A new method for assessing arteriolar diameter and hemodynamic resistance using image analysis of vessel lumen

To characterize the nonuniform diameter response in a blood vessel after a given stimulus (e.g., arteriolar conducted response), frequent serial diameter measurements along the vessel length are required. We used an advanced image analysis algorithm (the "discrete dynamic contour") to develop a quick, reliable method for serial luminal diameter measurements along the arteriole visualized by intravital video microscopy. With the use of digitized images of the arteriole and computer graphics, the method required an operator to mark the image of the two inner edges of the arteriole at several places along the arteriolar length. The algorithm then "filled in" these marks to generate two continuous contours that "hugged" these edges. A computer routine used these contours to determine luminal diameters every 20 microm. Based on these diameters and on Poiseuille's law, the routine also estimated the hemodynamic resistance of the blood vessel. To demonstrate the usefulness of the method, we examined the character of spatial decay of KCl-induced conducted constriction along approximately 500-microm-long arteriolar segments and the KCl-induced increase in hemodynamic resistance computed for these segments. The decay was only modestly fitted by a simple exponential, and the computed increase in resistance (i.e., 5- to 70-fold) was only modestly predicted by resistance increase based on our mathematical model involving measurements at two arteriolar sites (Tyml K, Wang X, Lidington D, and Oullette Y. Am J Physiol Heart Circ Physiol 281: H1397-H1406, 2001). We conclude that our method provides quick, reliable serial diameter measurements. Because the change in hemodynamic resistance could serve as a sensitive index of conducted response, use of this index in studies of conducted response may lead to new mechanistic insights on the response.     

Experimental measurements of the temperature variation along artery-vein pairs from 200 to 1000 microns diameter in rat hind limb

Theoretical studies have indicated that a significant fraction of all blood-tissue heat transfer occurs in artery-vein pairs whose arterial diameter varies between 200 and 1000 microns. In this study, we have developed a new in vivo technique in which it is possible to make the first direct measurements of the countercurrent thermal equilibration that occurs along thermally significant vessels of this size. Fine wire thermocouples were attached by superglue to the femoral arteries and veins and their subsequent branches in rats and the axial temperature variation in each vessel was measured under different physiological conditions. Unlike the blood vessels < 200 microns in diameter, where the blood rapidly equilibrates with the surrounding tissue, we found that the thermal equilibration length of blood vessels between 200 microns and 1000 microns in diameter is longer than or at least equivalent to the vessel length. It is shown that the axial arterial temperature decays from 44% to 76% of the total core-skin temperature difference along blood vessels of this size, and this decay depends strongly on the local blood perfusion rate and the vascular geometry. Our experimental measurements also showed that the SAV venous blood recaptured up to 41% of the total heat released from its countercurrent artery under normal conditions. The contribution of countercurrent heat exchange is significantly reduced in these larger thermally significant vessels for hyperemic conditions as predicted by previous theoretical analyses. Results from this study, when combined with previous analyses of vessel pairs less than 200 microns diameter, enable one estimate the arterial supply temperature and the correction coefficient in the modified perfusion source term developed by the authors.     

Total root length as estimated from small sub-samples

A method is proposed for estimating the total length of a root system from sub-samples. This method is based on the measurement of the length and diameter of small pieces of roots, and on the measurements of the bulk density of root sub-samples. It is assumed that roots are cylinders with a given bulk density. The length and diameter of small root pieces are measured by image analysis. A weighted quadratic mean (W.Q.M.) root diameter is then calculated and used in estimating the root length. This W.Q.M. diameter is defined as the real mean diameter of an equivalent single root with the same length and volume as the tested root system. The accuracy of prediction is demonstrated for one theoretical root system. The standard deviation of estimation can be calculated using sampling simulations.     

Visible spectroscopic technique for flowing erythrocytes in capillary

An optical spectroscopic system for determining the rate of oxygen release from flowing erythrocytes in microvessel is developed. The apparatus consists of following units attached to an inverted microscope. 1) A scanning spectrophotometer, equipped with a grating and a photon counter, was connected to an eyepiece of the microscope through a narrow light-guide, as to obtain the absorption spectrum (wave length range: 450-650 nm) of a focused spot (phi = 7 microns). 2) The velocity of erythrocyte flow was measured by dual-spots cross-correlation method, using two photomultipliers (connected to A/D converter and microcomputer) with two light-guides inserted into another eyepiece. 3) The diameter of vessel was estimated from digitized video-images, using a color image-processor. The ability of the apparatus was tested with (a) hemoglobin solution, (b) flowing erythrocyte suspension and (c) capillaries of rat mesentery. The rate of oxygen release through the vessel wall was calculated.     

A comparison of two methods for measuring vessel length in woody plants

Vessel lengths are important to plant hydraulic studies, but are not often reported because of the time required to obtain measurements. This paper compares the fast dynamic method (air injection method) with the slower but traditional static method (rubber injection method). Our hypothesis was that the dynamic method should yield a larger mean vessel length than the static method. Vessel length was measured by both methods in current year stems of Acer, Populus, Vitis and Quercus representing short‐ to long‐vessel species. The hypothesis was verified. The reason for the consistently larger values of vessel length is because the dynamic method measures air flow rates in cut open vessels. The Hagen–Poiseuille law predicts that the air flow rate should depend on the product of number of cut open vessels times the fourth power of vessel diameter. An argument is advanced that the dynamic method is more appropriate because it measures the length of the vessels that contribute most to hydraulic flow. If all vessels had the same vessel length distribution regardless of diameter, then both methods should yield the same average length. This supports the hypothesis that large‐diameter vessels might be longer than short‐diameter vessels in most species.     

A computational algorithm addressing how vessel length might depend on vessel diameter

The objective of this method paper was to examine a computational algorithm that may reveal how vessel length might depend on vessel diameter within any given stem or species. The computational method requires the assumption that vessels remain approximately constant in diameter over their entire length. When this method is applied to three species or hybrids in the genus Populus, vessel length is sometimes a linear function of vessel diameter and sometimes an exponential function of vessel diameter within a stem, based on R² values. Our results give within‐species variation of vessel length versus diameter, and we compare this to between‐species variation of mean diameter versus mean length.     

Wood anatomy reflects the distribution of Krascheninnikovia ceratoides (Chenopodiaceae)

Wood anatomy characters of the Eurasian/North American half-shrub Krascheninnikovia ceratoides were investigated on plants from different parts of the wide distribution area. The secondary xylem is developed as result of anomalous secondary thickening. The vessels are small (commonly <50 μm) and the secondary xylem is rayless. Differences in vessel diameter exist between the main axis, basal branches and flowering shoots. The maximum as well as the minimum vessel diameter in the flowering shoots is significantly smaller compared to the basal branches, as well as in the main axis in all provenances. The length of thick-walled libriform fibre cells varies between the main axis and the basal branches of the same plant individual, but in the short main axis the length of libriform fibre cells is relatively constant (193-217 μm), independent of provenance and climate. Obviously, the length of libriform fibre cells is a conservative character and dependent on plant size. The vessel diameter is a more sensitive parameter for ecological studies. It varies more clearly, considering the vessel position in the plant axis system, in relation with climate differences of the provenances of the examined plants.Plants from a temperate, semi-humid climate have the largest vessel diameter in basal branches and flowering shoots, while the vessel diameter of plants from an arid temperate climate in Central Asia is smallest. The vessel diameters depend not only on climate, but also on the position in the axial system of the plants. There is a trade-off between average vessel number and their maximum diameter in flowering shoots of populations from Russia and Mongolia. Thus, vessel diameters depend on both, position in the axial system of the plant and climatic conditions.     

中国中生代鸟类的体重估计及其演化趋势

体重是一项重要的生物学指标,生物的体重受到发育、繁殖和进化等诸多因素的影响。对于灭绝生物体重的估计有助于进一步恢复它们的各种生物学信息。本研究采用统计学的方法,对422件现生鸟类(分属于21目229种)的体重和18项骨骼量度指标分别进行一元回归分析,结果显示判定系数的分布范围在0.5～0.91之间,多数指标的判定系数均集中在0.8～0.9之间。采用另外64件测量有体重数据和骨骼量度的鸟样本对回归方程的估算准确率进行检验,发现前肢中肱骨长度和尺骨宽度以及后肢中胫跗骨宽度3项指标的估算准确率高于其他指标。分析结果还表明前肢两项指标对于估算鸣禽、猛禽和攀禽类等树栖鸟类的体重准确率较后肢显著;后肢指标对于估算陆禽类等地栖鸟类体重的准确率高于前肢指标。这一结果反映出与体重相关程度较高的骨骼量度指标在不同习性的鸟类当中存在着一定的差异。对于化石鸟类的体重估计,采用估算准确率较高并且便于测量的肱骨长度和胫跗骨宽度两项回归方程加以计算。通过对中国中生代鸟类的体重进行估算,结果显示中生代鸟类在系统发育过程中,反鸟类经历了体重逐渐减轻的过程,而今鸟类的体重开始不断增大并且出现显著的分异。     

Noninvasive determination of perfused artery dimensions ex vivo using a pressure-diameter relationship

Based on the decisive effects of the hemodynamic and mechanical environments on the development and remodeling of arteries in vivo, several groups have cultured tissue-engineered vessels and excised vessels in various mechanically active perfusion systems. To facilitate the interpretation and design of such studies, accurate estimates of the applied forces and resulting stresses are required, which in turn require an accurate estimate of vessel dimensions. The measured pressure drop along the length of the vessel could be used to calculate the average inner diameter, but practical considerations, including the modest accuracy of many pressure transducers, limit this approach. Using nine porcine arteries harvested from pigs weighing between 25 and 100 kg, we show that when real-time measurements of the pressure drop and the outer diameter during a vasoactive event are fit to a theoretical model, offset errors in the pressure measurement can be compensated for and estimates of vessel wall transverse area with an average error of 4.1% (not exceeding 8.3%) are achieved.     

Maximum likelihood estimation of population growth rates based on the coalescent.

We describe a method for co-estimating 4Nemu (four times the product of effective population size and neutral mutation rate) and population growth rate from sequence samples using Metropolis-Hastings sampling. Population growth (or decline) is assumed to be exponential. The estimates of growth rate are biased upwards, especially when 4Nemu is low; there is also a slight upwards bias in the estimate of 4Nemu itself due to correlation between the parameters. This bias cannot be attributed solely to Metropolis-Hastings sampling but appears to be an inherent property of the estimator and is expected to appear in any approach which estimates growth rate from genealogy structure. Sampling additional unlinked loci is much more effective in reducing the bias than increasing the number or length of sequences from the same locus.     

Validation of OPS imaging for microvascular measurements during isovolumic hemodilution and low hematocrits

Orthogonal polarization spectral (OPS) imaging is a new technique that can be used to visualize the microcirculation with reflected light. It uses hemoglobin absorption to visualize the red blood cells (RBCs). Thus the method could fail at low hematocrit (Hct). The aim of this study was to validate OPS imaging for quantitative measurements of diameter and functional capillary density (FCD) under conditions of hemodilution of varying degrees to achieve a wide range of Hcts. The validation was performed in the dorsal skinfold chamber of nine awake Syrian golden hamsters. Measurements of vessel diameter and FCD were performed off-line using Cap-Image on video sequences captured using OPS imaging and standard intravital fluorescence microscopy at baseline, 85, 70, 55, and 40% of the initial Hct. For hemodilution, isovolumic exchange of blood for 6% Dextran 60 was performed. Bland-Altman plots for the vessel diameter and FCD show good agreement between the two methods for both parameters at all studied Hcts. As expected, there was a systematic bias of approximately 4 microm in the diameter measurements since the RBC column was measured and not the intravascular diameter. In conclusion, OPS imaging can be used to measure diameter and FCD at a wide range of Hcts.     

Improvement of fish length estimates for underwater visual census of reef fish biomass

Accuracy and precision are of great importance in the assessment of reef fish biomass when conducting an underwater visual census (UVC). Quantification and subsequent correction of the bias is required in order to standardize the estimates and correct for underwater distortion. To optimize the UVC, the observer should receive length‐measurement training in order to obtain in situ‐measurements that are as accurate and precise as possible. The objective of this study was to quantify the bias of fish length measurements made by divers with and without training in order to enhance reef fish biomass estimates. Adaptation of the diver to estimate fish lengths was analysed as a part of reef fish biomass monitoring in the Karimunjawa National Park, a national marine sanctuary in the Java Sea, Indonesia. Two divers practiced estimating a variety of fish in a natural environment by using styrofoam models attached to strings and sinkers. Analyses showed that by training the diver, his/her accuracy and precision improved substantially. Proving its reliability, an underwater visual census (UVC) becomes a useful and reliable method to assess the biomass of reef fishes.     

A comparison of methods for converting rhizotron root length measurements into estimates of root mass production per unit ground area

Rhizotrons provide valuable information about plant root production, but measurements are usually made in units of root length per unit surface area of observation window surface. These measurement units are not easily comparable to above-ground plant growth. To address this deficiency, several techniques have been developed to convert rhizotron measurement units into root mass production per unit ground area. In this study, four different conversion methods were applied to the same dataset of rhizotron measurements. This data was used to reveal the effect of conversion method upon estimates of the temporal variation in, and annual magnitude of, gross root mass production. Application of four different conversion methods resulted in gross root production estimates ranging between 2.1 and 11.4 t ha⁻¹ year⁻¹. Temporal variation in gross root mass production also varied between methods. All current methods for quantifying root production are likely to cause some disturbance and bias. Based upon a comparison of the sources of error present in each conversion method, we assess which methods are likely to produce the most reliable estimates of root biomass production per unit ground area, and propose additional measurements which could further improve accuracy.     

Bayesian analysis of serial dilution assays

In a serial dilution assay, the concentration of a compound is estimated by combining measurements of several different dilutions of an unknown sample. The relation between concentration and measurement is nonlinear and heteroscedastic, and so it is not appropriate to weight these measurements equally. In the standard existing approach for analysis of these data, a large proportion of the measurements are discarded as being above or below detection limits. We present a Bayesian method for jointly estimating the calibration curve and the unknown concentrations using all the data. Compared to the existing method, our estimates have much lower standard errors and give estimates even when all the measurements are outside the "detection limits." We evaluate our method empirically using laboratory data on cockroach allergens measured in house dust samples. Our estimates are much more accurate than those obtained using the usual approach. In addition, we develop a method for determining the "effective weight" attached to each measurement, based on a local linearization of the estimated model. The effective weight can give insight into the information conveyed by each data point and suggests potential improvements in design of serial dilution experiments.     

Leukocyte adhesion and microvessel permeability

To investigate the direct effect of leukocyte adherence to microvessel walls on microvessel permeability, we developed a method to measure changes in hydraulic conductivity (L(p)) before and after leukocyte adhesion in individually perfused venular microvessels in frog mesentery. In 19 microvessels that were initially free of leukocyte sticking or rolling along the vessel wall, control L(p) was measured first with Ringer-albumin perfusate. Blood flow was then restored in each vessel with a reduced flow rate in the range of 30-116 microm/s to facilitate leukocyte adhesion. Each vessel was recannulated in 45 min. The mean number of leukocytes adhering to the vessel wall was 237 +/- 22 leukocytes/mm(2). At the same time, L(p) increased to 4.7 +/- 0.5 times the control value. Superfusion of isoproterenol (10 microM) after leukocyte adhesion brought the increased L(p) back to 1.1 +/- 0.2 times the control in 5-10 min (n = 9). Superfusing isoproterenol before leukocyte adhesion prevented the increase in L(p) (n = 6). However, the number of leukocytes adhering to the vessel wall was not significantly affected. These results demonstrated that leukocyte adhesion caused an increase in microvessel permeability that could be prevented or restored by increasing cAMP levels in endothelial cells using isoproterenol. Thus cAMP-dependent mechanisms that regulate inflammatory agent-induced increases in permeability also modulate leukocyte adhesion-induced increases in permeability but act independently of mechanisms that regulate leukocyte adhesion to the microvessel wall. Application of ketotifen, a mast cell stabilizer, and desferrioxamine mesylate, an iron-chelating reagent, attenuated the increase in L(p) induced by leukocyte adhesion, suggesting the involvement of oxidants and the activation of mast cells in leukocyte adhesion-induced permeability increase. Furthermore, with the use of an in vivo silver stain technique, the locations of the adherent leukocytes on the microvessel wall were identified quantitatively in intact microvessels.     

Segmental barrier properties of the pulmonary microvascular bed.

We determined liquid flux across single pulmonary microvessels of dog, ferret, and rat by our split-drop technique (J. Appl. Physiol. 64: 2562-2567, 1988). Data are reported from 58 lungs excised under halothane or pentobarbital sodium anesthesia and then blood perfused. We stopped blood flow at known vascular pressures and then micropunctured microvessels to inject oil, which we split with albumin solution. From measurements of vessel diameter and split oil drop length, we calculated Jv, the liquid transport rate per unit surface area [x 10(-6) ml/(cm2.s)]. At constant vascular pressure, Jv was not significantly different after different periods of oil-endothelium contact and at different sites within a single vessel. From measurements of Jv at different vascular pressures, we determined Lp, the hydraulic conductivity [x 10(-7) ml/(cm2.s.cmH2O)], and Pzf, the zero filtration pressure. From determinations of Pzf at different albumin concentrations, we quantified sigma alb, the albumin reflection coefficient. Lp and Pzf did not differ among venules of the same lung. However, in venules, Lp was 40% higher and sigma alb 25% lower than in arterioles (P less than 0.01). We conclude that 1) micropuncture procedures incidental to our split-drop technique do not progressively deteriorate the experimental microvessel and 2) in lung, permeability is higher in venules than in arterioles.     

Estimating genetic drift and effective population size from temporal shifts in dominant gene marker frequencies

Measurement of temporal change in allele frequencies represents an indirect method for estimating the genetically effective size of populations. When allele frequencies are estimated for gene markers that display dominant gene expression, such as, e.g. random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers, the estimates can be seriously biased. We quantify bias for previous allele frequency estimators and present a new expression that is generally less biased and provides a more precise assessment of temporal allele frequency change. We further develop an estimator for effective population size that is appropriate when dealing with dominant gene markers. Comparison with estimates based on codominantly expressed genes, such as allozymes or microsatellites, indicates that about twice as many loci or sampled individuals are required when using dominant markers to achieve the same precision.     

TECHNIQUES FOR MEASURING VESSEL LENGTHS AND DIAMETERS IN STEMS OF WOODY PLANTS

Results were compared between the latex paint and compressed air methods for determining total vessel lengths, and between the sectioning and maceration methods for determining vessel diameters. The minimum, mean, median, and maximum vessel diameters were less with the sectioning method than with the maceration technique. Vessel diameter distributions were always nonnormal and had roughly similar patterns with the two techniques, but were statistically different from one another. In all six species where the paint and air methods for determining vessel length were compared, both methods showed a similar skewed vessel length distribution, with many short vessels and few long ones. Although there was no consistent pattern to the difference in results with these two methods, the vessel length frequency distributions were statistically different from one another. With the paint method, many vessels, especially many of the narrowest ones, were not paint-filled at the paint infusion port. The air method utilized the paint method, in part, and, in addition, is based upon the incorrect assumption that all vessels in the stem are the same diameter. Both techniques tended to exclude vessel lengths of the narrowest vessels. However, the narrow vessels, although numerous, contributed an insignificant amount to the total theoretical hydraulic conductance in stems.     

Forest community analysis and the point-centered quarter method

The point-centered-quarter (PCQ) method has been applied in community analysis since the publication of the method nearly 50 years ago. This and other distance methods offer increased sampling efficiency over fixed-area plots (FAP), but have long been known to produce biased density estimates when plant distribution deviates from random spatial patterns. Spatial indices have been developed to quantify the direction of this bias when plant distributions are aggregated or evenly distributed. Its continued use, especially in community analysis, requires additional scrutiny in measurements of community structure. We measured 14 forest stands of varying age, elevation and disturbance regime using FAP and PCQ methods. Density estimates were biased, with the point-centered quarter method lower than fixed-area plot estimates when stems were aggregated and higher when stems were evenly spaced. In general the PCQ method underestimated species richness. The efficiency of the PCQ method makes it popular for ordination studies, although comparison of community structure varied from 18% to 90% similarity between the measurements of species basal area in the same stands using the two different methods. The bias observed in calculations of stem density, species abundance and community similarity indicate that use of the PCQ method should be approached with caution when used in community level analysis.     

Assessing bias in diameter at breast height estimated from tree rings and its effects on basal area increment and biomass

Above-ground forest productivity can be reliably estimated from tree-ring width measurements. In doing so, annual growth is linked to the tree’s basal area increment (BAI), which is the change in cross-sectional area associated with each annual ring. When BAI is estimated from ring-width series, a value for the diameter of the tree is required. This diameter is ideally measured in the field, but can also be estimated as the sum of the annual ring widths. Tree biomass can also be estimated directly from the diameter estimates derived from tree-rings. Summing the ring widths, however, typically underestimates the tree’s true diameter. To evaluate this potential bias in diameter, we compared field-measured diameter and diameter estimated from the sum of the ring widths using tree-ring chronologies for seven common species in the eastern United States. We then evaluated the impacts of using the biased diameter estimates on derived BAI and biomass values. To simulate field-sampling error (i.e., failure to reach the pith when obtaining a core sample), we re-calculated BAI and biomass after removing a portion of the innermost rings from each tree. Comparisons of these various methods quantify the substantial and consistent underestimations in forest productivity estimates. To reduce the bias in diameter when using ring widths, we developed a regression model to adjust the diameter using core samples. This model is predicated on having some field-measured diameter values available at a site to calibrate and validate the model, but it can then be used to produce estimates at similar sites with similar species where no field-measured diameter values are available. Values of BAI and biomass derived from model-estimated diameter were more accurate at representing absolute growth than values produced by using the sum of the ring widths. Assessing the interannual variations in tree-growth is dependent on having metrics that accurately reflect the area and mass of wood produced. Our results suggest that published estimates of BAI and biomass using the sum of the ring widths to estimate diameter have substantially underestimated these productivity metrics. Our new procedure allows for more reliable estimates of productivity metrics that use diameter-at-breast height derived from tree rings.