.1. Because subunit interactions are conserved, these studies have broad implications for gamma heterogeneity, function and subunit association with voltage-gated calcium channels.     

Neutron imaging versus standard X-ray densitometry as method to measure tree-ring wood density

Neutron imaging is a new non-destructive testing method in wood science. It is similar to X-ray methods but with differing sensitivities for different elements. In this study, neutron imaging was used to ascertain the density profiles of thin spruce samples and compared with results generated with standard X-ray microdensitometry. Data obtained through neutron imaging were similar to those resulting from the X-ray method. The advantage of neutron imaging is its higher sensitivity to some elements such as hydrogen. Together with the high neutron-sensitivity of the applied detectors (imaging plates) this makes shorter exposure times possible, and yields more detailed information on the inner composition of wood. X-ray film, which is still most commonly used in X-ray densitometry, has the disadvantage that the relationship between the optical density of the film and the density of wood is non-linear. This means that corrections and calibration with step wedges are necessary, whereas with neutron imaging the digital values can be used directly to calculate the density at a certain point of the specimen. Thus neutron imaging appears to be an appropriate method, which can be used as complement to established X-ray methods for fast and straightforward investigations of tree rings, growth zones and wood density.     

A fluorometric assay for the measurement of endothelial cell density in vitro

Summary A fluorometric assay for determining endothelial cell numbers based on the endogenous enzyme acid phosphatase is described. In preliminary studies, three substrates—p-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, and 2′-[2-benzthiazoyl]-6′-hydroxy-benthiazole phosphate (AttoPhos™)—were compared with respect to their kinetic, optimum assay conditions, sensitivity, and detection limits. Only AttoPhos™ was found to have a high degree of sensitivity, reliability, and reproducibility for measuring both high and low cell numbers in the same plate. In subsequent experiments, assay conditions were validated for measuring endothelial cell density in response to basic fibroblast growth factor and fumagillin. Furthermore, the AttoPhos™ assay revealed a linear correlation between acid phosphatase activity and cell number in many cell types, including BALB/3T3, CHO-K1, A431, MCF7, 2008, SK-OV-3, T47-D, and OVCAR-3. This assay is potentially valuable for use in many in vitro systems in which the quantitation of cell density and proliferation is necessary. The practical advantages of AttoPhos™ assay for measuring endothelial cell numbers include (1) nonradioactivity, (2) simplicity, (3) economy, (4) speed of assessment of proliferation of large number of samples, and (5) amenability to high-throughput drug screening.     

Electrophoresis with continuous scanning densitometry: separation of cells in a density gradient.

An instrument and procedure for electrophoresis with continuous optical scanping densitometry, automated data processing, and related methodology are described for the continuous analysis of electrophoresis and unity gravity sedimentation of macromolecules or cells in a static density gradient system. The instrument consists of a dual-beam spectrophotometer, a scanning stage and scanner control unit, an electrophoresis cell cassette, a filling/purging/cooling module, an analog-to-digital converter, and a digital data-logger. The distribution of cells is monitored repetitively during migration by absorbance measurements at any wavelength in the 200–800-nm range. A computer program provides the statistical analysis of each peak (baseline correction, smoothing, area, mean, standard deviation, skewness, and kurtosis) which can be further utilized for computing additional parameters, such as resolution and heterogeneity. A mixture of human and rabbit erythrocytes were used as a model system to evaluate the performance of the instrument and demonstrate some of its capabilities.     

Radiologic densitometry in the evaluation of disorders of bone mineral density in patients with diabetes mellitus]

Presents a quantitative assessment of the total content of minerals in bones of 50 diabetics and 50 controls, carried out by a modified photodensitometric procedure. The data evidence a significant reduction of the mineral content in the bones of diabetes mellitus patients, this permitting an earlier detection of osteoporosis in the course of diabetes.     

Computerized tomography and skeletal density of coral skeletons

In this paper I describe and discuss the use of medical X-ray computerized tomography (CT) in the study of coral skeletons. CT generates X-ray images along freely chosen sections through the skeleton and offers, as well, the possibility of density measurements based on X-ray attenuation. This method has been applied to measure the skeletal density of the Caribbean reef-building coral Montastrea annularis, from Curaçao, Netherlands Antilles. The observed, non-linear increase of skeletal density with depth can be attributed to decreasing photo-synthetic rates with increasing water depth. A comparison with extension rate measurements shows the inverse relationship between extension rate and skeletal density. CT proves to be aquick and non-destructive method to reveal growth structures (density banding) since it measures skeletal density.     

Optical density measurement of ultramicrovolumes of liquid

Measurement of the absorbancy of ultramicrovolumes of liquids in necessary to perform in the ultramicro-scale important analytical procedures like determination of the concentrations of substances, of enzymatic activities, etc. The proposed procedure of serial analysis is based on the application of capillary flow cells and of sensitive double-wavelength microphotometer designed in this Institute. Droplets of the sample solution, of standard solution and of the solvent are sucked by means of precision microsyringe into an Uviol glass capillary tube 50–100 μm i.d. with extended tips of smaller diameter which serve the flow cells. the droplets are separated from each other with air or pentadecane layers. During the extrusion of the droplets through the capillary across light probe of the instrument, the absorbancy is recorded. Minimum length of the droplets is 150 μm, and the minimum amount of nucleotide material necessary for the determination is 1·10^?10–5·10^?11 g.     

Automatic measurement of stomatal density from microphotographs

Key message

An automated process using a cascade classifier allowed the rapid assessment of the density and distribution of stomata on microphotographs from leaves of two oak species.

Abstract

Stomatal density is the number of stomata per unit area, an intensively studied trait, involved in the control of CO₂ and H₂O exchange between leaf and atmosphere. This trait is usually estimated by counting manually each stoma on a given surface (e.g., a microphotograph), usually repeating the procedure with images from different parts of the leaf. To improve this procedure, we tested the performance of a cascade classifier to automatically detect stomata on microphotographs from two oak species: Quercus afares Pomel and Quercus suber L. The two species are phylogenetically close with similar stomatal morphology, which allowed testing the reuse of the cascade classifier on stomata with similar shape. The results showed that a cascade classifier trained on only 100 sample views of stomata from Q. afares was able to rapidly detect stomata in Q. afares as well as in Q. suber with a very low number of false positives (5 %/1.9 %) and a small number of undetected stomata (14.8 %/0.74 %), when partial stomata near the edge of the microphotographs were ignored. The remaining undetected stomata were due to obstacles such as trichomes. As an example of further applications, we used the positions detected by the cascade classifier to assess the spatial distribution of stomata and group them on the leaf surface. To our knowledge this is the first time that a cascade classifier has been applied to plant microphotographs, and we were able to show that it can dramatically decrease the time needed to estimate stomatal density and spatial distribution.     

Direct measurement of the influx of sodium in frog skeletal muscle

When a frog's sartorius is immersed in sodium-free lithium-substituted solution at 0 degree C, the tissue sodium content declines in two distinct phases. The rate of the slow phase has a temperature dependence expected for a process dependent on metabolism (Q10, ca. 3), and sodium content (51.5 mmol/kg dry weight) equal to that measured by others using electron microprobe microanalysis. The rate of the rapid phase has a temperature dependence (Q10, 0.3-1) expected for a passive process, and a sodium content equal to that in the sorbitol space. It was concluded that incubation of a muscle at 0 degree C for 45 min in sodium-free solution will wash out almost all of the sodium in the extracellular space but will leave almost all the sodium in the intracellular space. The unidirectional sodium influx was measured by incubating a muscle in 22Na-containing Ringer's solution for a timed interval at 23 degrees C, then in sodium-free lithium-substituted solution at 0 degree C for 45 min, before analysis for ion content and radioactivity. The ratio of the specific activity of sodium in the muscle to that in the radioactive bathing solution was calculated, and the time course of its rise was used to calculate an influx rate coefficient. The use of the specific activity minimizes the error due to the loss of intracellular sodium and radiosodium which occurs during the wash in cold solution. It was found that the rate of the radiosodium uptake varied as the uptake proceeded, in a manner similar to that previously shown for the rate of the radiosodium efflux and attributed to the existence of a diversity of cell size in this muscle.